Isolation Optimization and Biotransformation of Nitrilase from Thermophilic Bacterial Isolates for Mandelic Acid Production

Abstract

newline ABSTRACT newlineThe importance of thermostable biomolecules has led to an upsurge of research into organisms from thermophilic sites. The present study was aimed to isolate mandelonitrile degrading microbes from hot water springs of Manikaran and Kheerganga (District Kullu, Himachal Pradesh). Extensive screening by indicator plate assay method resulted in obtaining nitrile degrading thermophiles. Identification of these thermophilic isolates was done on the basis of biochemical tests and 16s rRNA gene sequencing and were designated as Geobacillus icigianus, Geobacillus stearothermophilus, Bacillus licheniformis, Lysinibacillus macroides and Bacillus pumilus. Two isolates namely Geobacillus stearothermophilus MAC1 and Geobacillus icigianus MAC VI have been deposited at IMTEC and assigned accession numbers MTCC 12615 and 12616 respectively. The nitrilase enzyme was inducible in nature and was activated in presence of mandelonitrile. Optimization of various culture and process parameters of Geobacillus icigianus by OVAT resulted in enhancement of nitilrase activity. Further, RSM was used to generate a process model for enhanced nitrilase activity. A delayed inducer feeding resulted in better utilization of mandelonitrile as inducer. A 7.4 fold increase of nitrilase activity was observed after complete optimization. The enzyme was exceedingly thermostable as it showed activity (0.098 U/mgdcw) at 100°C even after 5h of incubation and has been utilized for mandelic acid synthesis. Scale-up of mandelic acid production was carried out by bench scale process using phosphate buffer with pH 7.0 at 60°C resulting in production of R-mandelic acid with 74% ee. newlineThermophiles have also evoked a great interest due to the ability to clone the genes from these thermophiles into mesophilic production strains thus facilitating the growth of microorganisms harbouring enzymes having better thermo stability at normal temperatures. Majority of known nitrilases reported are mesophilic in nature which are unstable at high temperature. A 730 bp gene encoding enantioselective nitrilase was identified in genomic DNA of G. stearothermophilus MAC 1 and G. icigianus MAC VI. The nitrilase gene of G. icigianus MAC VI was cloned into vector pGEX 4T2 and successfully expressed in E. coli BL21(DE3). The recombinant E. coli BL21(DE3) did not require an inducer for inducing nitrilase activity which is economical. The clone displayed nitrilase activity at temperature ranging from 60-100°C and was thermostable for 5 h at 100°C, characteristics that were found in the newlinex newlineoriginal strain. Further, in silico studies showed conservation of catalytic triad. Docking experiments resulted in gaining a better insight into the functioning of the nitrilase enzyme. Keywords: Nitrilase, thermophiles, 16S rRNA, mandelonitrile, mandelic acid, Response surface methodology, enantioselective