Studies on nitrile hydratase of microbial isolate Nit 36 and its application in Lactamide synthesis

Abstract

newline ABSTRACT newlineMicroorganisms particularly bacteria are a unique source of a variety of newlineenzymes. In this study, nitrile hydratase producing microbial isolate R. pyridinivorans newlineNIT-36 was isolated from the hot water spring of Tattapani (District: Mandi, newlineHimachal Pradesh). Though isolated from a thermal site this isolate does not show newlineactivity at high temperatures therefore it is categorized as a mesophile. Isolate NIT-36 newlinewas confirmed as Rhodococcus pyridinivornas NIT-36 on the basis of colony newlinemorphology, stained preparation, biochemical tests as well as 16S rRNA sequencing newlineand showed 99% similarity to Rhodococcus pyridinivornas SB 3094. Response newlineSurface Methodology was used to generate a process model for obtaining optimal newlineconditions for important parameters influencing NHase activity of R. pyridinivorans newlineNIT-36. Further scale up studies for acrylamide and lactamide production was done newlineby both batch and fed-batch methods. newlineThis study also incorporates the partial purification and characterization of NHase newlineprotein. Further the immobilization of R. pyridinivorans NIT-36 was done on natural newlinepolymer chitosan. The chitosan microspheres were prepared by suspension crosslinking newlinetechnique which resulted in formation of microspheres of 10 and#956;m size. The newlinemorphology, structure and properties of synthesized microspheres were characterized newlineby SEM, FTIR and XRD. The morphological characterization of microspheres newlineindicates that the chitosan N,N methylene-bis-acrylamide cross-linked cells forms newlineporous structure with smooth and uniform surface. The immobilized cells are newlinethermally stable compared to its free counterpart when exposed to thermal newlinedegradation studies. The immobilized NHase exhibited greatly effective catalytic newlineactivity, thermal stability and increased tolerance to the varied pH conditions for newlinenitrile hydration of lactonitrile. Furthermore, the operational stability of the newlinebiocatalyst was improved significantly by immobilization and it retained good activity newlineup to seven successive batches of reaction.