Proteolytic Activity of Bacillus amyloliquefaciens UEF01 Endophytic to Carnivorous Plant Utricularia exoleta R. Br.

Abstract

Aims: The endosphere of the carnivorous plant Utricularia exoleta R. Br. represents a unique niche for the study of microbial diversity. This study was aimed at to isolate and enumerate the protease producing potential of bacteria endophytic to U. exoleta R. Br. Study Design: Extracellular proteolytic activity of the cell-free culture filtrate of the bacterial endophyte was determined following standard colorimetic assay using casein as the substrate. Place and Duration of Study: The study was undertaken in the Microbiology Laboratory, Department of Botany, University of Calcutta during September 2015 and March 2016. Methodology: A total of 36 phenotypically distinguishable bacteria endophytic to leaf, stem, bladder and fruit of U. exoleta were isolated and evaluated for protease production. The best protease producing isolate, UEF01 was selected, characterized and the conditions for protease activity were optimized. Results: The selected isolate was characterized following morphological, physio-biochemical and 16S rRNA gene sequence analysis and identified as Bacillus amyloliquefaciens UEF01 (GenBank Accession No. KX816572). The crude protease of the cell-free culture filtrate of UEF01 was maximum at 35° C, pH 9 with 1.5% (w/v) casein. The enzyme appeared to be thermolabile with loss of >80% activity at 70°C (15 min incubation). Kinetic studies indicated the Km and Vmax values as 9.21 mg/ml and 71.43 U/mg of protein respectively. The enzyme was sensitive to Na and Mn ions as well as some selective protease inhibitors such as phenyl methyl sulfonyl fluoride, β-mercaptoethanol, and EDTA. Conclusion: This proteolytic study will help in understanding the role of endophytes in digestion of prey within the bladders of the carnivorous plants.