Microbial cellulases are the enzymes widely studied due to their enormous applications in biochemical industry. Among 12 fungal isolates isolated from mangrove plant debris and soil sample collected from Valanthakad Mangroves, Kerala, India, 3 of them were found to exhibit cellulolytic activity. Among them, the most potent isolate which exhibited maximum cellulolytic activity was identified as Trichoderma viride VKF3 [Gene bank accession number- JX683684.1] based on colony morphology, microscopic observation and molecular centeracterization using D1/D2 region amplification. The isolate T. viride VKF3 was found to be non-phytopathogenic against the selected plants. Neighbour joining tree depicted its least divergence rate from the root taxon HM466686.1. T. viride VKF3 was grown under dynamic carbon, nitrogen sources, pH and temperature of the medium to draw out the optimum conditions for cellulase production. Protein stability kinetics and biomass production was also studied upto 11th day of incubation. It was evident from the study, that dextrose and beef extract could be used as major carbon and nitrogen sources in submerged fermentation at pH 9.0 and incubation temperature of 25°C to get maximum CMCase yield. Optimum enzyme recovery period was identified between 5th to 9th days of incubation beyond which the enzyme activity was reduced. By comparing two fermentation methods, submerged fermentation was found to be the best for maximum enzyme production. But utilization of substrates like sugarcane bagasse and cassava starch waste in the SSF offers a better scope in biodegradation of solid waste contributing to solid waste management.
Cellulase is one of the most widely distributed enzymes with wide application. They are involved in conversion of biomass into
simpler sugars. Cellulase of Trichoderma longibrachiatum, a known cellulolytic fungus was compared with Clostridium thermocellum
[AAA23226.1] cellulase. Blastp was performed with AAA23226.1 as query sequence to obtain nine similar sequences from NCBI
protein data bank. The physicochemical properties of cellulase were analyzed using ExPASy’s ProtParam tool namely ProtParam,
SOPMA and GOR IV. Homology modeling was done using SWISS MODEL and checked quality by RMSD values using VMD1.9.1.
Active sites of each model were predicted using automated active site prediction server of SCFBio. Study revealed instability of
cellulase of two eukaryotic strains namely Trichoderma longibrachiatum [CAA43059.1] and Melanocarpus albomyces [CAD56665.1]. The
negative GRAVY score value of cellulases ensured better interaction and activity in aqueous phase. It was found that molecular
weight (M. Wt) ranges between 25-127.56 kDa. Iso-electric point (pI) of cellulases was found to be acidic in nature. GOR IV and
SOPMA were used to predict secondary structure of cellulase, which showed that random coil, was dominated. Neighbor joining
tree with C. thermocellum [AAA23226.1] cellulase as root showed that cellulases of Thermoaerobacter subterraneus [ZP_07835928] and
C. thermocellum [CAA4305.1] were more similar to eukaryotic cellulases supported by least boot strap values. Pseudoalteromonas
haloplanktis cellulase was found to be the ideal model supported by least RMSD score among the predicted structures. Trichoderma
longibrachiatum cellulase was found to be the best compared to other cellulases, which possess high number of active sites with
ASN and THR rich active sites. CYS residues were also present ensuring stable interaction and better bonding. Hydrophilic
residues were found high in active sites of all analyzed models and template.
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