Production of Cocktail Enzymes by Three Cladosporium Isolates and Bioconversion of Orange Peel Wastes into Valuable Enzymes

The current research demonstrates the biotechnological economization of accumulated and inefficiently used agro-industrial orange peel wastes to generate amylase, endoglucanase, exoglucanase, pectinase, and xylanase, industrially essential enzymes with growing demands in enzyme markets, from three Cladosporium isolates. In submerged fermentation (SmF) at 10°C, the isolate AUMC 10865 produced the highest level of amylase (4164 IU/gram dry substrate). Endoglucanase, exoglucanase and xylanase had development peaks (923 IU/gds, 2280 IU/gds, and 1646 IU/gds, respectively in case of Cladosporium sp. AUMC 11366. Pectinase produced the most (7840 IU/gds) in the strain AUMC 11340. At 30°C, the strain AUMC 11340 secretes the most amylase (4120 IU/gds), endoglucanase (2700 IU/gds) and xylanase (3220 IU/gds). Exoglucanase development reached the peak (8750 IU/gds) in the isolate AUMC 10865. The overall production (5570 IU/gds) was instead enhanced by pectinase in the AUMC 11366 isolate. In solid-state fermentation (SSF) at 10°C, the isolate AUMC 10865 outperformed the other two isolates producing 640.0 IU/gds amylase, 763.3 IU/gds endoglucanase, 771.0 IU/gds exoglucanase, 1273.23 IU/ gds pectinase and 1062.0 IU/gds xylanase, while the isolate AUMC 11366 produced the least amount of 399.7 IU/gds, 410.0 IU/gds, 413.3 IU/gds, 558.7 IU/gds, and 548.0 IU/gds, respectively. At 30°C, the isolate AUMC 11340 was superiorly producing higher levels of amylase (973.3 IU/gds), endoglucanase (746.0 IU/gds), exoglucanase (1052.0 IU/gds), pectinase (1685.3 IU/gds) and xylanase (1340.0 IU/gds), whereas isolate AUMC 10865 generated the least amounts of amylase (556.7 IU/gds) and exoglucanase (452.7 IU/gfs), and the isolate AUMC 11366 produced the least endoglucanase (256.3 IU/gds), pectinase (857.7 IU/gfs) and xylanase (436.3 IU/gds) amounts.


INTRODUCTION
Cladosporium is amongst the largest and diverse hyphomycetes, with more than 772 names. 1 Cladosporium was previously used to refer to all unrelated dematiaceous hyphomycetes with amero-to-phragmosporous conidia produced in acropetal chains. Cladosporium species are well suited to disperse in vast numbers over long distances, so they are cosmopolitan and commonly found in all forms of plants, and other debris, often isolated from air, soil, crops, grains, fruit, paint, textiles, and other organic matter. [1][2][3][4][5][6][7][8][9] Some species of this genus are plant pathogenic causing leaf spots and other lesions, 10 or they occur as hyper parasites on other fungi. 11 Active research on converting agricultural by-products into value-added products has been purposed to produce different enzymes of great economic value. Orange juice is now one of the best drinks in the world 12,13 and orange is one of Egypt's important commercial crops. 14 The production of orange juice is made with a large percentage of citrus fruits, of which around 50% to 60% is converted into citrus peel wastes, 15,16 which leads to the accumulation of large quantities of peel waste in the citrus processing industries. Important quantities have been collected of orange peel waste along with environmental issues to ensure that health risks arising from unsatisfactory treatment practices meet the essential needs for identifying alternative biotechnological waste recycling options. 17,18 In accordance with existing law on the environment, any wastes can be used as an ingredient for recycling. 19 The use of orange peel as a potentially useful low-cost resource will provide high quality goods. 12,18,20 The literature extensively describes a variety of promising approaches for effective use of orange peel wastes. Consequently, three Cladosporium isolates were used for biosynthesis of some important enzymes in submerged fermentation (SmF) and their enzymatic ability was used through solid-state (SSF) methods in order to bio-convert orange peel wastes as a low-cost substrate into valuable enzymes.

MATERIALS AND METHODS Isolation and Maintenance of Cladosporium Isolates
Three Cladosporium isolates involved in the current study, of which two were isolated from air of Beni Suef and Qena cities and one from grapevine fruits in Sohag city, Egypt. Exposure method 21 was employed for isolation of Cladosporium from air and direct plating technique 22 for isolation from grapevine fruits.
Czapek's Dox agar was used as an isolation medium. The medium is composed of (g/L): Sucrose, 30 For DNA isolation, a small portion from fungal growth of 7-day-old colonies of Cladosporium isolates AUMC 10865, AUMC 11340 and AUMC 11366 grown on potato dextrose agar (PDA) at 25°C were collected and transferred individually to 2 ml-Eppendorf tube. The DNA extraction was performed following CTAB method described in Moubasher et al. 24 The universal primers ITS1 and ITS4 25 were used for amplification of the internal transcribed spacer (ITS) region. 26,9 Alignment and Phylogenetic Analysis The sequences of Cladosporium isolates in this study were compared to the closely related sequences in GenBank as well as sequences of the type and ex-type Cladosporium species. Sequences of Cladosporium isolates in this study and those retrieved from GenBank were aligned together using MAFFT (version 6.861b) with the default options. 27 Alignment gaps and parsimony uninformative characters were cleaned using BMGE. 28 Maximum-likelihood (ML) and Maximum parsimony (MP) phylogenetic analyses were performed using PhyML 3.0. 29 The robustness of the most parsimonious trees was evaluated by 100 bootstrap replications. 30,31 The best optimal model of nucleotide substitution for the ML analyses was determined using Smart Model Selection (SMS) version 1.8.1. 32 The phylogenetic tree was drawn using MEGA X, 33 and edited using Microsoft Power Point (2016) and saved as TIF file. 9

Assessment of Enzymatic Potential in Submerged Fermentation (SmF)
Cladosporium AUMC 10865, AUMC 11340 and AUMC 11366 were cultivated, separately in 250 ml Erlenmeyer conical flasks containing 50 ml sucrose-free Czapek's broth medium supplemented with 1% soluble starch (for amylase production) or 1% pectin (for pectinase) or 1% xylan (for endoglucanase, exoglucanase and xylanase production). The flasks were inoculated, individually with 1 ml containing 1.8 x 10 8 spore/ ml of spore suspension obtained from 7-day-old cultures of the Cladosporium isolates tested. The flasks were then incubated for 7 days at 10°C and 30°C in shacking condition at 120 rpm. After incubation, the fermented media were filtered through Whatman No.1 filter paper and the cellfree supernatants obtained by centrifugation (10000 xg at 4°C for 10 min) were used as enzyme sources.

Enzymes Assay and Protein Estimation
Starch, carboxymethyl cellulose (CMC), microcrystalline cellulose (avicel), pectin, and oat spelts xylan were used for assays of amylase, endoglucanase, exoglucanase, pectinase and xylanase respectively. The reaction mixture consists of 0.5 ml of the filtered crude enzyme and 0.5 ml of previously-mentioned substrates at a concentration of 1% (prepared in 50 mM Nacitrate buffer, pH 5.0). The reaction was carried out in water bath at 50°C for 20 min. Afterwards, the process was stopped by introducing 2 ml of 3, 5-dinitrosalicylic acid (DNS) and the tube contents were placed in a boiling water bath for 10 min. 34 After cooling, the color absorbance was measured at 540 nm using UV-Visible spectrophotometer (T80+; UK). The amount of reducing sugar liberated was quantified using standard curves of glucose (for amylase, endoglucanase, exoglucanase, and pectinase), or xylose (for xylanase). One unit of the enzyme is defined as the amount of enzyme that liberates 1 µmol of the reducing sugar (glucose or xylose) equivalent per minute under the standard assay conditions. 35 Total protein content was measured by using bovine serum albumin (BSA) as the standard. 36 Calculations of enzyme activities were done. 37,38

Bioconversion of Orange Peel Wastes into Cocktail Enzyme Determination of Moisture Content (MC) of Orange Peels
Twenty grams of orange peels were weighed and dried in an oven at 105°C over-night, then cooled in a desiccator for 30 minutes and reweighed. The moisture content was calculated as follows: x 100 wt.of wet sample-wt.of dry sample wt.of wet sample

Solid-State Fermentation
Cladosporium isolates were grown separately in Erlenmeyer flasks containing 10 transparent supernatant was used as a source of cocktail enzymes. As previously stated, the amount of reducing sugar liberated, as well as enzyme activity and soluble protein were calculated.

Phylogenetic Analyses
The ITS sequences obtained from 32 strains produced 566 characters of which 494 were complete characters (no gaps, no N), 114 variable characters (23.1% of the complete characters) and 29 characters were parsimony informative (5.9% of complete). Tamura-Nei (TN93) was the best nucleotide substitution model. The dataset for maximal parsimony yielded 2 best parsimony trees with a tree length of 136 steps. The strongest ML tree score with a final ML probability optimization value of -1662.15949 and a tree size of 0.40191 was monitored to explain and explore the phylogenetic relationship between taxa (Fig. 1). The ITS sequences of Cladosporium isolates AUMC 10865, AUMC 11340, and AUMC 11366 from this study were uploaded to GenBank as MN826828, MN826919 and MN826822, respectively.

Enzymatic Capability of Cladosporium Isolates in SmF
Using SmF at 10°C and 30°C, the three Cladosporium isolates could develop amylase,  Fig. 2). On the other hand, using SmF at 30°C, the strain AUMC 11340 secretes the most amylase (4120 IU/gds), endoglucanase (2700 IU/ gds) and xylanase (3220 IU/gds) with the peak of specific activity of endoglucanase (27.8 IU/mg) and xylanase (28.1 IU/mg), while the maximum specific activity of amylase (43.7 IU/mg) was recorded in the strain AUMC 10865. Exoglucanase development reached the peak (8750 IU/gds) in the strain AUMC 10865 and specific activity (16.8 IU/mg) in the strain AUMC 11366. The overall production (2860 IU/gds and 5570 IU/gds) was instead enhanced by chitinase and pectinase  Fig. 3).

DISCUSSION
All living systems contain enzymes, which act as biological catalysts. 39,40 Enzymes are proteins in origin and catalyse a wide range of biological processes. Enzymes have been employed in several industries such as wine, cheese, bread, beer, vinegar, leather and linen. 41,42 In this study, three Cladosporium species were isolated from air and grapevine fruits. It was determined that the three isolates could not be identified based just on ITS sequencing, and that they may require sequencing of other genes such as actin (ACT), elongation factor 1-alpha (EF1), and large subunit (LSU) in order to be positively identified.
The enzymatic activity of the three Cladosporium species was assessed. With the aid of SmF, all isolates were able to produce different quantities of amylase, endoglucanase, exoglucanase, pectinase, and xylanase enzymes. In this context, numerous studies have shown that Cladosporium species can generate a variety of enzymes, including glucoamylase by Cladosporium gossypiicola, 43 laccase, cellulases, and hemicellulases by C. cladosporioides, 44,45 alpha-amylase, cellulase, and xylanase from C. cladosporioides, 46 and amylase, endoglucanase, exoglucanase and xylanase by C. allicinum AUMC 14511. 9 Cladosporium isolates in this study were used in SSF to convert orange peel wastes into valuable extracellular enzymes by fermenting the wastes. At 10°C, the isolate AUMC 10865 was superior to the other two isolates in the ability to produce the five extracellular enzymes from orange peels in SSF, while the isolate AUMC 11340 was the strongest at 30°C for all enzymes assayed. Orange peel waste has been shown to contain 16.9% soluble sugar, 9.21% cellulose, 10.5% hemicellulose, and 42.5% pectin, 18 which may be the explanation for the high enzyme production utilizing orange peels.
Microorganisms that have evolved to cold temperatures have a lot of promise in biotechnological applications, such as waste treatment and bioremediation at lower temperatures, as well as the textile and food sectors. [47][48][49] Cold-adaptive enzymes produced by psychrophilic microorganisms such as Cladosporium species have significant goals in bioconversion processes because of their high activity at low and moderate temperatures, they have the potential to economize processes by saving energy, and thus offer potential economic and environmental benefits such as reducing heatsensitive substrate alteration and the production of harmful by-products due to mild industrial conditions at low temperatures, and using psychrophilic enzymes. The aforementioned will make industrial operations more easy and safe, as in the case of conventional industry. 48, 50 Using Cladosporium species as enzyme developers, the current work demonstrated a lowcost substrate that does not require pretreatment. The expense of enzymes in industrial processes is a major problem. In addition, the usage of enzymes in diverse industrial processes indirectly influences the final product's price. Plant equipment and installation expenses account for a substantial percentage of an enzyme manufacturing facility's annual operating costs. 51 As a means of producing industrially significant enzymes, SSF offers numerous economic benefits. For the synthesis of fungal enzymes 52 and 53 have indicated that the SSF method is the most suited due to its high productivity, ease of enzyme recovery, and cost efficiency. Due to their cheap cost of collection, agro-industrial wastes are not only inexpensive solid substrates, but they are also ideal for extracellular enzyme synthesis. 54 The current study, on the other hand, revealed that SmF was the best for the synthesis of all enzymes examined. The increased productivity of submerged fermentation over solid state fermentation may be ascribed to more substrate availability in SmF due to improved aeration, and the surface of the substrate particles is routinely more susceptible to enzyme action than in solid state. Furthermore, the heat transmission and homogeneity of the submerged fermentation media make it preferable to the non-homogeneous solid state.

CONCLUSION
As part of this study, three Cladosporium isolates from air and grapevine fruits in Egypt were evaluated for their enzyme activity, as well as the bioconversion of orange peel wastes as a low-cost substrate for SSF's extracellular enzymes. Amylase, endoglucanase, exoglucanase, pectinase and xylanase were all produced at varying quantities by the isolates when grown in SmF and SSF.