Journal of Environmental Biology

Production of an extracellular neutral protease by Bacillus aerius UB02 endophytic to carnivorous plant Utricularia stellaris

M. Chaudhuri¹, A.K. Paul¹ and A. Pal²* 

¹Microbiology Laboratory, Department of Botany, University of Calcutta, Kolkata-700 019, India

²Post Graduate Department of Botany, Serampore College, Hooghly-712 201, India

*Corresponding Author Email : arundhatipalcu@gmail.com

Received: 06.08.2020                                                                  Revised: 04.02.2021                                                     Accepted: 03.05.2021

Abstract

Aim: Endophytic bacteria indigenous to carnivorous plants have been explored for production of novel bioactive metabolites including extracellular enzymes. Bacillus aerius UB02, an extracellular neutral protease producing isolate endophytic to bladder of Utricularia stellaris L. f. was used in this study.

Methodology: The bacterial isolate UB02 was identified following morpholological, physiological, biochemical and 16S rRNA gene sequence analyses. The media as well as the cultural conditions for production of protease were optimized. The extracellular protease was isolated and purified from the cell-free culture filtrate by ammonium sulphate precipitation, dialysis and DEAE Sephadex ion exchange column chromatography and the optimum conditions for its activity were determined.

Results: The isolate Bacillus aerius UB02 (GenBank accession no. MK 696417, MCC accession no. 4132), produced significant amount of extracellular protease (38.29 U mg^-1 protein) during growth in casein supplemented synthetic medium. However, peptone yeast extract glucose medium appeared to be the best for the synthesis of enzyme. Production of enzyme was enhanced by the inoculum density of 1.5% (v/v), culture volume: flask volume (CVF) ratio of 1:10, substrate concentration of 2.5% (w/v) with temperature and pH adjusted at 37°C and 7.4, respectively. Glucose (2.2%, w/v) and ammonium chloride (1.2 g/L) as carbon and nitrogen sources also favoured the enzyme production. The neutral protease with a molecular weight of approximately 35 kDa showed maximum activity at 40°C, pH 7.8 with 2% (w/v) casein. The enzyme exhibited Km and V_max values of 6.81 mg ml^-1 and 62.5 U mg^-1 of protein, respectively, and was moderately thermostable. The protease activity was inhibited by Pb and Cd as well as 1,10-phenanthroline and β-mercaptoethanol.      

Interpretation: These findings will help not only in understanding the role of endophytic bacteria and the enzymes produced by them in the digestion of prey by carnivorous plant but could also be explored for application in the field of biotechnology.

Key words: Bacillus aerius, Bacterial endophytes, Carnivorous plants, Enzyme kinetics, Extracellular protease, Utricularia stellaris 

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