In vitro and in silico analysis unravelled clinically desirable attributes of Bacillus altitudinis L-asparaginase.

Abstract

To identify a marine L-asparaginase with clinically desirable attributes and characterize the shortlisted candidate through in silico tools. Marine bacterial strains (number=105) isolated from marine crabs were evaluated through a stepwise strategy incorporating the crucial attributes for therapeutic safety. The results demonstrated the potential of eight bacterial species for extracellular L-asparaginase production. However, only one isolate (Bacillus altitudinis CMFRI/Bal-2) showed clinically desirable attributes, viz. extracellular production, type-II nature, lack of concurrent L-glutaminase and urease activities, and presence of ansZ (functional gene for clinical type). The enzyme production was 22.55±0.5µM/mg protein/min within 24 h without optimization. The enzyme also showed good activity and stability in pH 7-8 and temperature 37°C, predicting the functioning inside the human body. The Michealis-Menten constant (Km) was 14.75µM. Detailed in silico analysis based on functional gene authenticating the results of in vitro characterization and predicted the nonallergenic characteristic of the candidate. Docking results proved the higher affinity of the shortlisted candidate to L-asparagine than L-glutamine and urea. Comprehensively, the study highlighted B. altitudinis type II asparaginase as a competent candidate for further research on clinically safe asparaginases.