Biochemical and Biotechnological studies on Xylanase and β‐xylosidase Enzymes Produced by Trichoderma viride Under Solid State Fermentation

The current study concerns with enhancement of xylanase and β‐xylosidase activities from lignocellulosic materials by soil derived fungi Trichoderma viride under solid state fermentation (SSF). Xylanase and β‐xylosidase activities were found to affected by variety of factors. In this study, some agricultural wastes were selected and used as sources for carbon. Sugar beet pulp (SBP) was at the forefront of these types. Also, various sources for nitrogen were selected to determine the best one. Yeast extract was the best organic source. Maximum xylanase activity took place by using inoculum size 1.8 × 105 spores / ml at 30°C when the pH was 5.5 for eight days of incubation with the addition of 0.1 % of Tween 40. Moreover, spores of Trichoderma viride were irradiated with gamma–rays. The maximum activity was observed upon using 0.7 kilo‐gray (kGy). Furthermore, mixed cultures of Trichoderma viride and Penicillium janthenellum (wt / wt) were enhanced xylanase degrading capability. From another standpoint, ammonium sulphate and gel filtration chromatography were the best methods for xylanase purification. Characterizations of the purified enzyme were also selected and studied. It was found that, β‐xylosidase enzyme exhibited its maximum activity and stability when the pH was 6 at 40°C by the addition of CaCl2 metal ion. On the other hand, total protein contents and volatile constituents of Trichoderma viride and Penicillium janthenellum were separated and investigated using high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) techniques. It was found that, total protein contents for Trichoderma viride and Penicillium janthenellum were represented by fifteen and sixteen amino acids respectively. Also,29 compounds of the total volatile compounds for both Trichoderma viride and Penicillium janthenellum were identified. On the practical and applied field, crude enzyme was a good analyzer for agricultural residues as well showed antifungal and antibacterial effects.

a vast range of secondary metabolites such as enzymes, vitamins, antibiotics, polysaccharides and other organic acids which equivalent to that chemically synthesized 10 . The production of secondary metabolites by Trichoderma and Penicillium shows great variety and application potential in this field pharmaceutical and human health applications. Fungi are capable of producing several important secondary metabolites which are used as antifungal and antibacterial agents 23 . Fungi are capable of synthesize the bioactive metabolites from few precursors using unique biochemical path-way 7 . Trichoderma viride and Penicillium janthenellum recorded antifungal and antibacterial effects against Candida sp. and E. coli respectively 2 .

Aims and Highlights of This Study
The aim of this work is to use Trichoderma viride and Penicillium janthenellum for xylanase production during growth on solid substrates and describes approaches for biotechnological applications.
We carried out this study in the following areas: 1. Quantitative survey for xylanase and βxylosidase activities. 2. Optimization of the fermentation parameters affecting enzymes production. 3. Enzyme purification by different methods. 4. Characterizations of the purified enzyme produced. 5. HPLC and GC/MS analysis of the crude extract and the volatile constituent. 6. Some agricultural wastes treatment and pharmaceutical applications.

Strains selection and media used
Trichoderma viride (RCMB) 017002 and Penicillium janthenellum (RCMB) 001033 [1] were selected to produce xylanase and β-xylosidase under solid state fermentation. These strains were obtained from Al Azhar University, The Regional Center for Mycology and Biotechnology, Cairo, Egypt. Media used for screening must contain micronutrient elements such as carbone and nitrogen sources in addition to other metals (Table 1).

Xylanase production and estimation
Trichoderma viride and Penicillium janthenellum were grown on modified agar media followed by observing zones around colonies. After the colonies reached around 3 mm, iodinepotassium iodide solution (1.0 g iodine, 5.0 g potassium iodide, and 330 ml distilled water) was added to detect clearance zones. The fermentation was done in 125 ml flasks contained 5 g of lignocellulosic materials at 30°C when pH was 5 8 . A nutrient solution used for keeping the moisture level at 70% (Table 2).
After the experimental time run out, 50 ml of distilled water was added to the fermentation flask and the mixture was stirred at 100 rpm for 1 h. Solid phase was removed by the vacuum filtration and the liquid phase was centrifuged at 10.000 xg. The supernatant was used as crude enzyme solution. In this study, 0.2 ml of the crude enzyme solution was added to flask contained 4 ml of 0.5% (w/v) substrates dissolved in 200 mM citrate solution with pH 5.5 at 25°C. For enzyme estimation, optical density was measured at zero time and after 1 min by used spectrophotometer (Hitachi model U-2000 with electronic temperature control unit) at 235 nm.

Xylanase optimization and purification
Xylanase production was induced by various factors such as carbon sources, nitrogen sources, inoculum sizes, incubation periods, pH values, incubation temperatures, surfactants, gamma irradiation doses and mixed cultures of Trichoderma viride/Penicillium janthenellum. In this study, centrifugation and gel filtration chromatography were selected for xylanase purification. In centrifugation method, ammonium sulfate was added to 500 ml of cultural filtrate to reached to 60% saturation, and leave for 60 min at 4°C, then centrifuged for 20 min at 9000 rpm to separate the pellets. Ammonium sulfate was added again to supernatant to bring to 70% saturation and leave overnight, then centrifuged at 9000 rpm for 20 min, then 50 ml of sodium acetate solution was added to dissolve the active fraction with maintained the pH to 6 and dialyzed against the same buffer 1 . In gel filtration chromatography method, Sephadex G-100 column was loaded with dialyzed enzyme and eluted with sodium acetate buffer at pH 5.9 and flow rate 20 ml/h. Total 30 fractions of 5 ml were subsequently collected and its protein content was measured at optical density 280 using spectrophotometer (UV-VIS 1601 Shimadzu, Japan). The fractions that showed  higher enzymatic activity were pulled together for characterization 29 . β-xylosidase activity measurement Sodium carbonate solution was added to 4-nitro phenyl β-D-xylopyranoside as a substrate followed by addition of enzymatic extract with adjust the pH to 5 at 60° C for 10 min 8 . The unit of enzyme activity (U) was defined as the amount of enzyme required to release 1 μ mol of reducing sugar per minute 19 .

Characterization of β-xylosidase enzyme
In this study, the activity and stability of β-xylosidase enzyme were induced by various factors such as pH values, temperatures, and metal ions within concentration of 10 mM 26 .

Total protein content and investigation of the volatile constituents
Fungal mycelia were extracted with 70% ethanol. Separation of proteins were done according to Bellomonte et al. 1987 4 . In this method, 2.5 g of extract was dissolved in mixture of (10 ml distilled water, 23 ml ethanol and 2.5 ml sulphuric acid) and leave for 20 min at 27°C. Then, 25 ml of distilled water and 125 ml ethanol were added again. Ammonium hydroxide was added to adjust the pH to 3 and the mixture was filtrated. Acetone was added till precipitate was formed and centrifuged at 4000 rpm, then collected, dried and weighed. Then, HPLC analysis was detected. On the other hand, 25 g of fresh mycelia was distilled with pentane as organic solvent. The pentane layer was collected, dried over sodium sulphate anhydrous and stored at 4°C for GC/MS analysis. Then, investigation of the volatile constituents was also detected.

Xylanase applications
Xylanase was used for treatment of some agricultural residues such as wheat, rice straw and sugar cane bagasse 21 . In this study, 50 ml of crude enzyme was mixed with each residue in shaking flasks with 120 rpm for 24 and 48 h at 50°C and pH 7. The supernatant used to determine the reducing sugar. Also, crude extraction from fresh fungi Trichoderma viride and Penicillium janthenellum were evaluated as antimicrobial agents (antibacterial and antifungal) using antibiotic assay method. In this method, 50, 100, 150, and 200 µg/disc crude extract of Penicillium janthenellum and Trichoderma viride were tested against Candida sp. and E. coli using Sabouraud dextrose and nutrient agar media respectively. Fungal plates were incubated under aseptic conditions for 72 h at 28°C while, bacterial plates were incubated for 24 h at 30°C. The diameter of inhibition zone (mm) for each pathogen was determined exactly and then, the mean of the triplicates was calculated.

Screening to choose the most potent fungal strain
Trichoderma viride and Penicillium janthenellum were compared for xylanase production because of their higher ability to produce the enzyme. They showed xylanase activities represented by 330.3 and 220.5 U/gds for Trichoderma viride and Penicillium janthenellum respectively (Table 3).

Xylanase optimization produced by Trichoderma viride
In this study, we focused on Trichoderma viride for xylanase production. The organism ingested nutrients to live and produces secondary metabolites. Secondary metabolites were found to affect by various factors. Carbon source played an important role in the production of xylanase. To reduce the cost of enzyme production, an inexpensive lignocellulosic substrate should be preferred. The results showed that, sugar beet pulp (SBP) was the best source for carbon that exhibited the maximum xylanase activity while, other carbon sources gave lower activities. Wheat bran and (SBP + broad bean) exhibited un-noticeable change Table 3. Screening of the most potent fungal xylanase producers Enzyme Strains and enzymatic activities (U / gds)

Penicillium janthenellum
Trichoderma viride (RCMB) 001033 [1] (RCMB) 017002 Xylanase 220.5 ± 0.5 330.3 ± 0.06 and (SBP + banana) which gave enzymatic activities represented by 220, and 224 U/gds respectively (Fig. 1). Nitrogen source also was optimized  for maximum xylanase production under SSF. It was found that, the most potent nitrogen sources were yeast extract as organic source and ammonium sulfate as inorganic one which the activities represented by 330.3 and 328.6 U/gds respectively. Malt extract was the third nitrogen source that gave noticeable change of enzymatic activity after yeast extract and ammonium sulphate. Other nitrogen sources including organic and inorganic gave lower xylanase activity (Fig. 2). The maximum xylanase activity produced at inoculum size 1.8 × 10 5 spores/ml. As inoculum size increased, xylanase activity decreased (Fig. 3). The period of incubation was also optimized to find out the time period for maximum xylanase production. Trichoderma viride started xylanase activity from the second day of incubation period with slightly enzymatic activity. The enzyme activity increased as the incubation period increased and reached its maximum activity on the eighth day of the incubation period, then began to decrease significantly as the incubation period increase. However, xylanase activity still moderately high even on the ninth day of the incubation period (Fig. 4). The pH values of the culture medium also affect the production of enzyme. Several fungi are able to produce xylanase in acidic medium. Trichoderma viride was able to produce xylanase at all tested pH values. It was shown that, at low pH ranges (3 -4), the activity was very low, then began to increase gradually until reached its maximum activity at pH ranges (5 -6). The maximum activity was achieved at pH 5.5, then the activity started to decrease at pH ranges (6.5 -7.5) (Fig. 5). Temperature is one of the most essential parameters that found to affect the production of xylanase under SSF. Trichoderma viride started xylanase activity at 20°C and reached its maximum rate at 30°C, then the activity started to decrease as the incubation temperature increased. Above the optimum temperature (30°C), the enzyme denatured and thus the production decreased (Fig.  6). Surfactants were found to make a dispersing effect on the media and result in enhanced the aeration and increase in the enzyme secretion. The highest xylanase activity was observed upon addition of 0.1% of Tween 40. Tweens 20 and 60 caused slightly increase on activities as compared to Tween 40, while no effect occurred upon using triton X-100 (Fig. 7). Spores of Trichoderma viride were irradiated with slightly doses of gamma-rays. The maximum xylanase activity was achieved upon using 0.7 kGy of gamma irradiation dose then, the activity decreased as gamma irradiation doses increased until reached to 2 kGy of gamma irradiation doses, the enzyme activity faded (Fig.  8). Also, mixed cultures of Trichoderma viride and Penicillium janthenellum were found to increase xylanase degrading capability by about 101.75% higher than Trichoderma viride only and 202.2% higher than Penicillium janthenellum only (Fig. 9). Data in (Table 4) showed summarize of the optimum conditions used for the xylanase production.

Xylanase purification and β-xylosidase characterization
Xylanase purification was done by the centrifugation followed by ammonium sulphate. When cultural filtrate was saturated with ammonium sulphate up to 60%, the enzyme activity decreased to 6.03 U/ml, while supernatant was saturated again up to 75%, the enzyme activity raised to 13.47 U/ml. While, enzyme without ammonium sulphate exhibited activity determined  (Fig. 10). From the results, βxylosidase exhibited its maximum activity and stability when the pH was 6 at 40°C within addition of Cacl 2 (Fig. 11,12,13,14 and 15).

Total protein content and investigation of the volatile constituents
Trichoderma viride exhibited protein content represented by 12% (wt/wt), while      Penicillium janthenellum exhibited 10% (wt/ wt). HPLC analysis of total protein content of Trichoderma viride showed seven essential amino acids represented by 40.39% and eight nonessential amino acids represented by 59.61% (wt/ wt) ( Table 5). On the other hand, HPLC analysis of total protein content of Penicillium janthenellum showed eight essential amino acids represented by 41.49% and eight non-essential amino acids represented by 58.51% (wt/wt) (Figs 16 and 17). The volatile constituents of Trichoderma viride and Penicillium janthenellum were represented by 0.14% of fresh mycelia for each strain. GC/ MS analysis identified 29 compounds which represented by 84.17% and 79.18% of the total volatile compounds for Trichoderma viride and Penicillium janthenellum respectively (Table 6).

Xylanase applications
Wheat and rice straw were more efficiently to xylanase hydrolysis but, sugarcane bagasse was the more intensive one. As the incubation period increased, the reducing sugars released increased (Fig. 18). Results in (Table 7) illustrated that no inhibition zones were detected when disks were loaded with 50 µg/disc of the extract. While, 100 µg/disc concentration showed remarkable inhibition zone against Candida sp. and E. coli which represented by 10.93 mm and 10.16 mm respectively. This concentration known as minimum inhibition concentration (MIC). The inhibition zone increased as concentration of crude extraction increased. The crude extraction recorded high inhibition zone at 200 µg/disc which represented by 12.67 mm and 12.17 mm for Candida sp. and E. coli respectively.

DisCUssiON
In this study, Trichoderma viride was selected to produce xylanase and β-xylosidase enzymes under solid state fermentation conditions. Agriculture wastes like (sugar beet pulp, (sugar beet pulp + wheat bran), wheat bran, (sugar beet pulp + banana), (sugar beet pulp + broad bean) and broad bean) were used as sources of carbon for xylanase production. It was shown that, the maximum xylanase activity was achieved when sugar beet pulp (SBP) was used as a sole carbon source and yeast extract as a nitrogen source. These results were found to agree with Yardimci and Cekmecelioglu 2018; Ketipally and Ram 2018 13,38 who reported that, xylanase production was increased significantly when arabinose, xylose and cellulose were used as a sole carbon source. Otherwise, they were also reported that, yeast extract, malt extract and peptone with beef extract were the best sources for nitrogen that gave  higher xylanase activity. In this study, Trichoderma viride showed its maximum xylanase activity on the eighth day of the incubation period. This result was agree with SIPRIYADI et al. 2020 33 who reported that, the maximum activity of xylanase was obtained on the eighth day of incubation. Also, Richhariya et al. 2020 28 reported that, the maximum xylanase production was moderately high when wheat bran was used as a carbon source after 8 days of incubation. Temperature and pH value are essential factors to find out the maximum xylanase production under solid state fermentation. The optimum temperature and pH for xylanase production produced by Trichoderma viride achieved at 30°C when the pH was 5.5. These results were found to agree with Richhariya et al. 2020 28 who reported that the optimum xylanase production took place when the pH was (6-7) at 30°C for 8 days of incubation.
The enzyme production decreased as pH and temperature increased. Also, in this study, the maximum xylanase production took place at the inoculum size 1.8 × 10 5 spores/ml. This result disagree with Ezeilo et al. 2020 11 who reported that, 1 × 10 8 spores/g was the optimum inoculum size for xylanase production, while enzyme activity decreased as inoculum size increased. Also, Khanahmadi et al. 2018 14 reported that, the optimum inoculum level for xylanase production was 1 × 10 7 spores/ml. Surfactants played an important and positive role in enhancement of enzyme activity. Surfactants induced the enzyme production by increased the penetration of water into the solid substrate and increased the surface area for microbial growth which increased the permeability of the microbial cell membrane and increase the release of proteins into the medium. In this study, the highest xylanase activity was achieved upon addition of Tween 40. While, Tweens 20 and 60 caused slightly increase on the activity as compared to the control. While no effect occurred upon using triton X-100. These results were agree with Bala and Singh 2017 3 who reported that, different surfactants such as Tweens and triton X-100 enhanced production of all the hydrolytic enzymes as compared to other surfactants. Khanahmadi et al. 2018; Nanjundaswamy and Okeke 2020 14,22 reported that, the maximum xylanase production was achieved by the addition of 0.1% (w/v) of Tween 20. Gamma-mutagenesis including gamma irradiation has a great importance and advantages as a mutagenic agent. The optimum dose of gamma irradiation used to produce the highest quantity of xylanase was 0.7 kGy. Further increase of gamma irradiation doses results in a noticeable decrease of viability of spores. This result disagree with Kostyleva et al. 2018 16 who found that, the most aggressive irradiation dose that gave the highest levels target of the enzyme was 2.5 kGy. Xylanase and endoglucanase activities obtained by irradiated T. reesei strain were significantly higher than the activities observed in the original strain.
On the other hand, mixed culture of recombinant strains can reduce the cost of enzyme production and increase enzyme degrading capability.