Isolation and identification of marine Bacillus altitudinis KB1 from coastal Kerala: asparaginase producer.

Abstract

L-asparaginase is a target for many researchers as its properties against cancer, especially leukaemia, and protective agents reduce acrylamide in fried food. In this study, the water samples from Thumba Arattuvazhi Beach in Kerala were screened for l-asparaginase producing microorganisms. This was followed by colourimetric screening using modified M9 media with 0.009% Phenol red dye and using l-asparagine as a sole nitrogen source. Then, the Nessler assay was performed to quantify the enzyme. Molecular identification was made by 16SrRNA sequencing and aligned the sequence with GeneBank for phylogenetic tree construction using BLAST. Seawater was serially diluted for 10-1 to 10-6 using nutrient agar plates. A total of 19 bacterial colonies were isolated. The colonies were evaluated to produce l-asparaginase according to the pink zone around the colonies on the modified M9 medium using a red phenol indicator. The KB1 sample was selected for further studies according to plate colour assay. Nessler assay of L-asparaginase quantified as 2.537 IU/ml. Molecular characterisation showed the sequence association with Bacillus altitudinis the sequence submitted in Genebank as B. altitudinis KB1 strain. The l-asparaginase II gene (AnsB) was amplified based on the entire length of the hypothetical protein of annotated genome with accession number CP022319.2. The l-asparaginase activity in this study was 57% higher than the reference organism B. altitudinis BITHSP010. The l-asparaginase producing bacterium B. altitudinis KB1 from a marine source in Kerala can produce asparaginase, which can be utilised for biotechnology applications.