Agro-residues, chemicals and sample collection
Agro-residues obtained from local farms were washed thoroughly, followed by oven drying and grinding to obtain particles of uniform size 1 mm, to be used as carbon source during screening and fermentation process. Xylose (Sigma, USA), Oat spelts xylan, birchwood xylan, carboxymethyl cellulose (CMC), bovine serum albumin (BSA) and dinitrosalicylic acid (DNSA) were procured from HiMedia Laboratories Ltd. Mumbai, India. All the other media, chemicals, salts and reagents used were of analytical grade (Sigma-Aldrich, St. Louis, MO, USA; HiMedia, Mumbai, India; and Merck & Co., Inc.). Optical compound microscope (Make-Magnus MLX-M) was used for microscopic analysis.
Samples were collected aseptically in soil rich in decaying plant matter and garbage waste from different locations of Noida and Delhi, India such as Okhla Bird Sanctuary (28.5514° N, 77.3185° E), Challera (28.5545° N, 77.3372° E) and Tajpur Pahari (28.4948° N, 77.3059° E) and stored at 4°C until further study.
Isolation and screening of GHs producing microorganisms
Serially diluted samples inoculated on nutrient agar (NA) and potato dextrose agar (PDA, 50 µg ml-1 ampicillin) plates, were incubated at 20-60°C for up to 72 h for isolating indigenous glycoside hydrolase producing strains. Isolates were sub-cultured and stored at 4°C and as glycerol stocks. Czapek-Dox mineral salt agar medium and Remazol brilliant blue xylan supplemented medium was utilized for screening of cellulolytic (Teather and Wood, 1982) and xylanolytic (Shrivastava et al. 2011) microorganisms, respectively.
Screening through enrichment media and determination of xylanase and endoglucanase activity
Enrichment medium (Potato dextrose broth supplemented with 1% corn cob; agricultural residue; pH 5.5) was inoculated with pre-inoculum (106 spores/ml). Extracellular enzymes were produced through medium containing (g l-1) yeast extract (5), KH2PO4 (1) and MgSO4.7H2O (0.5) supplemented with 2% (w/v) corn cob (pH 5.5) at 40°C in a rotary shaker at 100 rpm for 10-12 days, with enzyme activity of cell free extract determined at interval of 24 h.
Xylanase activity was determined by monitoring the production of reducing sugars by 3,5-dinitrosalicylic acid (DNS) method (Miller 1959) with minor modifications using xylose as standard according to Bailey et al. (1992). One unit of xylanase activity was defined as the amount of enzyme that catalyzes the release of 1 µmol of xylose equivalent per minute under defined assay conditions. CMCase activity was determined according to Ghose (1987), with 1% CMC as substrate. One unit of CMCase activity was defined as the amount of enzyme that releases 1 μmol of glucose per ml per minute.
Identification of fungal isolates
Xylanolytic and cellulolytic microbial strains were examined microscopically using Lactophenol cotton blue (Leck 1999) and Grams stain (Bartholomew and Mittwer 1952) according to standard procedures. Four potential fungal strains with high titers of xylanase were identified based upon the ITS region, 18S rRNA, 28S rRNA, β-tubulin and actin gene sequencing at commercial facility provided by Chromous Biotech Pvt. Ltd., and Eurofins Genomics India Pvt. Ltd., Bengaluru, India. Genomic DNA was extracted from the selected fungal isolates according to Al-Samarrai and Schmid (2000) with slight modifications and used for amplification of ITS region, 18S rRNA, nLSU rRNA, β-tubulin and actin genes using primer pairs ITS1/ITS4 (White et al.1990), n-SSU-0817/nu-SSU-1536 (Borneman and Hartin 2000), LROR/LR7 (Vilgalys and Hester 1990; Rehner and Samuels 1994), BENA1/BENA3 (Davolos et al. 2012) and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. All nucleotide sequences of the genes were submitted to NCBI GenBank database. Similarity search was carried out for each gene sequence using online BLAST program (http://www.ncbi.nlm.nih.gov/) and analyzed (Table 1) using the ClustalW algorithm available in molecular evolutionary genetic analysis (MEGA X) software with default parameters (Kumar et al. 2018). The phylogenetic tree of the ITS region was generated by employing the maximum likelihood method using Tamura-Nei model (Tamura and Nei 1993) and the evolutionary distance was inferred using the bootstrap method (n = 1000 bootstrap replications).
Multigene phylogeny construction through alignment of relevant sequences and trimmed matrices construction of ITS and nLSU as well as ITS, nLSU and β-tubulin sequences was done through MEGAX. A Bayesian interference (BI) analysis was conducted via MrBayes v3.2.2 (Ronquist et al. 2012) using a GTR + I + Γ model (General Time Reversible model) for 3000000 generations with sampling every 100 generations. The settings were as follows: nst = 6, rates = invgamma, MCMC heated chain set with nchains = 4 and temp = 0.2, ngen = 3000000, samplefreq = 100. Two independent analysis with four chains each (one cold and three heated) were run until the average standard deviation of the split frequencies dropped below 0.01. The initial 25% generations of MCMC sampling were discarded as burn-in. Refinement of the phylogenetic tree was used for estimating BI posterior probability (BIPP) values (Zheng et al. 2020). The tree was viewed in FigTree v1.4 (Rambaut 2012).
Enzyme production, partial purification and molecular characterization
Enzyme production from strains AUMS60 and AUMS64 was carried out as mentioned above with fermentation media supplemented with 3% (w/v) corn cob (pH 7); at 40°C on a rotary shaker (110 rpm) for 7 days. Filtered cell free extract was concentrated 10-fold via ultrafiltration membrane with a molecular weight cutoff of 10-kDa (Millipore TFE system, Bedford, MA, USA). Xylanase activity of all samples were determined as mentioned previously and protein quantification was done through Lowry’s method using BSA as standard (Lowry et al. 1951).
Electrophoretic analysis of crude and partially purified samples was done through native and SDS PAGE (Ornstein and Davis 1964, Laemlli 1970, Shrivastava et al. 2013) with protein bands developed through silver staining (Blum et al. 1987) documented through Gel documentation system (Bio-Rad Laboratories, Hercules, CA, USA). Zymography was carried out with method of Royer and Nakas (1990) with slight modification. Reagents were procured from Biorad (Bio-Rad Laboratories, Hercules, CA, USA) and Himedia (HiMedia Laboratories Pvt Ltd., Mumbai, India).
Biochemical characterization of partially purified enzymes
Effect of pH on partially purified enzymes from AUMS60 and AUMS64 was studied at pH 5-8 at 40°C, 50°C and 60°C (Sodium acetate and Tris chloride buffer, 0.05 mol l-1). Stability at pH 5 and 6 were studied for 120 h at 40°C. Temperature optima was determined by studying catalytic efficiency from 30°C to 90°C. Thermostability was studied at 40°C, 50°C and 60°C. Effect of selective metal ions and chemicals (NaCl, KCl, CaCl2, CuSO4, MgSO4, FeSO4, EDTA and SDS) in dose of 1 and 10 mM was studied by incubation at 50°C for 30 min with respective analyte.
Effect of enzyme dose (20-100 U g-1 of substrate) was studied on saccharification of purified substrate (1% (w/v) birchwood xylan prepared in Acetate buffer, pH 6.0) upto 24 h with incubation at 40°C in rotary shaker at varying speed of 100-140 rpm.