Production Characterization and in Silico Studies C Chitinases from Actiomycetes and its Applications

Abstract

Chitinase is gaining importance in recent years due to its numerous applications newlinein biotechnology. The chitinases are potential alternative for chemical agents used in newlinecrop protection, medicine and genetic engineering. Among all chitinase sources, newlinemicrobial chitinases have many advantages because of their easy and economic newlineavailability. The marine and fresh water samples and rhizosphere soil samples were used newlinefor isolation of chitinolytic actinomycete species through enrichment in selective media. newlineThe potential chitinase producers were screened by qualitative and quantitative assay newlinemethods. The efficient chitinolytic actinomycete specie was identified by 16 S rRNA newlinesequencing as Streptomyces macrosporeus M1 and used for optimized production of newlinechitinase. The optimum chitinase production was found on third day of incubation, at newline40°C temperature and 6.0 pH. Potassium nitrate (0.2%) and calcium sulafate (10 mM) newlinewere found as effective source of nitrogen and metal salt respectively for chitinase newlineproduction. The optimized parameters gave 2.62 fold increase in production over control. newlineOptimum medium for chitinase production composed of colloidal chitin (1.5%), calcium newlinesulafate (10 mM) and potassium nitrate (0.5%) when incubated at 40°C temperature for 3 newlinedays gave 9.2 U/ml yield of chitinase by Streptomyces macrosporeus M1. Purification of newlinethe crude S. macrosporeus M1 chitinase was done by ammonium sulfate precipitation, newlinedialysis and DEAE column chromatography. The purification process gave 6.81 fold newlinepurified enzyme with 18.78% yield. The molecular mass of purified chitinase was 47 newlinekDa as determined by SDS PAGE. The chitinase gene of S. macrosporeus M1 was found newlineof 740 bp after sequencing. The structure of chitinase was determined by homology newlinemodeling and S. macrosporeus M1 chitinase showed 61.20% structural similarity with newlineexisting chitinase model of Streptomyces thermoviolaceus. The purified chitinase newlineproduced chitooligosaccharides mixture of degree of polymerization 4 to 17 from chitin. newlineThe chitooligosaccharides mixture showed 72% antioxidant activity determined by newlineDPPH assay and 61% anti inflammatory activity was estimated by inhibition of protein newlinedenaturation assay. The protoplast of S. cerevisiae leu- auxotroph cells were prepared newlinefrom chitinase and further applied for leu- gene transformation and regeneration. The S. newlinemacrosporeus M1 also chitinase showed antifungal activity against Ustilago maydis, newlineRhizopus oryzae, F. oxysporium, and A. flavus showing possibility of exploring it as newlinebiocontrol agent against phytopathogens. newline