Optimized chromogenic dyes-based identification and quantitative evaluation of bacterial l-asparaginase with low/no glutaminase activity bioprospected from pristine niches in Indian trans-Himalaya

Abstract

Here, we report on the isolation of bacterial isolates from Himalayan niches, which produced extracellular l-asparaginase with low/no glutaminase activity. From the 235 isolates, 85 asparaginase positive bacterial isolates were identified by qualitative screening using optimized chromogenic dyes assay. Optimized concentration of different dyes revealed maximum color visualization in phenol red (0.003%). The diversity analysis of asparaginase positive isolates revealed that Proteobacteria (83%) are the most dominant, followed by Actinobacteria (12%), Firmicutes (3%), and Bacteriodetes (2%). Eleven isolates, which represented seven Pseudomonas species, one species each of the genus Arthrobacter, Janthinobacterium, Lelliottia, and Rahnella, were selected for further studies based on highest zone ratio and novel aspects for l-asparaginase production. Of these, five isolates, namely, Pseudomonas sp. PCH133, Pseudomonas sp. PCH146, Pseudomonas sp. PCH182, Rahnella sp. PCH162, and Arthrobacter sp. PCH138, produced l-asparaginase without glutaminase activity after 55 h of growth with the former isolate showing the highest l-asparaginase activity (1.67 U/ml). Interestingly, this is the first report of l-asparaginase production by members of the genera Janthinobacterium, Rahnella, and Lelliottia.

Keywords: Anti-leukemic; Extracellular; Glutaminase free; Microbial diversity; l-asparaginase.

Fig. 1

Diversity analysis of total bacterial isolates (235) and their percent distribution at phylum level (indicated in parentheses)

Fig. 2

Phylogenetic tree based on 16S rDNA sequences of asparaginase positive bacterial isolates (85) using the neighbor-joining method and their percent distribution at phylum level (indicated in parentheses). Bootstrap values, indicated at the nodes, were obtained from 1000 bootstrap replicates. Bootstrap values > 94.99% were only included in the phylogenetic tree. The scale bar corresponds to 0.05 nucleotide changes per site