pectinase ph experiment

Production of cold-active pectinases by three novel Cladosporium species isolated from Egypt and application of the most active enzyme

• Ahmad Mohamed Moharram 1 , 2 ,
• Abdel-Naser Ahmed Zohri 1 ,
• Abd El-Latif Hesham 3 ,
• Hossam E. F. Abdel-Raheam 4 ,
• Mohamed Al-Ameen Maher 1 &
• Osama Abdel-Hafeez Al-Bedak 2  

Scientific Reports volume  12 , Article number:  15599 ( 2022 ) Cite this article

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• Biotechnology
• Microbiology

An Author Correction to this article was published on 22 September 2023

This article has been updated

Cladosporium parasphaerospermum , Cladosporium chlamydosporigenum , and Cladosporium compactisporum have all been discovered and characterized as new Cladosporium species. The three new species seemed to generate cold-active pectinases with high activity at pH 6.0 and 10 °C, pH 6.0 and 15 °C, and pH 5.0 and 15 °C, respectively, with the most active being C. parasphaerospermum pectinase. In submerged fermentation (SmF), C. parasphaerospermum produced the most cold-active pectinase with the highest activity and specific activity (28.84 U/mL and 3797 U/mg) after 8 days. C. parasphaerospermum cold-active pectinase was isolated using DEAE-Cellulose anion exchange resin and a Sephadex G 100 gel filtration column. The enzyme was purified 214.4-fold and 406.4-fold greater than the fermentation medium using DEAE-cellulose and Sephadex G 100, respectively. At pH 7.0 and 10 °C, pure pectinase had the highest activity (6684 U/mg), with K m and V max determined to be 26.625 mg/mL and 312.5 U/min, respectively. At 5 mM/mL, EDTA, MgCl 2 , and SDS inhibited the activity of pure pectinase by 99.21, 96.03, and 94.45%, respectively. The addition of 10 U/mL pure pectinase enhanced the yield of apple, orange, apricot, and peach juice by 17, 20, 13, and 24%, respectively, and improved the clarity and colour of orange juice by 194 and 339%, respectively. We can now add cold-active pectinase production to the long list of Cladosporium species that have been identified. We also report three new species that can be used in biotechnological solutions as active microbial pectinase producers. Although further research is needed, these distinct species might be used to decompose difficult and resistant pectinacious wastes as well as clear fruit juices.

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Introduction.

Cladosporium is one of the largest and most complex genera of hyphomycetes, which currently includes more than 728 names. Until recently, all types of unrelated dematiaceous hyphomycetes characterized by amero-to-phragmosporous conidia formed in acropetal chains had been referred to Cladosporium 1 . Species of Cladosporium are well adapted to spread easily in large numbers over long distances, therefore they are cosmopolitan and widely present in all various types of plants and other debris, mostly isolated from air, soil, seeds, grains, food, paint, textiles and other organic matter 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 . Several species of this genus are plant pathogenic causing leaf spots and other lesions 10 , or they occur as hyperparasites on some fungi 11 .

Cladosporium species are also known to be common endophytes 12 , 13 , 14 as well as phylloplane fungi 15 , 16 , 17 , 18 . Some Cladosporium species including C . cladosporioides , C . chlorocephalum and C . uredinicola were recorded as entomopathogens of Aphids and whiteflies 19 , 20 . The Genus Cladosporium is considered to be a rich source of diverse and bioactive natural compounds 21 . Some species were reported to produce anticancer compounds such as L-asparaginase 22 , paclitaxel 23 and useful enzymes including cellulases 24 and pectinases 25 , 26 , 27 .

Enzymes are nowadays in great demand in a variety of industrial applications, including food, detergent, paper, textiles, and organic chemical synthesis, due to their high efficiency and environmental friendliness 28 , 29 , 30 . Furthermore, these enzymes are part of a well-established worldwide industry that is expected to grow to US$6.3 billion by few years 28 , 29 , 30 , 31 . The current tendency is to utilize cold-active enzymes to lower the temperature of industrial processes, allowing for energy savings and reduced carbon footprint, as well as the productive capacity that operate better at ambient or lower temperatures 32 , 33 , 34 . Because they are (i) cost-effective, (ii) energy saving, (iii) capable of catalyzing processes without additional heat aid, and (iv) selectively inactivated by mild heat input 35 . In biotechnology, cold-active enzymes are used to prevent a range of unwanted reactions and restrict the loss of volatile components 36 , 37 , 38 , 39 . As a result of these inevitable uses, the function of cold-active enzymes is expected to increase dramatically in the next years 39 , 40 .

Pectin is one of the most numerous and complicated polymers that make up the plant cell wall. It is a group of polysaccharides that contain at least seven structural components, the most well-known of which are homogalacturonan, xylogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II 41 , 42 . Pectin is made up of a main chain of galacturonic acid residues bound by (14) links (homogalacturonan), or a mixture of galacturonic acid and rhamnose (rhamnogalacturonans) or galacturonic acid and xylose (rhamnogalacturonans) (xylogalacturonan). Various molecules, such as methyl, ethyl, and various sugar moieties (arabinose, rhamnose, galactose, and others), can then be replaced for the main chain 43 . Pectin biodegradation necessitates the coordinated activity of multiple enzymes known collectively as pectinases, due to its complicated structure. Pectin methyl esterases, pectin acetyl esterases, polygalacturonases, polymethylgalacturonases, polygalacturonate lyases, polymethylgalacturonate lyases, rhamnogalacturonase, arabinases, and xylogalacturonases are all examples of pectinases 42 .

Around 10% of the enzyme market is made up of pectinolytic enzymes, which are used in the juice, food, paper, and textile industries 44 , 45 , 46 . Low temperatures (15 °C) are employed in the juice industry to minimize cloudiness and bitterness in fruit juices in order to prevent the spread of harmful bacteria, preserve labile and volatile flavor components, and save energy 34 , 45 . Researchers have been looking for pectinases that can act at low temperatures but also at low pH, because the pH of fruit juices and grape must be between 2.5 and 3.5 42 . Pectinases are presently derived from mesophilic filamentous fungi, mostly Aspergillus species, however they work poorly below 35 °C 47 . Cold-active enzymes, on the other hand, have higher enzymatic activity than mesophilic enzymes at lower temperatures 48 . As a result, the current research sought to develop cold-active pectinases from three newly-discovered Cladosporium species from Egypt, as well as purify, characterize, and use the most active pectinase in fruit juice production.

Molecular studies

Cladosporium parasphaerospermum aumc 10865.

ITS:  Based on a megablast search of NCBIs GenBank nucleotide database, the closest hits using ITS sequence are Cladosporium cladosporioides , Cladosporium parahalotolerans and Cladosporium halotolerans [(GenBank KJ767065, MK262909 and MK258720; identities = 554/556 (99.64%); Gaps = 1/556 (0%)]. ACT: the closest hits using ACT sequence are Cladosporium halotolerans and Cladosporium omanense [(GenBank MF084398 and MH716046; identities 127/132 (96.21%); Gaps = 1/132 (0%)]. LSU: the closest matches using LSU are Digitaria exilis [(GenBank LR792838; identities = 1142/1152 (99.13%); Gaps = 10/1152 (0%)] and Cladosporium delicatulum [(GenBank JQ732984 and JQ732983; identities = 1140/1152 (98.96%); Gaps = 10/1152 (0%)].

Cladosporium chlamydosporigenum AUMC 11340

ITS:  Based on a megablast search of NCBIs GenBank nucleotide database, the closest hits using ITS sequence are Cladosporium subcinereum [(GenBank OK510262; identities = 551/554 (99.46%); Gaps = 2/554 (0%)] and Cladosporium floccosum [(GenBank MK460809; identities = 549/553 (99.28%); Gaps = 3/553 (0%)]. ACT: the closest hits using ACT sequence are Davidiella tassiana [(GenBank EU730605; identities = 229/230 (99.57%); Gaps = 0/230 (0%)] and Cladosporium herbarum [(GenBank EF679510 and EF679511; identities = 228/230 (99.13%) and 227/230 (98.70%); 0/230 (0%)]. While compared to the type materials, the closest hits are Cladosporium macrocarpum [(GenBank EF679529; identities = 226/230 (98.26%); Gaps = 0/230 (0%)], Cladosporium herbarum [(GenBank EF679516; identities = 225/230 (97.83%); Gaps = 0/230 (0%)] and Cladosporium versiforme [(GenBank KT600613; identities = 216/227 (95.15%); Gaps = 3/227 (1%)]. LSU: the closest hits using LSU sequences are Cladosporium herbarum and Cladosporium allicinum [(GenBank MH047193 and GU214408; identities = 1184/1199 (98.75%); Gaps = 13/1199 (1%)].

Cladosporium compactisporum AUMC 11366

ITS:  Based on a megablast search of NCBIs GenBank nucleotide database, the closest hits using ITS sequence are Cladosporium cladosporioides [(GenBank ON045558 and MT367253; identities = 547/552 (99.09%) and 548/554 (98.92%); Gaps = 0/552 (0%) and 1/554 (0%)]. ACT: the closest hits using ACT sequence are Cladosporium cladosporioides [(GenBank KY886457; identities = 232/232 (100%); Gaps = 0/232 (0%)], Cladosporium proteacearum (ex-type) [(GenBank MZ344213; identities = 223/229 (97.38%); Gaps = 0/229 (0%)], Cladosporium devikae (ex-type) [(GenBank MZ344212; identities = 212/216 (98.15%); Gaps = 1/216 (0%)] and Cladosporium cladosporioides (ex-type) [(GenBank HM148490; identities = 220/230 (95.65%); Gaps = 0/230 (0%)]. LSU: the closest hits using LSU sequence are Cladosporium delicatulum and Cladosporium uredinicola [(GenBank JQ732985 and EU019264; identities = 1206/1221 (98.77%); Gaps = 12/1221 (0%)], Toxicocladosporium irritans (ex-type) [(GenBank EU040243; identities = 1184/1221 (96.97%); Gaps = 12/1221 (0%)], and Rachicladosporium inconspicuum (type) [(NG_059443; identities = 1159/1224 (94.69%); Gaps = 16/1224 (1%)].

Phylogenetic analyses

Descriptive statistical parameters of phylogenetic analyses and calculated tree scores for each analyzed sequence locus are summarized in Table 1 . The constructed phylogenetic trees for ITS, ACT and LSU are shown in Figs. 1 , 2 , 3 , respectively.

Cladosporium parasphaerospermum sp. nov

Moharram AM, Zohri AA, Hesham A, Maher MA and Al-Bedak OA.

Name refers to globose to subglobose conidia near to that of C. sphaerospermum .

Egypt, Beni Suef, Air, Maher MA, AUMC 10865. Ex-type culture: EMCCN: 2062.

Macroscopic and microscopic characteristics

Colonies on PDA reaching 18–20 mm diameter after 7 days at 28 °C, raised at the center, radially sulcate, radially furrowed under the colony, olive, olive green at the center (2F6). Margin curled, paler than the colony center (3F8). Sporulation profuse. Exudates absent. Colonies on SNA reaching 17–19 mm in diameter after 7 days at 28 °C, flat, slightly raised at the center, circular, olive, olive green (3F4-6). Margin entire. Sporulation abundant. Exudates absent. On OA colonies attaining 16–19 mm in diameter after 7 days at 28 °C, circular, flat, somewhat lanuginose, dark olive, dark olive green (1F4-6). Margin entire. Sporulation abundant. Exudates absent. Mycelium abundantly formed, branched, 3–5 µm wide, septate, pale brown to brown, smooth. Conidiophores macronematous, micronematous, abundantly formed, arising terminally or laterally, more or less straight to flexuous, cylindrical, pale brown to brown, smooth, septate, commonly (− 35) 75–100 × 3–5 µm (av. 87.5 × 4) µm (n = 50), not constricted at septa. Ramoconidia integrated, terminal, intercalary, cylindrical, smooth, thick-walled, 0–1 septate, 8–18 × 3–5 µm (av. 13 × 4) µm (n = 50), with 1–3 loci per cell. Loci usually confined to small lateral shoulders, protuberant, conspicuous, short cylindrical, 1–2 µm wide, up to 1–2 µm high. Conidia brown to dark brown, smooth, thick-walled, globose, subglobose, lemon-shaped, 0–septate, 4–6 × 3–5 µm (av. 5 × 4) µm (n = 50). Chlamydospores not formed (Fig. 4 ).

Maximum likelihood phylogenetic tree generated from ML/MP combination analysis based on alignment of ITS sequences of C. parasphaerospermum AUMC 10865, C. chlamydosporigenum AUMC 11340 and C. compactisporum AUMC 11366 with the most similar sequences belonging to Cladosporium in GenBank database. Sequences of species in this study are in blue color. Bootstrap support values (1000 replications) for ML/MP combination equal to or greater than 50% are indicated at the respective nodes. The tree was rooted to sequence of Cercospora beticola CBS 116456 as outgroup (in red color).

Maximum likelihood phylogenetic tree generated from ML/MP combination analysis based on alignment of ACT sequences of C. parasphaerospermum AUMC 10865, C. chlamydosporigenum AUMC 11340 and C. compactisporum AUMC 11366 with the most similar sequences belonging to Cladosporium in GenBank database. Sequences of species in this study are in blue color. Bootstrap support values (1000 replications) for ML/MP combination equal to or greater than 50% are indicated at the respective nodes. The tree was rooted to sequence of Cercospora beticola CBS 116456 as outgroup (in red color).

Maximum likelihood phylogenetic tree generated from ML/MP combination analysis based on alignment of LSU sequences of C. parasphaerospermum AUMC 10865, C. chlamydosporigenum AUMC 11340 and C. compactisporum AUMC 11366 with the most similar sequences belonging to Cladosporium in GenBank database. Sequences of species in this study are in blue color. Bootstrap support values (1000 replications) for ML/MP combination equal to or greater than 50% are indicated at the respective nodes. The tree was rooted to sequence of Cercospora beticola CBS 116456 as outgroup (in red color).

Cladosporium parasphaerospermum (AUMC 10865). ( A–C ) 7-day-old colonies on PDA, SNA and OA at 25 °C. ( D–F ) Macronematous conidiophores and conidial chains. ( G ) Globose to subglobose conidia. Scale bar = 20 µm ( D ), 10 µm ( E–G ).

Cladosporium chlamydosporigenum sp. nov

Refers to the formation of chlamydospores in culture.

Egypt, Sohag, Grapevine fruits, Maher MA, AUMC 11340. Ex-type culture: EMCCN: 2332.

Colonies on PDA reaching 15–17 mm diameter after 7 days at 28 °C, raised at the center, wrinkled, irregular, olive, olive green (3F6-7). Margin undulate, narrow, paler than the center (3E5-6). Sporulation abundant. Exudates absent. On SNA colonies attaining 9–11 mm in diameter after 7 days at 28 °C, flat, filamentous, olive, olive green (3E2). Margin filiform, narrow, (3E6-7). µm (av. 250 × 5) µm (n = 50), not constricted at septa. Ramoconidia integrated, terminal, intercalary, cylindrical, 11–22 × 6–8 µm (av. 16.5 × 7) µm (n = 50), verruculose to finely roughened, 0–2 septa with 1–3 loci per cell, Loci usually confined to small lateral shoulders, protuberant, conspicuous, short cylindrical, 1–2 µm wide, up to 1 µm high. Conidial chains unbranched or branched, conidia pale brown, straight, subglobose, obovoid to ellipsoid, verruculose to finely roughened, 0–septate, 4–11 × 5–7 µm (av. 7.5 × 6) µm (n = 50). Chlamydospores produced in hyphae, intercalary, aggregated, brown to dark brown, thick-walled, globose, subglobose, 15–25 × µm (Fig.  5 ).

Cladosporium chlamydosporigenum (AUMC 11340). ( A–C ) 7-day-old colonies on PDA, SNA and OA at 25 °C. Mycelium with abundant chlamydospores. ( D–E ) Macronematous conidiophores and conidial chains. ( F ) Aggregated chlamydospores. Scale bar = 20 µm.

Cladosporium compactisporum sp. nov

Refers to the compact conidial chains.

Egypt, Qena, Air, Maher MA, AUMC 11366. Ex-type culture: EMCCN: 2358.

Colonies on PDA attaining 25–28 mm after 7 days at 28 °C, raised, umbonate, circular, olive to olive green (3E3-3F4). Margin entire, narrow, about 3.0 mm in width, paler than the colony center (3E1-3). Sporulation profuse. Exudates absent. Colonies on SNA attaining 17–20 mm diameter after 7 days at 28 °C, raised, umbonate, olive to olive green (3F6-7). Margin entire, about 3.0 mm in width, paler than the colony center (3E2-3). Sporulation abundant. Exudates absent. Colonies on OA attaining 19–23 mm in diameter after 7 days at 28 °C, raised, umbonate, lanuginose, olive grey (2E1-2). Margin undulate, narrow, dark olive grey (2F2). Sporulation abundant. Exudates lacking. Mycelium abundantly formed, branched, 3–5 µm wide, septate, swollen, pale brown to brown, smooth. Conidiophores macronematous and micronematous, abundantly formed, arising terminally or laterally, more or less straight to flexuous, nodulose, geniculate at the upper part, cylindrical, pale brown to brown, smooth, septate, branched, 100–300 µm × 3.0–6.0 µm (av. 200 × 4.5) µm (n = 50). Ramoconidia integrated, terminal, intercalary, cylindrical, 7–22 × 3–4 µm (av. 14.5 × 3.5) µm (n = 50), smooth, 0–1 septa with 1–3 loci per cell. Loci usually confined to small lateral shoulders, protuberant, conspicuous, short cylindrical, 1 µm wide, up to 1–2 µm high. Conidia formed in compact and branched chains, pale brown, subglobose, obovoid to ellipsoid, smooth, 0-septate, 4–6 × 3–4 µm (av. 5 × 3.5) µm (n = 50). Chlamydospores not formed (Fig.  6 ).

Cladosporium compactisporum (AUMC 11366). ( A–C ) 7-day-old colonies on PDA, SNA and OA at 25 °C. ( D–F ) Geniculate and swollen conidiophore bearing compact chains of conidia. Scale bar = 20 µm.

Optimization of cold-active pectinases production by the three Cladosporium strains

Effect of ph and temperature on pectinase production.

Cladosporium parasphaerospermum AUMC 10865, Cladosporium chlamydosporigenum AUMC 11340, and Cladosporium compactisporum AUMC 11366 cold-active pectinase production was investigated in this work by altering the pH of the fermentation medium between pH 3.0 and 10.0 each at 5°, 10°, and 15°. Cladosporium parasphaerospermum AUMC 10865 produced the most pectinase (26.3 ± 2.1 U/mL) at pH 6.0 and 10 °C (Fig.  7 ), whereas C. chlamydosporigenum AUMC 11340 produced the most (24.63 ± 2.5 U/mL) at pH 6.0 and 15 °C (Fig.  8 ), and C. compactisporum AUMC 11366 (21.93 ± 2.3 U/mL) at pH 5.0 and 15 °C (Fig.  9 ).

Effect of pH at 5, 10, and 15 °C on the pectinase production by Cladosporium parasphaerospermum AUMC 10865.

Effect of pH at 5, 10, and 15 °C on the pectinase production by Cladosporium chlamydosporigenum AUMC 11340.

Effect of pH at 5, 10, and 15 °C on the pectinase production by Cladosporium compactisporum AUMC 11366.

Effect of nitrogen source and incubation time

Pectinase production by Cladosporium parasphaerospermum AUMC 10865 was enhanced (28.84 ± 2.7 U/mL) after 8 days incubation using sodium nitrate as a nitrogen source (Fig.  10 ). While, ammonium chloride was found to be best for pectinase production by Cladosporium chlamydosporigenum AUMC 11340 (26.6 ± 1.28 U/mL), and Cladosporium compactisporum AUMC 11366 (24.01 ± 1.76 U/mL) after 9 days (Figs. 11 , 12 ).

Effect of nitrogen source and incubation time on the pectinase production by C. parasphaerospermum AUMC 10865 at pH 6.0 and 10 °C after 8 days.

Effect of nitrogen source and incubation time on the pectinase production by C. chlamydosporigenum AUMC 11340 at pH 5.0 and 10 °C after 9 days.

Effect of nitrogen source and incubation time on the pectinase production by C. compactisporum AUMC 11366 at pH 7.0 and 5 °C after 9 days.

Production of cold-active pectinase by C. parasphaerospermum AUMC 10865 in SmF

In submerged fermentation at the optimum conditions, the three fungi produced pectinases at a rather high output. Cladosporium parasphaerospermum generated 5.6 g of pectinase powder per liter of fermentation media, followed by C. chlamydosporigenum at 3.65 g and C. compactisporum at 2.85 g (Fig.  13 ). For purification and use, the C. parasphaerospermum AUMC 10865 pectinase that was the most cold-active was chosen.

Pectinases powder produced by Cladosporium strains in SmF.

Purification of pectinase produced by Cladosporium parasphaerospermum AUMC 10865

The Cladosporium parasphaerospermum AUMC 10865 pectinase was homogenized using a number of techniques. Initial partial purification of the enzyme involved adding solid ammonium sulphate to the cell-free supernatant. The fraction with a salt saturation of 70% showed pectinase activity. This fraction was dialyzed with citrate buffer (pH 6.0) and freeze-dried before being applied to the anion exchanger (DEAE-Cellulose), which was pre-equilibrated with 50 mM citrate buffer (pH 6.0). The proteins were extracted using a gradient of NaCl (0–1.5 M).

Purification profile of pure pectinase

Pectinase activity was discovered in fractions 60–150 of a DEAE-Cellulose column (Fig.  14 ), which were pooled, concentrated, and dialyzed against citrate buffer (pH 6.0). This cycle of purification increased pectinase purity by 214.4-fold, with a specific activity of 1005.55 U/mg protein (Table 2 ). The fractions with the highest pectinase activity were pooled, condensed with a freeze drier, and loaded onto a Sephadex G-100 column (Fig.  15 ). The pectinase activity-highest fractions were pooled, concentrated, and dialyzed against citrate buffer (pH 6.0). With a specific activity of 1906 ± 65 U/mg protein, this phase of purification resulted in a 406.4-fold improvement in pectinase purity (Table 2 ).

Purification of pectinase by ion exchange chromatography using DEAE-cellulose.

Purification of pectinase by size exclusion chromatography using Sephadex G-100.

Effect of pH and temperature on the pure pectinase activity

The activity of pure pectinase was further tested in the presence of various physical and chemical factors. The purified pectinase has an optimal pH of 7.0. The purified pectinase had the highest activity (4553 ± 124 U/mg) at pH 7.0 and 5 °C, which increased to 6684 ± 173 U/mg at 10 °C (Fig.  16 ).

Effect of pH and temperature on activity of the pure pectinase.

Effect of some ions and inhibitors on the pure pectinase activity

The purified enzyme was sensitive to all salts tested at a concentration of 5 mmol/mL. Pectinase activity was significantly reduced by 99.21, 96.03, and 94.45% using EDTA, MgCl 2 , and SDS, respectively in the reaction (Table 3 ).

Kinetic constants of the pure pectinase

The current findings revealed that Michaelis–Menten constant (K m ) and the maximum reaction velocity (V max ) values for the pure pectinase were calculated as 26.625 mg/mL and 312.5 U/min (Fig.  17 ).

Line-weaver-Burk equation used for K m and V max calculation.

Fruit juice production by the pure pectinase

When compared to the control, the enzyme treatment for all of the fruit pulp used resulted in a significant improvement in juice yield, clarity, and colour. It was determined how enzyme treatments affect the extraction of apple, orange, apricot, and peach juices. Enzyme addition increased juice recovery in all fruits. The enzyme treatment of apple, orange, apricot, and peach resulted in a significant increase in juice yield of 16.45, 16.43, 15.93, and 8.73%, respectively. The results also revealed a significant improvement in the clarity and colour of the juice derived from the pulp of the orange fruit, reaching 194.0% and 338.6%, respectively, above the control, while the rest of the fruits employed showed just a modest rise (Table 4 ).

Identification of novel species is at the heart of biodiversity research, and in recent years biodiversity efforts have been favouring DNA based method to morphology-based ones 49 , 50 , 51 , 52 , 53 . Understanding of the microbial composition aids awareness of host–microbe interactions and their environmental function, revealing a complex and delicate balance that can be easily upset 54 , 55 , 56 . Due to the complexity of fungal genomes and the lack of verified databases documenting appropriate biodiversity, such metagenomic studies in fungi are still limited. Identification of novel species is crucial in biodiversity research, which has lately adopted DNA-based methodologies. In this work, we introduced three new Cladosporium species as Cladosporium parasphaerospermum , Cladosporium chlamydosporigenum , and Cladosporium compactisporum based on the morphological characteristics as well as phylogenetic analyses of ITS, ACT, and LSU loci.

Cladosporium parasphaerospermum , C. chlamydosporigenum , and C. compactisporum species clades were supported by ITS rDNA and partial actin gene analyses, with C. parasphaerospermum and C. compactisporum separated in the ACT tree each by a single long branch, and C. chlamydosporigenum separated by a single short branch. Because the three strains occupied discrete lineages, the LSU tree validated their uniqueness. C. parasphaerospermum can be recognized from C. halotolerans 57 , and C. parahalotolerans 57 by its smaller ramoconidia (8–18 µm), which measure 15–37 and 24–37 µm, in both species, respectively. C. chlamydosporigenum is distinguished from other Cladosporium species in ITS clade by smaller conidia (4–11 µm) and ramoconidia (11–22 µm), as well as the formation of chlamydospores and the absence of head-like swellings with additional intercalary swellings. C. compactisporum was discovered in ITS tree as part of a moderately supported clade alongside C. cladosporioides and C. tenuissimum , and in the ACT tree as part of the C. salinae clade on a lengthy distinct branch. It produces smaller ramoconidia (7–22 µm) with 1–3 loci than C. cladosporioides (15–50 µm), which has up to 4 loci packed at the tip. C. compactisporum differs from C. tenuissimum 57 in that it has geniculate and nodulose conidiophores with compact conidial chains, whereas most C. tenuissimum conidiophores are neither geniculate nor nodulose. On Oat agar, C. tenuissimum has a longer conidiophore (up to 900 µm) than C. compactisporum . Conidiophores of 100–300 × 3–6 µm in length and ramoconidia of 7–22 µm in length distinguish C. compactisporum from C. salinae 58 , which has weakly differentiated conidiophores (25–50 × 2.5–3 µm) and smaller ramoconidia (9.5–13.5 × 2.5–3.5 µm).

The current research aimed to isolate cold-active pectinases from three novel Cladosporium species that could function at low temperatures. The three strains produced a considerable amount of cold-active pectinases, which were active at temperatures as low as 5 and 10 °C. This is the first report of cold-active pectinase production from psychrotolerant Cladosporium species that we are aware of.

Pectinase has lengthy been used in commercial food processing to degrade pectin and aid in various processing steps such as liquefaction, clarification, and juice extraction 59 . Pectinases are among the most widely used enzymes, accounting for 40% of all food enzymes 44 , 60 . It has been demonstrated that certain Cladosporium species generate active pectinases 9 , 25 , 26 , 61 , 62 , 63 . However, the synthesis, optimization, purification, and application of a cold-active pectinase in this study is groundbreaking. Due to minor changes in methodology, it is difficult to compare the values of enzyme activity reported by different researches. As a result, comparisons should be made with care.

Because of their biodegradability, non-toxicity, high selectivity, and high yields, microbial enzymes are superior to chemical synthesis 64 . The global enzyme market was valued at $9.9 billion in 2019 and is expected to grow at a 7.1% annual rate from 2020 to 2027 65 . The majority of commercial enzymes, including pectinase, are now mesophilic or thermophilic. In the food sector, and particularly in the fruit processing sector, there has been an increasing desire to replace high-temperature procedures with low-temperature processes. Specific economic and environmental benefits, such as energy savings, retention of biologically inert and aromatic fragrance components, contamination mitigation, and eradication of any residual enzyme activity, which cause deactivation of enzyme when temperature is raised, are driving this shift in trend 34 , 59 , 65 , 66 , 67 .

Pectinases released by microorganisms account for approximately 25% of global food enzyme sales. The vast majority of which is derived from filamentous fungi, specifically Aspergillus niger 68 , 69 . It is uncommon for filamentous fungi to produce pectinase activity below 40 °C. This is true even for filamentous fungi that are psychrophilic or psychrotolerant. Sclerotinia borealis , a pathogenic fungus prevalent in extremely cold locations that does not grow over 20 °C, generates pectinases with optimal activity at 40 °C 70 . Mucor flavus is another example of a psychrotolerant fungus that generates pectinases with optimum activity at 45 °C 71 .

To the best of our knowledge, there is just one case of a filamentous fungus generating pectinases with optimal activity below 40 °C in the literature. Botrytis cinerea , a phytopathogenic fungus, generates pectinases with optimum activity between 34 and 37 °C 72 . In this investigation, Cladosporium parasphaerospermum produced high quantity of pectinase with the maximum activity at pH 7.0 and 10 °C. Thus, this is the first study to purify and exploit cold-active pectinase from Cladosporium species, which might be great candidates for cold-active enzyme synthesis. Industrial pectinases generated from fungi are a blend of pectinolytic enzymes and other proteins. Other commercial processes, such as fruit juice clarity, require only one kind of pectinase activity. As a result, other sources of pectinase must be investigated. The catalytic properties and stability of an enzyme in diverse physio-chemical conditions are important for commercialization.

Pectinase activity in a pure preparation of Cladosporium parasphaerospermum was described and evaluated in this work for its potential application in fruit juice clearing. The use of Cladosporium parasphaerospermum pure pectinase improved juice recovery in all fruits used. The enzyme treatment of apple, orange, apricot, and peach resulted in a considerable increase in juice output. The results also demonstrated a considerable improvement in the purity and colour of the juice obtained from orange fruit.

In the current study, three novel Cladosporium species were introduced and described as Cladosporium parasphaerospermum , C. chlamydosporigenum , and C. compactisporum . The three novel species appeared to produce cold-active pectinases that had high activity at pH 6.0 and 10 °C, pH 6.0 and 15 °C, and pH 5.0 and 15 °C, respectively, of which C. parasphaerospermum pectinase was the most active. The enzyme was purified by 214.4-fold and 406.4-fold by DEAE-Cellulose and Sephadex G 100, respectively. The highest activity of the pure pectinase was gained at pH 7.0 and 10 °C. K m and V max were calculated to be 26.625 mg/mL and 312.5 U/min, respectively. The use of pure pectinase boosted the yield of apple, orange, apricot, and peach juice and improved the clarity and colour of orange juice. We can now add cold-active pectinase production to the long list of Cladosporium species that have been identified. We also report three new species that can be used in biotechnological solutions as active microbial pectinase producers. Although further research is needed, these distinct species might be used to decompose difficult and resistant pectinacious wastes as well as clear fruit juices.

Materials and methods

Isolation and maintenance of cladosporium strains.

Three Cladosporium isolates involved in the current study, of which two were isolated from air of Beni Suef and Qena cities and one from fruits of grapevine cultivated in Sohag city, Egypt. Settle plate method 73 was employed for isolation of Cladosporium from air and direct plating technique 74 for isolation from grapevine fruits. Czapek’s Dox agar was used as an isolation medium. The isolation medium contained (g/L): Sucrose, 30; Na 2 NO 3 , 2; K 2 HPO 4 , 1; KCl, 0.5; MgSO 4 .7H 2 O, 0.5; FeSO 4 , 0.01; ZnSO 4 , 0.01; CuSO 4 , 0.005; Rose Bengal, 0.05; chloramphenicol, 0.25; agar, 15 and the final pH 7.3. The newly discovered strains were preserved as frozen and lyophilized cultures and added to the culture collections of the Assiut University Mycological Centre (AUMC) and the Egyptian Microbial Culture Collection Network (EMCCN) as AUMC 10865 = EMCCN 2062 (Air, Beni Suef, Egypt), AUMC 11340 = EMCCN 2332 (Grapevine fruits, Sohag, Egypt), and AUMC 11366 = EMCCN 2358 (Air, Qena, Egypt). The new species were catalogued in the MycoBank with accession numbers MB844532, MB844533, and MB844534, respectively, along with their descriptions. Nexus file of the sequence alignments for all data sets were uploaded to Tree BASE http://purl.org/phylo/treebase/phylows/study/TB2:S30171?x-access-code=a18696d1afcf659925a63a31c3ebd045&format=html (Study no. 30171).

Morphological studies of the Cladosporium strains

For growth rate determination and phenetic description of colonies, strains were point inoculated on potato dextrose agar (PDA), synthetic nutrient agar (SNA) and oat meal agar (OA) 75 , 76 , and incubated at 25 °C for 14 days in darkness. Surface colours were rated using the colour charts 77 .

Molecular identification of the Cladosporium strains

Dna extraction, pcr and sequencing of its, act and lsu.

DNA isolation of Cladosporium isolates AUMC 10865, AUMC 11340 and AUMC 11366 was performed following CTAB method 78 . The universal primers ITS1 and ITS4 79 were used for amplification of the internal transcribed spacer (ITS) region, ACT783R and ACT512F for amplification of ACT gene 80 , and LROR and LR7 81 for amplification of the large subunit (LSU). PCR was done following Al-Bedak and Moubasher 82 .

Alignments and phylogenetic analyses

Sequences of Cladosporium species (ITS, ACT, LSU) in this study were compared to the type and ex-type species in GenBank. MAFFT (version 6.861b) with the default options 83 was used for alignment of the three sequence sets (ITS, ACT, LSU) in this study. Cercospora beticola CBS 116456 was used as outgroup. Alignment gaps and parsimony uninformative characters were optimized by BMGE 84 . Maximum-likelihood (ML) and Maximum parsimony (MP) phylogenetic analyses were performed using PhyML 3.0 85 . The robustness of the most parsimonious trees was evaluated by 1000 replications 86 . The best optimal model of nucleotide substitution for the ML analyses was determined using Akaike Information Criterion (AIC) as implemented in Modeltest 3.7 87 . The phylogenetic tree was drawn and visualized using MEGA X 10.2.6 88 , 89 . The resulting tree was edited using Microsoft Power Point (2016) and saved as TIF format 9 .

Optimization of cold-active pectinase production by the Cladosporium strains

In a previous study, the three Cladosporium strains (AUMC 10865, AUMC 11340 and AUMC 11366) were found to be capable of producing cold-active pectinases in SmF at 10 °C 27 . For maximization of pectinase production, pH, temperature, nitrogen source and fermentation time influencing pectinase production were optimized by varying parameters using two factors at a time (TFAT) for the three strains. The experiments were conducted in 250 mL Erlenmeyer flasks each with 50 mL fermentation medium (sucrose-free Czapek’s broth) supplemented with 1% citrus pectin as a sole carbon source. The flasks were inoculated separately with spore suspension (1%; v/v) obtained from 7-day-old of Cladosporium strains, and incubated under different operating conditions such as pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0), each at 5, 10, and 15 °C, and nitrogen source (peptone, yeast extract, sodium nitrate, ammonium sulfate, and ammonium chloride; each at 0.2%), at 1–10 days of incubation. Three replications of the experiment were performed.

Pectinase assay

The colorimetric approach was used to measure pectinase activity 90 . Under static circumstances, 0.5 mL of adequately diluted cell-free supernatant was incubated with 0.5 mL of 1.0% citrus pectin (prepared in 50 mM Na-citrate buffer, pH 6.0) for 20 min at 10 °C. The mixture was boiled for 15 min after 2.0 mL of 3, 5-Dinitrosalicylic acid (DNS) was added. The colour created was evaluated at 540 nm for absorption. The quantity of enzyme that catalyses the synthesis of 1 µmol of galacturonic acid per minute at the standard assay conditions was defined as one unit of pectinase.

Production of cold-active pectinase by Cladosporium parasphaerospermum in SmF

For pectinases production by Cladosporium parasphaerospermum AUMC 10865, the fungus was employed in Erlenmeyer flasks (500 mL) in SmF at the optimum conditions using the fermentation medium. Cladosporium species was inoculated with 1.5 × 10 8 spore/mL spore suspensions obtained from 7-day-old cultures. The incubation period lasted at 10 °C and 150 rpm.

Purification of the cold-active pectinase

Ammonium sulfate precipitation and dialysis.

Following the incubation time, cell-free supernatant was recovered by centrifuging at 10,000 rpm for 10 min. At 4 °C, total protein was isolated using 70% saturated solution of ammonium sulphate. A freeze dryer (VirTis, model #6KBTES-55, NY, USA) was used to separate and lyophilize the precipitated protein. Lyophilized protein was dissolved in citrate buffer (pH 6.0) and dialyzed twice for 2 h at room temperature (cutoffs: 12–14 KD) against deionized water, eliminating the water each time, before being refrigerated overnight at 4 °C to remove salts and small molecules. The dialyzed protein was then lyophilized, and used in enzyme characterization experiments as partially purified fungal pectinase.

Ion exchange chromatography

A glass column (30 × 2.0 cm; 75 cm 3 bed volume) was filled with DEAE-Cellulose anion exchanger. After equilibrating the column with citrate buffer (50 mM, pH 6.0), a 6.0 mL sample was loaded onto it. With NaCl concentrations of 0, 0.1, 0.25, 0.5, 1.0, and 1.5 M, the enzyme was eluted with citrate buffer. The volume of the fractions was 5.0 mL. The pectinase activity was assessed using the previous approach. The fractions with the highest pectinase activity were mixed, concentrated, and kept for further study.

Gel filtration chromatography

In a glass column, Sephadex G 100 was packaged (55 × 2.5 cm; bed capacity 270 cm 3 ). The protein was eluted using citrate buffer (50 mM, pH 6.0) after this column was loaded with the concentrated sample (15 mL). Pectinase activity were evaluated using the techniques described previously in fractions of 5.0 mL volume. The pectinase-positive portions were mixed together, concentrated, and kept for future research.

Impact of pH, temperature and some ions and inhibitors on the pure pectinase activity

The impact of pH (3.0–10.0) at 5–15 °C on pure pectinase activity was investigated. The reaction mixture contained 100 µL pure enzyme and 900 µL pectin (dissolved in 50 mM buffer solution). After the reaction time (20 min), the reaction was terminated by introducing 2.0 mL of 3,5-dinitrosalicylic acid (DNS) 90 , and the pectinase activity was determined as previously mentioned. The buffers used were citrate buffer (pH 3.0–6.0), phosphate buffer (pH 7.0–8.0), and glycine/NaOH buffer (pH 9.0–10.0). Also, some ions (Na + , K + , Ca +2 , Co +2 , Ni +2 , Cu +2 , Fe +2 , Mg +2 , Mn +2 , and Zn +2 ) were evaluated by introducing them at 5 mM/mL concentrations as NaCl, KCl, CaCl 2 , CoCl 2 , NiSO 4 , CuSO 4 , FeSO 4 , MgSO 4 , MnSO 4 , and ZnSO 4 . A 5 mM/mL ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) were also used to evaluate an enzyme inhibitor. Under standard conditions, the activity of the microbial pectinase in the absence of metal ions or EDTA or SDS was evaluated to define 100% activity. Three replications of the experiment were performed.

Determination of kinetic constant (K m and V max )

K m (Michaelis–Menten constant) and V max (maximum reaction velocity) values of the purified pectinase were determined by measuring enzyme activity at different concentrations of citrus pectin (1–16 mg/mL), using the Line-weaver-Burk equation 91 .

Application of the pure pectinase in fruit juice production

Apple, orange, apricot, and peach pulps were examined for juice production, clarity, colour, and pH using Cladosporium parasphaerospermum AUMC 10865’s pure pectinase. Each fruit pulp was treated with 10 U/mL pectinase enzyme (v/v), with untreated fruit pulps serving as controls. The processed fruit pulps were then incubated at 10 °C for 60 min. After inactivating the enzyme by boiling for 5 min, samples were recovered by centrifugation at 5000× g for 10 min. for clarity measurements. The juice yield was estimated by dividing the juice mass by the fruit mass 92 .

Statistical analysis

Data were subjected to analysis of variance (ANOVA: two-factor with replication) followed by the Duncan’s multiple range test 93 .

Data availability

The newly discovered strains were preserved as frozen and lyophilized cultures and added to the culture collections of the Assiut University Mycological Centre (AUMC) and the Egyptian Microbial Culture Collection Network (EMCCN) as AUMC 10865 = EMCCN 2062 (Air, Beni Suef, Egypt), AUMC 11340 = EMCCN 2332 (Grapevine fruits, Sohag, Egypt), and AUMC 11366 = EMCCN 2358 (Air, Qena, Egypt). The new species were catalogued in the MycoBank with accession numbers MB844532 (MycoBank Typification; MBT 10007647), MB844533 (MycoBank Typification; MBT 10007648), and MB844534 (MycoBank Typification; MBT 10007649), respectively, along with their descriptions. Nexus file of the sequence alignments for all data sets were uploaded to Tree BASE http://purl.org/phylo/treebase/phylows/study/TB2:S30171?x-access-code=a18696d1afcf659925a63a31c3ebd045&format=html (Study no. 30171). The datasets generated and/or analyzed during the current study are available in the GenBank repository ( https://www.ncbi.nlm.nih.gov/genbank ) and MycoBank ( https://www.mycobank.org/ ).

Change history

22 september 2023.

A Correction to this paper has been published: https://doi.org/10.1038/s41598-023-42490-7

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Ahmad Mohamed Moharram, Abdel-Naser Ahmed Zohri & Mohamed Al-Ameen Maher

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A.M.M., A.-N.A.Z.: Supervision, revising; M.A.M.: Fungal isolation, enzymes production; A.E.H.: Editing, revising; O.A.A.-B.: Molecular work, data analysis, writing, revising; O.A.A.-B., M.A.M.: Enzyme purification and characterization; H.E.F.A.-R., M.A.M.: Enzyme application. All authors have read and agreed to the published version of the manuscript.

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Moharram, A.M., Zohri, AN.A., Hesham, A.EL. et al. Production of cold-active pectinases by three novel Cladosporium species isolated from Egypt and application of the most active enzyme. Sci Rep 12 , 15599 (2022). https://doi.org/10.1038/s41598-022-19807-z

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Pectinases: Production, Harvest, Recovery, and Potential Industrial Application

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• Riya Sahu 2 &
• Surajbhan Sevda 2  

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Pectinases are a group of enzymes that lyse pectin. Pectins are polysaccharides, and they are found abundantly in plant cell walls. The feature of pectinolytic activity is exploited by the industry. With the advances, the industry has developed a focus on various microbial sources for the efficient production of pectinolytic enzymes. The bioprocess principles are applied extensively for the production of pectinase enzymes for efficient commercial production and harvest. Many factors affect the yield of pectinases, which are overlooked, and the shortcomings are improved; this is done with the help of understanding and the research made on studying the biochemical properties of pectinases. The purification and characterization of pectinase enzymes have a key role in controlling the purity and standards. With the help of studies on the mechanism of action of pectinases, it is found to have wide applications, which range in the field of the brewery, juice making, jam making, retting, plant fiber making, paper making, and so on.

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Fungal Pectinases: Production and Applications in Food Industries

Origins and features of pectate lyases and their applications in industry

Microbial Pectinases and Their Applications

Abbreviations.

Homogalacturonan

Pectinesterase

Polygalacturonases

Polygalacturonate lyase

Pectin lyase

Polymethyl galacturonase

Rhamnogalacturonan I

Rhamnogalacturonan II

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Dr. Surajbhan Sevda thanks NIT Warangal for the Research Seed grant (P1128) for a support of this work.

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Sahu, R., Sevda, S. (2022). Pectinases: Production, Harvest, Recovery, and Potential Industrial Application. In: Verma, P. (eds) Industrial Microbiology and Biotechnology. Springer, Singapore. https://doi.org/10.1007/978-981-16-5214-1_10

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Effect of temperature, pH, pectin concentration, and incubation time on pectinase activity

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Isolation and characterization of pectinase-producing bacteria ( Serratia marcescens) from avocado peel waste for juice clarification

• Setegn Haile 1 ,
• Chandran Masi 1 , 2 &
• Mesfin Tafesse 1 , 2  

BMC Microbiology volume  22 , Article number:  145 ( 2022 ) Cite this article

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Bacterial pectinase is an enzyme that could be employed in numerous sectors to break down pectin polysaccharide compounds. The goal of this study is to find pectinase-producing bacteria in avocado peel waste and see if the pectinase enzyme produced can be used to make fruit juice clarification.

The researchers isolated four different bacterial strains from avocado peel waste samples. The potential two bacterial isolates that were identified as being Serratia marcescens and Lysinibacillus macrolides. Finally, the analysis of pectinase production and its application in fruit juice clarification were performed using one of the bacterial strains of Serratia marcescens . The clear apple, lemon, and mango juices were further processed to assess each juice's properties. The highest antioxidant activity was recorded in lemon juice samples. The lemon juice showed the highest total titratable acidity and total phenol content. Apple juices contained the highest total soluble solids, reducing sugar content, and viscosity and the mango juices have the maximum pH value recorded.

Conclusions

The pectinase isolated from the bacterium  Serratia marcescens  could clear fruit juices. This pectinase needs to be studied more to make sure it works better in the fruit industry and other businesses.

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Introduction

Enzymes are biological catalysts that help chemical reactions occur under various physicochemical conditions. All enzymes are protein in nature, but each has a unique performance function [ 1 , 2 , 3 ]. Enzymes were first identified in the mid-nineteenth century and the first to recognize the technical potential of cultivated enzymes and commercialize primarily using fungal enzymes, but 20 years later, Boidin and Affront in France pioneered the synthesis of bacterial enzymes [ 4 , 5 ].

Today, industrial enzyme technology relies on microbial sources such as bacteria and yeasts. These microorganisms are essential in the production of pectinase enzymes which find applications in biotechnological processes that use pectin as a carbon source [ 6 , 7 ]. Pectin is a component of the plant's cell wall and middle lamella, and a very thin extracellular layer that connects the young cells [ 8 , 9 ].

The pectin substances are complex colloidal acid polysaccharides with a long galacturonic acid pillar chain and glycoside bonds. Seven polysaccharides and 17 monosaccharides, such as d-Glucuronic acid, l-Fucose, d-Glucose, d-Mannose, and d-Xylose, are present in these chains of pectin compounds [ 6 , 10 , 11 ]. Pectic acid, pectinic acid, pectin, and protopectin are the four types of pectin substances used as substrates in pectinase processing [ 12 , 13 ]. The solubility of pectic substances in water was one of the most relevant criteria used to identify those [ 14 , 15 ].

Only 25% of the Microbial pectinase enzyme was used in the food and industrial sectors around the world, even though the market kept growing [ 16 , 17 ]. Pectinase finds applications in the extraction of fruit juice, clarification of juice, refining of vegetable fibers, degumming of natural fibers, and wastewater treatment [ 18 , 19 ]. It also speeds up tea fermentation and eliminates the foam-forming property of instant tea powder by destroying the pectin present in tea powder. Even though they aren't just used to make coffee, they're also used to remove the mucilaginous layer from coffee beans [ 15 , 20 ].

Fungal organisms produce the vast majority of pectinase used in the industrial environment. The enzyme is used mostly for the degradation of pectic compounds in a variety of industrial sectors because of which their demand has increased in recent years [ 11 , 21 ]. To address this demand for pectinase, a bacterial source of pectinase could be used. Pectinases break down pectin, causing a decrease in viscosity and the formation of clusters, making centrifugation or filtration easier. As a result, the juice has a more transparent appearance and a more intense taste and color [ 22 , 23 ]. The efficiency of Pectinolytic enzymes in fruit juice clarity is, however, reliant on the amount of pectin and pectinases available in the substrate, resulting in better juice extraction and clarification [ 14 , 24 ]. The major sources of microorganisms used to produce pectinase are pectin-containing fruits and their peels, such as orange pulp, avocado peel, potatoes, tomatoes, sugar beet pulp, peaches, strawberries, lemon carrots, and banana peels. A bacterium has not been found in avocados, but pectic and pectin compounds have been found in a lot of research on avocados [ 7 , 12 ].

Pectinases are used in acidic and alkaline environments and are especially useful in the food and textile industries [ 25 , 26 ]. Pectinases and their uses are being studied in global research sectors to get optimal fastened activity with enzymes. Pectinase has a wide range of applications, which has increased global demand. The uses of these enzymes are depicted in Fig.  1 . There is a lot of value in the pectinase enzyme because it makes fruit juice clear. The main goals of this research were to find and study pectinase-producing bacteria from avocado peel wastes ( Serratia marcescens ), and improve the clarity of the fruit juice.

Isolation of pectinase bacteria, production, and application of pectinase

Isolation of bacteria from avocado peel

It was used to isolate bacteria from avocado peel waste using serial dilution, pour plating, and streak isolation methods. They were sub-cultured into a new growth medium to obtain a pure isolate. The four pure isolates were obtained after extensive isolation techniques. The pure bacterial isolates were labeled as white colony (W3, W32) red colony (R5), and yellow colony Y31 to make it easier to distinguish between them [Fig.  2 ].

Single Colony isolation by Pour Plate Technique using pectin agar plate

Primary and secondary screening

There was a strong (hydrolysis) zone among all four bacterial isolates, indicating the existence of pectinase activities. The diameter of each hydrolysis zone was calculated to determine the potential bacterial isolate [Fig.  3 ]. Isolate R5 measured the largest diameter around the colony at 20.54 ± 1.32 mm [Table 1 ]. The activities of crude pectinase were measured in the secondary screening. The selected isolates from the primary screening method were subjected to fermentation in a suitable medium, and their behaviors were assessed to be further screened. A liquid sample (0.4 ml) was taken to assess pectinase activation by using phosphate buffer after 24 h of incubation in the production media. Isolate R5 had the highest pectinase activity of 5.410 ± 14 mol/ml/min, isolate W3 had the second highest at 3.090 ± 17 mol/ml/min, and isolate Y31 had the lowest at 2.490 ± 23 mol/ml/min [Table 2 ].

Primary screening of Pectinase producing bacteria isolates using pectin agar plate

Morphological and biochemical identification of bacterial isolates

Using Bergey's Manual of Determinative Bacteriology [ 27 ] isolates R5 and Y31 were discovered to be Gram-negative bacteria, whereas isolate W3 was confirmed to be Gram-positive bacteria. The isolates were tentatively described as  Bacillus species, Serratia species, and  Erwinia species [Table 3 ] using ABIS-online software. We chose gram-positive bacteria from W3 and gram-negative bacteria from R5 for further research.

Molecular identification of isolates

The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using genomic DNA from selected bacterial isolates (R5 and W3) as templates. The genomic DNA and PCR amplification products were analyzed using agarose gel electrophoresis compared with DNA marker isolate R5, and W3, as shown in Fig.  4 . To obtain the right sequences, the PCR products were filtered and sequenced. Each isolate's sequences were uploaded to the NCBI database and compared to previously published sequences. The closest neighbors of the isolates R5 (MN932109.1)  Serratia marcescens  and W3 (MN932110.1)  Lysinibacillus macrolides were queried using NCBI BLAST (HTTP://  www.ncbi.nlm.nih.gov/Blast ). The nucleotide  Serratia marcescens  strain 16S ribosomal RNA gene sequences from R5 quest (mBLAST, NCBI) showed 99% homology (Fig.  5 ). The sequences of the W3 search (mBLAST, NCBI) showed 86% similarity to the nucleotide of Lysinibacillus sp ., and 85% similarity to the nucleotide of Lysinibacillus macrolides in the phylogenetic tree (Fig.  6 ).

A gel documentation system was used to acquire an image of an agarose gel dyed with ethidium bromide - DNA marker (M), Genomic DNA extracted - 3(R5) and 4 (W3), and PCR amplification product of bacterial genomic DNA - 7 (R5) and 8 (W3)

Phylogenetic tree constructed based on 16S rRNA gene sequences of Serratia marcescens _R5 with other Serratia species obtained from GenBank database

Phylogenetic tree constructed based on 16S rRNA gene sequences of Lysinibacillus macroides _W3 with other Lysinibacillus species obtained from Gene Bank database

Production of pectinase and its optimization

The development of a broad hydrolysis zone on pectin agar plates was used to characterize the screened Pectinolytic bacterial isolates. Fermentation and quantitative screening of isolate R5- Serratia marcescens were performed, measured by pectinase activity. After 48 h of fermentation, the maximum pectinase activity was 5.41 ± 0.14 µmol/ml/min [Table 2 ].

Effect of fermentation time on pectinase production

To get the most pectinase production from isolate R5- Serratia marcescens , we selected five incubation times (hours) that were used. Pectinase activity increased gradually over 24 h of incubation until optimum pectinase activity was achieved. After the optimum incubation period, the pectinase activities began to decrease. In this analysis, the optimum fermentation period for pectinase development was found to be 72 h, and the highest pectinase activities were found to be 6.86 ± 0.32 µmol/ml/min of released glucose [Table 4 ] when pectin was used as a substrate.

Effect of pH on pectinase production

The effect of different pH of fermentation medium on pectinase production by bacterial isolate R5- Serratia marcescens was studied. The pectinase production could be affected due to the variations in pH medium. The pectinase activity increased when we reached the optimum pH and decreased after the optimum pH value. The production medium, which was adjusted to pH 8 produced maximum pectinase activity (8.84 ± 0.34 µmol/ml/min), followed by pH 7 (6.66 ± 0.73 µmol/ml/min) [Table 5 ].

Effect of temperature on pectinase production

The effects of temperature on pectinase production by bacterial isolate R5- Serratia marcescens indicated that maximum pectinase activity was obtained at 35 °C. As a result, pectinase activity increased as temperature increased and slightly decreased after crossing the optimal temperature of 35 °C. However, pectinase activity was not completely lost even if the temperature increased to 50 °C. Although the maximum pectinase activity was 7.76 ± 56 µmol/ml/min of glucose released at 35 °C, pectinase activities above the optimal temperature were frequently higher than those below the optimal temperature [Table 6 ].

Effect of substrate concentration on pectinase production

The effect of substrate concentration on pectinase production by bacterial isolate R5- Serratia marcescens showed antagonistic effects after 1% of pectin (8.91 ± 0.23 µmol/ml/min). As a result, observed in Table 7 , pectinase activity increased with increased substrate concentration (pectin) up to the optimal concentration and decreased after the optimal substrate concentration.

Purification of pectinase and determining protein concentration

Pectinase precipitation is the preferred concentration method and an ideal step in the purification process. One of the most well-known and widely used methods of purifying and concentrating pectinase, especially at the laboratory scale, is salting-out proteins, particularly ammonium sulfate. Pectinase was isolated from isolated R5- Serratia marcescens and subjected to various saturation levels of ammonium sulfate varying from 30–90%. The pectinase activities and protein content partially purified by ammonium sulfate are presented in Table 8 .

Application of pectinases in fruit juice clarification and yield

This study tried to determine the effect of pectinase on the volume of juice, juice yield, and juice clarity in terms of transmittance by using apple, lemon, and mango fruits. The experiments were carried out with crude pectinase, partially purified, and water as control. According to the results shown in Table 9 , the volume of lemon juice was enhanced by three folds from control to crude pectinase (10.0 -13.0 ml) and two folds increment from purified pectinase (13.0 -14.5 ml), and 86.67% of yield. Using mango juice, the same experiment was carried out. The amount of mango juice increased twofold from control to crude pectinase enzyme (8.0–10.0 ml) after 1 h of pectinase and water treatment and increased almost threefold from crude pectinase to purified pectinase (10.0 -13.5 ml), and 66.67% of yield.

The same experiment was performed to express the yield of juice in terms of percentages. As the above table indicates, the rate of juice yield was improved, starting from control to purified pectinase (Table 9 ). The yield variation of apple juice between control and crude pectinase was 20%. Similarly, the 20% of apple juice yield variation was recorded between crude pectinase and purified pectinase. Therefore, apple fruit showed consistent variation from control to crude and crude to purified pectinase. The yield of lemon juice variation between control and crude pectinase was 20%, but the divergence between crude pectinase and purified pectinase was recorded as 10%. As a result, there were no consistent differences in lemon juice yield from control to pure pectinase. In mango juice, there was a 13.34% difference in yield from control to crude pectinase and a 23.33% difference in juice output between crude pectinase and purified pectinase.

Effect of pectinase on juice clarification

The transmittances of the clarified juice determined the effect of pectinase on juice clarity in terms of percentage (Figs. 7 and 8 ). As shown three different fruits [apple, lemon, and mango] were subjected to crude and purified pectinase by taking water as control, the maximum clarity of juice was obtained was 95.82% of transmittance for lemon fruit. When compared to the result achieved with crude pectinase, which was 78.99% of juice clarity transmission, the transmittance of lemon juice treated with purified pectinase was 16.83% higher.

Effect of pectinase on juice clarity in terms of transmittances

The effect of pectinase on fruit juice clarification: Contol – C (Before), Test – T (After clarification)

Untreated and treated juice properties

The viscosity, pH and acidity, total soluble solids (TSS), total titratable acidity (TTA), reducing sugar content, pectin presence test, Total phenolic content (TPC), and Antioxidant activity of three different fruits [apple, lemon, and mango juice] were evaluated, and the results were compared to the raw sample (untreated juice is a control), as shown in Tables 10 and 11 . The pH of each fruit juice sample decreased with pectinase, and the total titrable acidity (TTA) increased after the juice was clarified by pectinase, as shown in Table 10 . These results were probably related to pectin degradation and liberation of galacturonic acids after pectinase treatment. The reducing sugar content of each fruit juice sample increased after treatment by pectinase (Table 10 ), which is probably related to the liberation of reducing sugar after the degradation of pectin compounds. Because pectinase degrades polysaccharides (pectin), the viscosity of each fruit juice sample was dramatically reduced after treatment with pectinase, as shown in Table 10 . It reduced the presence of cohesive network structure in fruit juice samples. The result of the total phenol content of each fruit juice sample also showed a decreasing trend after the treatment of the juice with pectinase, as shown in Table 11 . The hydrolysis of pectin led to the decomposition of phenolic compounds and decreased their content in fluids.

The hydrolysis of isolate R5 in primary screening is almost identical to that of previous studies [ 2 , 28 , 29 ], which obtained 22.8 mm and 20.5 mm, respectively. This may be due to the ability of bacteria to break down pectin. The hydrolysis zone of isolate (W3), in particular, was linked to the last tomato isolate [ 6 , 23 , 30 ]. Similarly, the results of each isolate's secondary screening method are more similar to the previous investigation of decomposing orange peels; this resulted in the sources of the samples mentioned by [ 22 , 31 ]. Pectinase-producing bacteria were isolated from coffee pulp [ 32 ], and the results of the secondary screening method were lower than isolate R5.

Biologists have used a series of biochemical tests to distinguish closely related bacteria in the detection of bacteria [ 27 ]. Bergey's Manual of Determinative Bacteriology, based on their physical and biochemical properties, the ABIS-online software was used to detect Pectinolytic bacteria [ 27 , 29 ]. In molecular identification, the neighbor-joining method was used to build the phylogenic tree based on the 16S rRNA gene sequences of isolates R5 and W3 and associated nucleotide sequences [ 2 , 22 , 33 ].

Different researchers investigated the molecular characterization of pectinase production with Serratia species.  Serratia rubidaea  (E9.HM585373) was isolated from tomato fruits and characterized using the 16S rRNA method. As reported by Abd-alla et al., [ 6 ] these bacterial species were found to be producing polygalacturonases at a temperature of 40 °C. Serratia rubidaea  and Serratia marcescens  are different from each other in species labels, but both are used to produce pectinase, even though their isolate sources are different. About 20 enzyme-producing bacterial strains were isolated from municipal solid waste using the 16S rRNA method. Among those bacterial strains, most of the strains were  Bacillus species . Sarreen et al., [ 34 ] discovered that only two bacterial strains, Serratia marcescens (MH194203) and Lysinibacillus species (MH194187), were associated with this discovery. Serratia oryzaestrain S32 (SOZ00000000.1) was also found in lake water. This showed that bacterial strains could make pectinase, which was found by Huguevieux et al., [ 35 ].

The pectinase, which was produced in just 24 h of incubation, was two times more potent than the 2.43 U/ml pectinase obtained by  Bacillus sonorensis  in the same incubation period [ 11 , 36 , 37 ]. The enzyme activity results obtained after 72 h were similar to those obtained by Jayani et al., [ 38 ] who obtained 7.88 U/ml of pectinase activity after 72 h of fermentation time using  Bacillus sphaericus . This study finding shows that this bacterial isolate, R5- Serratia marcescens , requires an alkaline condition in pectinase production processes [ 24 , 39 ]. According to the investigations by Mohandas et al., [ 36 ] the highest pectinase activity, 2.43 U/ml, was recorded at alkaline pH 8 by  Bacillus sonorensis . According to Sohail and Latif [ 40 ], the optimal poly galacturonase production of  Bacillus mojavensis  was at pH 8.0 . Streptomyces  species require a slightly alkaline condition of pH 8.5 for maximum pectinase production.

Temperature is one of the essential parameters essential for the success of pectinase production. According to Kothari and Baig [ 41 ], the maximum polygalacturonase activities were produced at a temperature of 35 °C by  Erwiniacarotovora . In the same bacterial species ( Erwinia spp ) , the highest pectinase (polygalacturonase and pectin lysate) activity was recorded at a temperature of 35 °C [ 5 , 42 ]. Other recent investigations indicated that the highest amount of pectinase produced by  Entero bactertabaci NR1466677 was at the optimal temperature of 35 °C [ 9 , 30 ].

In this study, various concentrations of pectin substrate were used as a carbon source for bacterial growth and were subjected to a fermentation medium to produce pectinase. The pectinase activity data in this study appear to be comparable to the work of Darah et al., [ 21 ]. They obtained maximum polygalacturonase activity at 1% of the pectin concentration for  Entero bacteraerogenes . Some bacterial species, such as  Entero bactertabaci NR14667 ,  could produce the highest pectinase activity at 0.3% of pectin concentration, as reported by Obafemi et al., [ 30 ]. More recently, maximum pectinase activity at 2% of citrus pectin concentration was also recorded for  Chryseo bacterium indologenes strain SD  [ 21 , 43 ].

The unique activities of pectinase increased from crude to dialyzed pectinase. With 10 mg of protein concentration, the maximal activity was 47.32 U/mg, suggesting that the protein molecules separated by ammonium mainly contained the enzyme pectinase and that the proportion of protein other than pectinase was higher in the crude form of the enzyme [ 11 , 31 , 44 ]. Purification steps also resulted in the removal of interfering materials found in the crude cell-free sample, allowing for increased enzyme activity [ 13 , 45 ]. From the application of pectinase, the volume of juice treated with both crude and purified pectinase varied with the type of fruit used in the process. As the results indicate, the highest juice volume was obtained from pectinase-treated lemon fruit, which might be due to the presence of solubility of pectin in lemon fruit [ 46 , 47 ].

Similarly, the volume of juice variation from fruit to fruit that was treated with pectinase was investigated by using different fruits such as strawberry juice (7.0 – 10.0 ml), grape juice (15.0 -21.5 ml), apple juice (12.0—18.5 ml), peach apple juice (12.0—10.5 ml), chary apple juice (8.0 – 10.0 ml) and orange juice (9.0 – 10.0 ml) [ 29 , 48 ]. The result of juice clarity treated with crude and purified pectinase was 1.99% and 18.82% more excellent than the previous report by Maktouf et al., [ 49 ], which was 77% of transmittance for pectinase using lemon fruit as the raw material for juice clarification. The result of mango juice transmittance obtained in this current experiment affirms the report made by Kumar et al., [ 50 ], who received 92.5% of transmittance after 150 min of incubation time. The effect of pectinase on juice clarification was also studied in apple juice. According to Yuan et al., [ 51 ], the clarity of apple juice treated with pectinase increased by 71.8% of transmittance. In their experiment, the clarity of apple juice rose to 84% transmittance.

Table 10 indicates that the total soluble solids decreased in each fruit juice sample after clarification. This can be due to the disintegration of solid compounds after the destruction network formed by pectinase [ 51 , 52 ]. This was similar to the report of de Oliveira et al., [ 14 ], for apple juice clarification by pectinase. Table 10 shows that pectin was present in all untreated fruit juice samples, but pectin was not found in treated fruit juice samples by pectinase. A similar finding was reported by Hosseini et al., [ 53 ], for pomegranate juice clarification by free pectinase.

This result was similar to that of [ 37 , 52 ], which was done on the clarification of apple juice by pectinase. In determining the antioxidant activity of each fruit juice sample, ABTS radical scavenging activities (ABTS-RSA) and DPPH radical scavenging activities (DPPH-RSA) of each fruit juice were evaluated, and the results were compared with the raw sample. As shown in Table 11 , pectinase activities can change the phenolic compound profiles and the decomposition of another antioxidant by pectin hydrolysis [ 35 , 52 , 53 ].

The fruits contain a high amount of pectin; the extraction of fruit juice has historically resulted in a cloudy, unappealing color and high viscosity. Researchers studied avocado wastes that were found to contain four distinct bacterial strains. One strain was classified as a Serratia marcescens based on morphological and biochemical characteristics. It's important to do more research on this enzyme to make sure and improve the efficiency of the bacteria for use in the fruit industry.

Materials and methods

The major laboratory instruments used in this study were gel documentation, balance, centrifuge, Spectrophotometer, autoclave, refrigerator, microscope, water bath, water bath with shaker, and incubator.

Sample collection

The avocado peel wastes were collected from the juice processing site of ECOPIA PLC, a private limited company, in Addis Ababa, Ethiopia. The samples were transferred into sterilized plastic bags and brought to the microbiology laboratory at AASTU. The sample containing bags were closed and stored in a 4℃ refrigerator until the analysis time [ 43 ].

Serial dilutions

Homogenized one gram (1 g) of the avocado peel wastes sample was suspended in 9 ml of sterilized distilled water and was properly mixed. The mixture of 1 g avocado peel waste sample and 9 ml of distilled water were serially diluted from 10 –1 to 10 –6 in test tubes [ 54 ]. Serial dilution and spread plate methods were the techniques used to isolate the target to pectinase enzyme-producing bacteria using Nutrient agar media [ 55 ].

Isolation and purification

The growth of bacterial colonies was observed after 24 h of incubation time. The next task was the purification and preservation of the culture. The individual colonies with similar character and size were isolated from culture plates and transferred to new agar plates to obtain pure colonies by the repeated streaking method. The purity of the test isolate was assessed using colony morphology and microscopy; pure colonies of bacteria were preserved with 20% of glycerol and stored at -80 °C for further study [ 56 ].

Primary screening of pectinase producing bacteria isolates

All pure colonies from overnight cultured bacteria [freshly activated plates] were transferred to new pectin agar media and incubated at 30 °C for 48 h. At the end of incubation, 0.3% of Congo red solution was flooded onto the Petri dishes and left for 10 min. This solution formed a clear zone around the colonies, which indicates that bacterial isolates can produce pectinase and the diameter of clear zones is proportional to the bacteria’s relative pectinase production capacity [ 16 ].

Secondary screening of pectinase producing bacteria isolates

The bacterial isolates showing maximum clear zone on primary screening media were considered the highest pectinase producer [ 54 ]. Those bacterial isolates with a higher clear area were subjected to submerged fermentation for pectinase production using the same medium as primary screening but without agar. The freshly cultured (stationary phase bacterial) isolates 0.2 ml in yeast extract broth were inoculated on 100 ml of sterilized production media (the media was fixed for 15 min at 121 °C) in 250 ml of the flask and incubated at 30 °C on a rotary shaker at 125 rpm for 48 h. At the end of incubation time, the media was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 10 min. The supernatant was used as crude enzymes to evaluate the efficiency of bacterial isolates on the production of pectinase activities by using sodium acetate buffer pH 6.8 [ 1 , 45 ].

Determination of pectinase activity

Pectinase activity was determined by measuring the amount of released reducing sugar under assay conditions or by enzymatic degradation of pectin as described Nelson-Somogyi methods. The procedure was started by mixing 1 ml of substrate solution prepared by Phosphate buffer (pH 7) and 0.4 ml of specific supernatant enzyme in test tubes. Then, the mixed solution was incubated at 40 °C in the water bath for 40 min. After adding 0.3 ml of Somogyi copper reagent and mixture of the test tubes was set in a boiling water bath for 10 min. After incubation, the tubes were cooled to room temperature, and 0.3 ml of the Nelson arseno molybdate reagent was added. The solution was cooled to room temperature and measured at 540 nm after centrifuging at 10,000 rpm for 10 min by taking supernatant as the enzyme. The amount of released glucose per milliliter per minute was calculated using D-glucose (10–100 micromoles) from the standard curves. One unit of pectinase activities was defined as the amount of glucose released in the term of μmol of reducing sugar per ml per minute under Standard assay conditions [ 43 , 57 ]. The pectinase activity was calculated using the following formula [ 58 ].

where RG is released glucose obtained from D-glucose standard curve.

TVA is the total volume of assay.

T is the Incubation time.

VEA is the volume of enzyme used to assay.

Morphological and biochemical tests for the identification of bacterial isolates

Single colonies grown on pectin agar media were smeared and examined under the microscope for morphological conformity using gram staining. The following biochemical tests were performed to identify bacterial isolates: carbohydrate fermentation tests, indole test, methyl red (MR) test, Vegas-Proskauer (VP) test, Citrate utilization test, hydrogen sulfide generation test, catalase test, and urease test [ 59 ].

Molecular identification and PCR amplification of screened isolates

Molecular approaches were used to identify the prospective isolates that were examined and selected utilizing the primary and secondary screening procedures (R5 and W3). The genomic DNA of the isolates was extracted using the Bacterial Genomic DNA Extraction Kit, which was modified slightly from the manufacturer's procedure (QIAGEN, QIAamp DNA Mini Kit). The amplification process took 2:35 total time. On a 0.8% agarose gel dyed with a DNA-safe stain, the PCR products were seen. Finally, the PCR products were sequenced, and the obtained sequence data were analyzed using the basic local alignment search tool (BLAST) software ( http://www.ncbi.nlm.nih.gov/blast ) against the 16S ribosomal RNA sequence database, with the mega X software ( http://www.ncbi.nlm.nih.gov/mahalik ) to generate the phylogenetic tree from the national center for biotechnology [ 44 ].

Production of crude pectinase by submerged fermentation

The pectinase production by submerged fermentation was conducted as described by Kumar A and Sharma R [ 50 ]. The bacterial isolate identified as a potential candidate was selected to produce this crude enzyme. About 2 ml of the three hours bacterial cultures (log phase bacteria) were inoculated to a pre-sterilized fermentation medium to maintain pH 8 [ 43 ] and incubated at 30 °C using a rotary shaker at 125 rpm for 48 h. At the closed fermentation time, the production medium was centrifuged at 10,000 rpm for 10 min. The clear supernatant was used for pectinases activities [ 47 ].

Optimization of pectinase production

The production of pectinase was optimized by using four parameters namely, fermentation time, temperature, pH, and substrate concentration. The relative activity of each parameter was calculated as the percentage by using the following formula:-

where As = the activities of sample in µmol/ml

MS = the maximum activities of the sample in µmol/ml.

Optimization of fermentation time on pectinase production

The production media was prepared at constant pH 7 and 1% substrate concentration to examine the influences of fermentation time on enzyme production by bacterial isolate. A Single colony of R5 isolate was inoculated in 15 ml of yeast extract pectin broth and incubated at a temperature of 30 °C overnight. The production media was sterilized at 121 °C for 15 min. This fixed production media was inoculated with 2 ml of overnight culture bacteria and incubated at a temperature of 30 °C using a rotary shaker with 125 rpm for 24 to 120 h. The activities were assayed in 24 h intervals [ 60 ].

Optimization of pH on pectinase production

To investigate the influences of pH variation on enzyme production by bacterial isolate, the pH level of production media was adjusted from a pH 5 to pH 10 using 0.1 M sodium acetate and 0.1 M Sodium hydroxide [ 4 ] with a few modifications. The production media was sterilized at 121 °C for 15 min and inoculation with 2 ml of overnight bacterial cultured bacteria. This inoculated media was incubated at 30 °C using a rotary shaker with 125 rpm for 72 h, after which the enzyme activities were assayed.

Optimization of temperature on pectinase production

Temperatures for enzyme production were maintained at the following temperatures to explore the effects of temperature change on enzyme production using bacterial isolate: 25, 30, 35, 40, 45, and 50 °C [ 61 ]. The production media (pH 8) was inoculated with 2 ml of overnight cultured bacteria and incubated using a rotary shaker at 125 rpm for 72 h. After the end of the fermentation times, the enzyme activities were assayed.

Optimization of substrate concentration on pectinase production

To study the influences of substrate concentration on enzyme production using the bacterial isolate, the enzyme was produced by various concentrations of substrates (pectin). The concentrations of substrate maintained for enzyme production were 0.25%, 0.5%, 0.75%, 1.0%, 1.25%, and 1.5% [ 55 ]. The sterilized production media (pH 8) was inoculated with 2 ml of overnight cultured bacteria and incubated at 30 °C using a rotary shaker at 125 rpm for 72 h. After the end of the fermentation time, the enzyme activities were assayed.

Purification of pectinase by ammonium sulfate precipitation

The crude enzyme was partially purified using ammonium sulfate precipitation methods described by Ramalingam et al., [ 62 ]. To avoid the denaturation of enzymes, all purification steps were carried out in a cold environment, utilizing an ice bath and temperatures of 40 °C. About 150 ml of the crude enzyme was precipitated by the addition of four different saturation levels of ammonium sulfate: 30%, 50%, 70%, and 90%. They then dissolved the enzyme proteins that had been frozen in saltwater and dialyzed them with dialysis membranes after the above steps were done [ 61 ].

Application of pectinases in fruit juice clarification

The purified pectinase was applied in the fruit juice-making process to test the clarity of the juice. Three different fruits which have the signs of physical damage (lemon, mango, and apple fruits) were bought from a fruit market (Akaki Kality, Addis Ababa) and brought to microbiology laboratories for juice preparation [ 61 ].

Juice preparation

Lemon, mango, and apple fruits were washed carefully and chopped into smaller sizes on the side with a sharp knife. Twenty grams (20 g) of each chopped fruit were weighed into separate beakers. Those chopped were treated with crude pectinase, purified pectinase, and untreated samples were kept as controls in which the enzyme was replaced by distilled water. The controls and enzyme-treated samples were incubated in a water bath at 40 °C for 1 h, and the activity was stopped by cooling in an ice bath. After that, each juice was filtered through filter paper before the volume of juice production was measured. After enzymatic treatments, the filtered fruit juice was pasteurized at 60 °C for 20 min. To figure out how much juice was made from each piece of fruit, Rai et al., [ 20 ] used the method. The formula is:

Clarification of juice

Using a UV spectrophotometer and the Shet et al., [ 26 ] procedure, the clarity of each fruit juice was measured in terms of percentages of transmittances. Around 8 ml of each fruit juice was taken and chilled in a water bath before adding the pectinase enzyme product, after heating at 40 °C to inactivate any natural fruit enzymes or bacteria present. The enzymes (2 mL) were added to 8 mL of fruit juice. After a 4-h incubation period, the samples were heated for 3 min at 40 °C. The juice was centrifuged for 20 min at 3000 rpm, the supernatant was filtered out with filter paper, and the clarity of the juice was calculated by measuring the absorbance at 660 nm with a UV spectrophotometer. Distilled water was used as a blank, and the clarity was expressed in percentages [ 28 ].

PH and acidity

The total titrable acidity of the juices was assessed by titration of the juice sample with 0.2 N sodium hydroxide and the pH of cleared and un-clarified fruits (Lemon, Mango, and Apple) juice samples were measured using a pH meter. The results were presented using the phenolphthalein reagent as an indicator and based on g citric acid per 50 ml of each fruit juice [ 46 ].

Total soluble solid and reducing sugar content

The total soluble solids (SST) of each fruit (Lemon, Mango, and apple) juice were recorded using a refractometer, and the reducing sugar content of each fruit juices sample was measured by the DNS method [ 63 ].

The viscosity of Lemon, Mango, and apple fruits juice samples was evaluated by viscometer at a share rate of 75 rpm and room temperature.

Pectin presence test

The pectin test was performed on Lemon, Mango, and Apple fruit juice samples by combining cold ethanol with each sample and storing it in the refrigerator overnight. The presence of supernatant indicates the presence of pectin in the sample, while the absence of supernatant suggests the absence of pectin [ 53 ].

Total phenol content

The Folin-Ciocalteu technique was used to determine each fruit juice sample [ 23 , 25 ]. The total phenol content of the sample was determined by comparing it to the Gallic acid standard curve, with the result represented in mg gallic acid equivalent per ml juice sample (mg GAE/ml)[ 49 ].

Determination of antioxidant activity

The ABTS and DPPH radical scavenging activities of each fruit juice sample were determined using a modified version of the method published by Hosseini et al., [ 53 ]. An ABTS solution (6.5 mM) was combined with a potassium persulfate solution (3.5 mM) and stored in darkness for 18 h to make an ABTS solution. After vortexing, the obtained mixtures were kept in darkness for 45 min to complete the antiradical reaction. 0.2 ml of each fruit juice sample was diluted 50 times with distilled water and added to 1.8 ml of the obtained ABTS solution and DPPH ethanolic solution (0.1 mM). After recording the absorbance at 734 nm for ABTS radical scavenging activity (ABTS-RSA) and 517 mn for DPPH radical scavenging activity (DPPH-RSA), these antiradical activities were calculated as follows:

Statistical analysis

Statistical analyses of data were conducted using IBM SPSS statistics 20 and origin 2019. All tests were performed in triplicates, and data were expressed as Mean ± standard deviation.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The genome sequence data for R5 and W3is available in the GenBank repository under the project of Addis Ababa Science and Technology University (Website: https://www.ncbi.nlm.nih.gov/nuccore/MN932109.1 and https://www.ncbi.nlm.nih.gov/nuccore/1796387721 ).

Abbreviations

Revolutions per minute

Microliters

Polymerase chain reaction

Basic Local Alignment Search Tool

Deoxyribonucleic Acid

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We thank the University President, Academic Vice President, College Dean, Head of Department, and Lab coordinator of the Department of Biotechnology, College of Biological and Chemical Engineering help us with this research work.

This research work was supported by the Directorate of Research and Technology Transfer, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia.

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All authors have carried out the avocado peel samples collected from the dumping site of ECOPIA PL randomly from a decayed avocado waste using a pre-sterilized spatula. MT &SH carried out the isolation of potential bacteria. SH & CM carried out the identification of bacteria. All authors are contributed by the final confirmation of potential Pectinase-producing bacteria and manuscript revisions. All authors have approved the final version of the manuscript and agree to be held accountable for the content therein.

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Haile, S., Masi, C. & Tafesse, M. Isolation and characterization of pectinase-producing bacteria ( Serratia marcescens) from avocado peel waste for juice clarification. BMC Microbiol 22 , 145 (2022). https://doi.org/10.1186/s12866-022-02536-8

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The Effect of pH on Pectinase

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Stevie Fleming

*** A well structured report which covers the basic criteria but is lacking depth in key sections. Research and rationale There is some background material referring to the action of pectinase but it is a little limited. The student should have included some references to provide a background for the project. Pectinase is used extensively industrially so there is a lot of additional material available. Planning A testable hypothesis has been formulated and stated concisely. The apparatus is suitable for the project but had not been justified. Insufficient thought had been given to the key variables. The range of pH values was not really adequate. For an A level experiment a minimum of five values of the independent variable is considered to be acceptable. The control would not work as expected since the apple puree must have a pH level which would allow the pectinase to work. The risk assessment is minimal and mentions procedures that were not in the stated method. Implementing The apparatus seemed to have been used competently but the data should have been recorded in a more suitably headed table. Analysing and Evaluation A summary table was presented but no graph was included. No statistical analysis of data was included e.g. calculation of standard deviation. The explanations were sound and related to basic biological knowledge. The analysis was not helped by the small range of data obtained which meant the candidate tried to "fill in the gaps". There was no evaluation of the method or results obtained Communication The layout was acceptable with suitable headings and subheadings. The data could have been presented more clearly in tables that follow scientific conventions. Spelling, punctuation and grammar were reasonable and scientific terms used correctly. No background reference were used

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Production of pectinases for quality apple juice through fermentation of orange pomace

M. mahmoodi.

Biotechnology Research Lab., Faculty of Chemical Engineering, Noshirvani University of Technology, Babol, Iran

G. D. Najafpour

M. mohammadi, associated data.

Production of pectinases by Aspergillus niger was successfully carried out through solid state fermentation. Orange pomace was used as substrate to produce pectinases using a wild type of A. niger isolated from a rotten orange texture. Some of the important parameters affecting exo- and endo-pectinases activities such as temperature, moisture, C/N ratio were optimized. The results indicated that the produced pectinases exhibited maximum activity in temperature range of 45–55 °C and the maximum enzyme productivity occurred at 70% moisture content and C/N ratio of 10. The enzyme kinetic was studied using Michaelis–Menten and Logistic model and the equation were fitted to experimental data for both exo- and endo-pectinases activities. In evaluation of kinetic model, it was found that Monod model presented perfectly fitted with experimental data. Monod kinetic parameters ( μ m a x and K S ) for exo-pectinase activities were 771.7 μM min -1 and 31.91 mM, respectively. The Monod kinetic parameters ( μ m a x and K S ) for endo-pectinase activity were 48.19 mP min -1 and 478.3 mM,  respectively. Finally, the performances of the produced pectinases were evaluated on natural apple juice. It was confirmed that concentration of soluble sugar, clarity and viscosity of the juice and the yield of extracted juice were significantly improved by the enzymatic hydrolysis activity of pectinases.

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Introduction

Enzyme production via solid state fermentation (SSF) has been proposed as an alternative method to submerged fermentation (SmF). SSF processes are interesting for countries with abundant agricultural and industrial solid wastes (Smits 1998 ). In Asian countries, application of SSF technology in industrial scale for enzyme production is considered as a reliable fermentation process. Nevertheless, there are still lots of challenges for implementation of SSF technology at large scale due to lack of suitable bioreactor with controlling units and accessories for SSF process which guarantee an acceptable production yield (dos Santos et al. 2004 ).

Pectinases are the most important groups of enzymes that can be obtained in SSF much effectively than SmF (Maldonado and De Saad 1998 ). Pectinases are responsible for the hydrolysis of pectinic chain found in the plant cell walls. Pectin is a galacturonic acid-rich polymer in primary cell wall and middle lamella of plant cells which plays an important role as a structural component. It is responsible for maintaining the integrity and safety of plant tissues (MacDougall et al. 2001 ; Palin and Geitmann 2012 ). Pectin is plentifully found and characterized in several fruits, vegetables and agricultural residues. If pectin is hydrolyzed by pectinases; then, the plant cell is ruptured and the cell components are released.

Pectinases are widely used in fruit juice industry for the improvement of the quality of extracted juice. The enzyme enhances the process yield and accelerates the clarification of extracted juice (Saxena et al. 2014 ; Shah et al. 2015 ).

However pectinases is not recommended to be used for orange juice, because pulp precipitation which forms a two phase configuration in orange juice is unattractive and unmarketable. Thus pectinases should be controlled in orange juice by thermal treatment (Baker and Bruemmer 1972 ).

Pectinase are also used for oil extraction, degumming of plant fibers in pulp and paper industry; and also involved in specialized food applications like extraction of bioactive compounds (Hoondal et al. 2002 ; Bisht et al. 2015 ).

Produced pectinases by Aspergillus species are commercially important for beverage industries. Several forms of pectinases have been produced by Aspergillus species (Antier et al. 1993 ). In this study, A. niger was isolated from orange which seems to be well adapted to pectic substrate. According to literature (Maldonado and De Saad 1998 ), it is confirmed that pectin induces pectinase production. Therefore, it would be suitable and preferable to use pectin as substrate for pectinase production. Table  1 summarizes pectin percentages of fruits and citruses peels which are the main sources of pectin in the nature. Citruses are plentiful and locally available in fruit markets. Furthermore, citrus peel can be used in its original form without any specific pretreatment in solid state fermentation.

Table 1

Composition of pectin in different fruits and vegetables

The structure of an orange has different parts including albedo, flavedo and lamella. All these parts contain a percentage of pectin; but albedo is the most important source of pectin in an orange (Liu et al. 2006 ). Therefore, selection of a species of orange for pectinase production, it should be remembered that high pectinases production yield can be achieved by species which contains more albedo part.

Several effective parameters can influence on the activity of obtained pectinase, the most important parameters are; temperature, pH, moisture content and C/N ratio. These parameters were extensively studied with different species of microorganisms as enzyme producers. In this work, we optimized these parameters and the effect of extracted enzyme at optimum condition was tested on the quality of apple juice. Furthermore, enzyme kinetics was studied to determine the produced enzyme’s affinity toward the substrate.

Materials and methods

Microorganism isolation.

A wild type Aspergillus niger was isolated from a rotten orange by streaking technique according to the features specified by Najafpour ( 2015 ). The screened and isolated strain (see appendix of supplementary material for cultured strain) was cultured and maintained on a solid medium containing (in g per 100 mL distilled water): pectin, 1; yeast, 0.5; peptone, 0.5; MgSO 4 , 0.01; K 2 HPO 4 , 0.2 and agar, 1.5. The cultured media was monthly re-cultivated.

Preparation of spore suspension

Spore suspension was prepared by washing a 5-day incubated plate agar media with isotonic saline solution (Martínez-Trujillo et al. 2011 ). After sporulation, the spores were added to media to reach a final concentration of 10 7 spores g −1 of dry solid (Acuña-Argüelles et al. 1995 ).

Substrate preparation and optimization of process parameters

Fresh orange pomace with initial moisture content of 30% was obtained from a local factory. Then ammonium sulfate and yeast extract (1:1) were added as nitrogen sources as proposed by Phutela et al. ( 2005 ). Five grams of sterilized substrates taken in 250 mL Erlenmeyer flasks was used for each experiment. The experiments were carried out in triplicate and the average values were reported. The parameters selected for optimization were temperature (30, 40, 45, 50, 55, 60, 70 °C), moisture content (60, 65, 70, 75 and 80 wt%) and carbon to nitrogen ratio (C/N) (5, 10, 15, 30). Substrate with desired C/N ratio was prepared by supplementation of the orange pomace (with carbon and nitrogen content of 32 and 1%, respectively) with ammonium sulfate and yeast extract as nitrogen sources.

Enzyme extraction

The produced pectinases were extracted by washing the 96 h incubated culture using acetate buffer solution (pH 5). The extracted solution was filtered and centrifuged at 4000 rpm for 20 min to separate suspended spores, and the supernatant was kept at 3 °C for further analysis (Minjares-Carranco et al. 1997 ).

Enzyme assay

Endo-pectinase activity was assayed in a reaction mixture containing 1 mL of extract and 18 mL of 2% pectin in acetate buffer solution (0.1 M, pH 4.5) and the mixture was incubated at 45 °C for 30 min. Finally, the reduction in viscosity was determined by Ostwald capillary viscometer (distilled water was used as reference). One unit of endo-pectinase activity was defined as the amount of enzyme required to reduce the viscosity by 50% in 1 min (Acuña-Argüelles et al. 1995 ).

Exo-pectinase activity was assayed in a reaction mixture containing 0.3 mL of suitably diluted enzyme extract, 0.7 mL acetate buffer solution (0.1 M, pH 4.5) and 1 mL of 0.9% pectin in acetate buffer solution. The mixture was incubated at 45 °C for duration of 30 min. Reducing sugars content of the solution were determined by DNS method (Miller 1959 ). One exo-pectinase unit was defined as the amount of enzyme that catalyses the formation of one μmol of galacturonic acid per minute (Solis-Pereira et al. 1993 ).

Determination of kinetic parameters

For the determination of kinetic parameters, enzymatic reactions were carried out at the same conditions as described above (for determination of exo- and endo-pectinases activities) but using a constant amount of enzyme concentration (15 and 5.2, % v/v for exo- and endo-pectinases activities, respectively) and different initial pectin concentrations (0.463, 4.63, 13.9, 23.7 and 46.36%, mM for exo-pectinase activity and 5.426, 54.25, 238, 579.89 and 644.3 mM for endo-pectinase activity). Kinetic parameters for exo- and endo-pectinases activities were expressed by determination of the initial velocity at different concentration of substrate; then, Monod and Logistic equation were fitted to experimental data using MATLAB software (2013).

Evaluation of enzyme activity on apple pulp

Apples were sliced into 5 mm cubic size. Equal amount of chopped apples (50 g) were put into two beakers at the same conditions. One mL of extracted pectinase was added to one beaker and 1 mL deactivated pectinase (incubated and heated in boiling water for duration of 5 min) was added to another beaker. Both beakers were kept at 45 °C for duration of 2 h and the produced juices were compared.

Result and discussion

Effect of temperature on enzyme activity.

The exo- and endo-pectinases activities of the extracted enzymes were measured at different temperatures in the range of 30–70 °C. The highest enzyme activities for both exo- and endo-pectinase were obtained in the temperature range of 45–55 °C. These results were in accordance with reported data by Acuña-Argüelles et al. ( 1995 ) for exo- and endo-pectinase activities of the pectinases produced by A. niger . In addition, Siddiqui et al. ( 2012 ) reported that the obtained polygalacturonase from Rhizomucor pusillus was optimally active at 55 °C.

Effect of moisture

Effect of initial moisture content of substrate as an influential parameter in SSF was investigated. The variation of exo- and endo-pectinase activities along with various moisture contents are shown in Fig.  1 . As result show, moisture content had profound impact on exo- and endo-pectinase activities. The results indicated that maximum activities were achieved at the initial moisture content of 70% (w/w). Moisture content is related to water activity which is one of the most important parameters for growth of microorganisms and its value increase with raising of water content, however in case of solid state fermentation presence of too much water in the solid may fill the inter particle spaces and as a result oxygen transfer may be limited in the microorganism environment. Although at the moisture content of above 70% the water activity increases; nevertheless, limited availability of oxygen to the microorganism hindered the cell growth and enzyme production. In a similar research, Patil and Dayanand ( 2006 ) reported the optimum moisture of 65% for production of pectinases through solid state fermentation of deseeded sunflower head by A. niger , which is nearly in accordance with our obtained results. In addition, they reported the optimum productivity of 34.2 U/g for exo-pectinase activity which is less than exo-pectinase productivity obtained in present work (45 U/g) at optimum moisture content. This is mainly due to the fact that orange pomace has more pectin in its structure than deseeded sunflower head and is more effective in stimulated pectinases production.

The effect of moisture content of solid media on production of exo- and endo-pecinases activity by A. niger (at 30 °C)

Effect of C/N ratio

Figure  2 depicts the obtained results which indicate that at C/N ratio of 10, maximum activity of exo- and endo-pectinase was achieved. In a similar research, Kurnar and Reddy ( 2008 ) obtained optimum C/N ratio of 5.96 for production of pectinase through solid state fermentation of Manihot utilissima by A. niger . This suggests that, among chemical and physical factors, the C/N ratio is an important parameter that needs to be optimized.

The effect of C/N ratio of solid media on production of exo- and endo-pectinase activity by A. niger (at 30 °C)

Enzyme kinetics

Kinetic parameters for exo-pectinase activity were determined by quantifying the initial velocity of galacturonic acid production at different concentration of substrate; then, Monod and Logistic equation were fitted to experimental data. These rate equations are defined, stated as follows:

where s is the concentration of substrate (citrus pectin) in the reaction mixture and μ max , K S and S m a x are constants. Kinetic parameters for exo-pectinase activity were determined by measuring the initial reaction rate of pectinases in production of soluble sugar in solutions contained different concentration of pectin. The kinetic parameters ( μ m a x and K S ) for Monod model were 771.7 μM min -1 and 31.91 mM,  respectively. Also the kinetic parameters ( μ max and S m a x ) for Logistic model were 4.438 × 10 -5 mP min -1 and - 1.776 × 10 4 mM,  respectively. For exo-pectinase, the regression coefficients (R 2 ) for Monod and Logistic models were 0.998 and 0.991, respectively. As can be inferred from the very high regression coefficient Monod model was suitable for describing the kinetics of exo-pectinase activity.

Similarly, kinetic parameters for endo-pectinase activity were determined by quantifying the initial reaction rate of pectinases in reduction of viscosity of different concentration of pectin. The kinetic parameters ( μ max and K S ) for Monod model were 48.19 mP min -1 and 478.3 mM,  respectively. The kinetic parameter ( μ max and S m a x ) for Logistic model were 4.438 × 10 -5 mP min -1 and - 1.776 × 10 4 mM,  respectively. For endo-pectinase, the regression coefficients (R 2 ) for Monod and Logistic models were 0.999 and 0.937, respectively. Results indicated that Monod model was very appropriate to describe the kinetics of endo-pectinase activity as implied by the high regression coefficient.

Acuña-Argüelles et al. ( 1995 ) reported K S values for exo- and endo-pectinase activities of the pectinases obtained through solid state fermentation were 2.05 and 270.4 mg mL -1 , respectively. Comparing these results with K S values obtained for exo- and endo pectinases activities in this study were 31.91 and 478.3 mM (6.19 and 194 mg mL -1 ), respectively. The obtained results reveal that pectinases obtained in this study have more affinity towards substrate in term of endo-pectinase activity; but it has a bit less affinity towards the substrate in term of exo-pectinase activity. Furthermore, Mutlu et al. ( 1999 ) found that the Michaelis–Menten parameters were μ max  = 0.0046 pectin % (w v −1 ) s −1 and K S  = 1.137% w v −1 pectin for endo-pectinase activity of commercial pectinase (Pectinex Ultra SP-L) at 35 °C (R 2  = 0.998).

Evaluation of enzyme on the quality of apple juice

Reduction in viscosity of apple juice by enzyme treatment.

In fruit juice industries low viscosity of juice accelerates movement of the fluid in the heat exchanger so that pumping energy requirement decreases. In order to decrease the viscosity of crude juice, usually enzymatic treatment is carried out. To quantify, the effect of produced pectinases on juice viscosity, variation in the viscosity of 18 mL of apple juice was considered. The procedure was similar to the method described for endo-pectinase activity measurement. The results showed that the enzymatic treatment of apple juice by the synthesized enzyme reduced the viscosity of apple juice about 7.2%. The reduction in viscosity of various kinds of fruit juices including Pear, Kiwi, Banana and etc., through enzymatic treatment by pectinases were reported in the literature (Sharma et al. 2015 ).

Enhanced sugar concentration of apple juice

Soluble sugar concentration increases due to action of pectinases on insoluble pectin which is suspended in apple juice. It was quantified by the described method for determination of exo-pectinase activity. The results indicated that suitable amount of enzyme can increase the soluble sugar by 7% (w/v), which implicate that sugar content of apple juice increased by the use of pectinases treatment. The beneficial point is enhancement of natural sugar; which is exactly contributed by its nature without addition of any synthetic sugar.

Pectate formation

Apple pectin is highly methylated. Pectin estrase is a kind of pectinases that strips methoxyl groups from pectin molecules as of the negative charge on pectin chains increases. In the presence of calcium ions calcium pectate is formed which is insoluble and gradually precipitate; finally, the clarified juice is produced. In order to evaluate the effect of extracted pectinase on apple juice, 1 mL of enzyme was mixed with 18 mL of apple juice ansd the mixture was filtered after 1 h (similar procedure was carried out for the deactivated enzymatic extract). The quantitative results revealed that pectate formation increased by 19% (see appendix of supplementary material for additional information).

Improvement of apple juice extraction

Enzymatic activity on natural apple was evaluated according to described method in materials and methods section. The results showed that extraction of juice in the beaker with active enzyme was 13 mL more than another beaker. This improvement in fruit juice extraction is very significant in industrial scale and demonstrates the important role of pectinases in fruit juice industry. The improvement in juice extraction for various kinds of fruits including apricot, pear, plum and etc., through enzymatic treatment by pectinases were reported in the literature (Sharma et al. 2015 ).

In this study, attempt was made to produce pectinases via SSF with implication of a pectinases producing strain of Aspergillus niger. Furthermore, for maximum enzyme productivities, effective parameters such as temperature, moisture and C/N ratio were defined and the kinetic parameters were determined in optimum condition. In evaluation of kinetic model, it was found that Monod model presented perfectly fitted with experimental data. The Monod kinetic parameters ( μ m a x and K S ) for exo-pectinase activity were 771.7 μM min -1 and 31.91 mM, respectively. The Monod kinetic parameters ( μ m a x and K S ) for endo-pectinase activity were 48.19 mP min -1 and 478.3 mM,  respectively. The produced pectinases also exhibited significant activity in natural pectinic environment. It was proved that the concentration of soluble sugar, clarity and viscosity of the juice as well as the juice extraction yield were significantly improved by pectinolytic activity. Therefore, it was concluded that pectinases are useful natural additives for juice industry.

Below is the link to the electronic supplementary material.

Acknowledgements

Authors are gratefully acknowledged Biotechnology Research Lab. Noshirvani University of Technology (Babol, Iran) for the facilities provided to make present work to be successful and useful research.

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