The chemicals and media were purchased from Himedia, Sigma and Merck.
Composition of carbohydrate utilization broth:
(NH4)2SO4 (0.2%), K2HPO4 (0.4%), Na2HPO4 (0.6%), FeSO4 (0.1%), MgSO4 (2%), CaCl2 (0.1%), MnSO4 (0.001%), ZnSO4 (0.007%), CuSO4 (0.005%), H3BO3 (0.001%) and MoO3 (0.001%) at pH 7.2 ± 0.2. Pectin, carboxy methyl cellulose (CMC) and starch @ 1% were added to the broth for preparing respective carbon source utilization broth.
Isolation of fungal culture:
Fungi were isolated from degrading organic substrate by serial dilution, and pour plating isolation techniques were used. The isolates were then sub-cultured into their respective selective growth media (Pectin, CMC and Starch) until pure cultures were obtained.
Primary screening of fungal isolates:
Primary screening of fungal isolates for pectinase, cellulase and amylase production was done on the basis of clear halo zone formation on pectin/CMC/starch utilization agar plate.
Secondary screening of fungal isolates:
The carbohydrate utilization broth with 1% substrate (Pectin/CMC/Starch) was inoculated with fungal isolates, showing positive results in primary screening, at pH 7.0 and temperature 30°C for 10 days. Culture filtrate was tested for pectinase, cellulase and amylase activities as per the following protocol:
Twenty ml of culture filtrate was centrifuged @ 10000 rpm for 10 minutes at 4°C. Five ml of the cell-free supernatant obtained was filtered through syringe filter (0.22µm) and was used for enzyme assay Miller (1959).
One volume of supernatant was precipitated with 4 volumes of cold acetone, and incubated at -20°C for 20 minutes. The samples were centrifuged @ 10000 rpm at 4°C for 10 minutes. The pellet was dissolved in the freshly prepared 0.2M acetate buffer (pH- 5.5) and assayed for pectinase, cellulase and amylase activities.
The enzyme reaction mixture contained 0.4 ml of substrate (Pectin 0.5%/ CMC-1.0%/ Starch-1.0% dissolved in 0.2 M of acetate buffer pH- 5.5), 0.1 ml of crude enzymes and 0.5 ml of distilled water. The tubes were incubated at 35°C for 1 hour in water bath. One ml of DNS (Dinitrosalicylic acid) reagent solution was added to stop the enzyme reaction. Subsequently, the reaction tubes were placed in water bath at 100°C for 10 minutes. Standards of D-galacturonic acid and glucose were taken in the range of 1mM to 10mM for pectinase and cellulase/amylase, respectively. The optical density was recorded at 550 nm using spectrophotometer (double beam UV-VIS). Enzyme activity was expressed as the amount of enzyme that liberated 1 µmol of reducing sugar per ml per min. Enzyme activity was calculated as per the following equation:
Enzyme activity ml-1minute-1=
Scanning electron microscopic (SEM):
Scanning electron microscopic image of selected fungal isolate was followed as per method of (Babu et al. 2018).
The fungus grown on mosambi peel was fixed with glutaraldehyde (2.5%) in phosphate buffer (10 mM; pH 7.4) for 4 h. The fixed samples were rinsed twice with deionized water and dehydrated with increasing concentrations of ethanol (10, 20, 40, 60, 80, 90 and 100%) for 10 min each. Samples were dried at room temperature for three hours then sputter-coated and visualized at 2000X.
Molecular identification of selected fungal isolate:
The highest enzyme-producing fungal isolate was subjected to molecular characterization on the basis of ITS region Specific Primer (ITS1 and ITS4) sequencing technique. DNA was isolated from the culture, evaluated on 1.2 % agarose gel, extracted DNA was amplified with ITS region Specific Primer (ITS1 and ITS4) using Veriti® 96 well Thermal Cycler (Model No. 9902). A single discrete PCR amplicon band of 600-800 bp range was observed. Further, the PCR amplicon was purified through enzyme treatment and subjected to Sanger sequencing. Bi-directional DNA sequencing reaction of PCR amplicon was carried out with ITS1 and ITS4 primers using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. The Consensus sequence of 610 bp of ITS region was generated from forward and reverse sequence data using aligner software. BLAST alignment search tool of NCBI Genbank database was done using ITS region sequence. Based on maximum identity score first fifteen sequences were selected and aligned using the multiple alignment software program ClustalW. The distance matrix was generated using RDP database and the Phylogenetic tree was constructed using MEGA 5 (Kumar et al. 2011).
Mosambi peel, collected from wholesale fruit market, Dubagga, Lucknow, India, was properly washed with water, chopped and air-dried. 100g of chopped piece (approx. 2mm size) was taken, autoclaved and inoculated with 1 ml of actively growing culture of selected fungal isolate @ 1.0*107spores/ml and incubated. Samples were withdrawn at different time intervals; the culture filtrate was used for enzyme assay. Samples were precipitated with acetone and analyzed for pectinase, cellulase and amylase activities (as described in enzyme assay section).
To study the effect of nutrient addition on production of pectinase, cellulase and amylase, ammonium sulphate (as source of nitrogen) @ 0.1% and potassium dihydrogen orthophosphate (as source of potassium and phosphorus) @ 0.01% was added to chopped mosambi peel, autoclaved, inoculated by Trichoderma asperellum NG 125 @ 107 spores/ml and incubated at 30°C for 5 days and enzymes activities were observed.
Selection of suitable natural substrate on enzyme immobilization:
Fibre was extracted from bagasse, rice husk, paddy straw and wheat straw by boiling with water, treating with 1N-HCl followed by 1N-NaOH treatment. It was then washed with water, oven-dried at 60°C and sieved in 60-150 mesh size. (Ranganna 2001).
The extracted fibre was autoclaved, the enzyme was added and incubated for 3 hours. The un-immobilized enzyme was washed away with acetate buffer, while activity was tested in enzyme immobilized on fibre.
Enzyme purification and characterization:
Pure substrates (1% pectin/ CMC/ starch in carbohydrate utilization broth) inoculated with Trichoderma asperellum were incubated at 30OC for ten days. The culture filtrate was precipitated with cold acetone, pellet was dissolved in acetate buffer and passed through gel filtration chromatography column (1.0*40cm) packed with Sephadex G-100 matrix pre-equilibrated with 0.2M acetate buffer (pH 5.5). Two ml fractions were collected in Eppendorf tubes at the flow rate of 20 ml/hour and enzyme activities were observed.
Characterization of enzymes with respect to temperature, pH and substrates was carried out as per method described by Coral et al. (2002) using temperatures of 20, 30 and 40°C and pH levels of 4.5, 5.5, 6.5 and 7.5. The heat stability of enzyme was determined by subjecting it to 65°C for one hour.
Relation between substrate concentration and enzyme production was also worked out. Enzymes were assayed in reaction buffer (pH 5.5 ) with substrate concentrations between 0.25- 5 mg/ml for pectin and 0.5-10 mg/ml for carboxymethyl cellulose and starch. The values of Km (Michaelis constant) and Vmax (maximum velocity) were calculated from Michaelis–Menten saturation curve.
In order to determine the molecular size of the pectinase, cellulase and amylase produced in respective pure substrate, enzyme precipitate was subjected to electrophoresis in 10% native polyacrylamide gel and 12% denaturating sodium dodecyl sulfate-polyacrylamide gel in discontinuous buffer as per described by Laemmli (1970). Coomassie brilliant blue R-250 staining was used to visualize protein bands after electrophoresis.
A comparative study of enzymatic mango juice extraction using multienzymes produced by Trichoderma asperellum against a commercial pectinase was conducted. The diluted mango pulp was incubated at 35OC for 180 min with 0.5 ml of crude enzyme extract/ commercial pectinase. The content was filtered through Whatman No. 1 filter paper and quantified.
All the experiments were carried out using completely randomized design in triplicate, repeated twice for reproducibility. The analysis of experimental data with two-way analysis of variance (ANOVA) was conducted followed by Fisher's multiple comparison test at p<0.05. The least significant difference (LSD) test was used to determine whether there was significant difference among the samples (Gomez and Gomez 1984)