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

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 Km and Vmax determined to be 26.625 mg/mL and 312.5 U/min, respectively. At 5 mM/mL, EDTA, MgCl2, 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.

Phylogenetic analyses.Descriptive statistical parameters of phylogenetic analyses and calculated tree scores for each analyzed sequence locus are summarized in

MycoBank. MB 844533.
Etymology.Refers to the formation of chlamydospores in culture.

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.        www.nature.com/scientificreports/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 1005.55U/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).
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 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).www.nature.com/scientificreports/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).
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   www.nature.com/scientificreports/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).

Discussion
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 coldactive 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 °C70 .Mucor flavus is another example of a psychrotolerant fungus that generates pectinases with optimum activity at 45 °C71 .
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 °C72 .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.

Conclusion
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. 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 °C27 .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-dayold 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.
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 .

Figure 1 .
Figure 1.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).

Figure 2 .
Figure 2. 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).

Figure 3 .
Figure 3. 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).

Figure 10 .
Figure 10.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.

Figure 11 .Figure 12 .
Figure 11.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.

Figure 16 .
Figure 16.Effect of pH and temperature on activity of the pure pectinase.

Table 1
. The constructed phylogenetic trees for ITS, ACT and LSU are shown in Figs. 1, 2, 3, respectively.

Table 1 .
Moharram AM, Zohri AA, Hesham A, Maher MA and Al-Statistical parameters representing phylogenetic studies performed on three distinct loci's sequence alignments.

Table 2 .
Purification profile of pure pectinase produced by C. parasphaerospermum AUMC 10865 at pH 6.0 and 10 °C in SmF.

Table 3 .
Effect of some ions and inhibitors (5 mmol/mL) on pectinase activity produced by C. parasphaerospermum (mean ± SD, n = 3).The results are expressed as the activity in the tested inhibitory conditions compared to the pectinase activity in the control without inhibitors (in bold).At the 0.05 level of probability, means in a column with the same letters are not statistically different.Significant values are in bold.

Table 4 .
Yield, clarity, colour and pH of apple, orange, apricot, and peach fruit juices treated with pure pectinase produced by Cladosporium parasphaerospermum AUMC 10865.At the 0.05 level of probability, means in a column with the same letters are not statistically different.Significant values are in bold.