Cloning, expression, purification and characterization of lipase from Bacillus licheniformis, isolated from hot spring of Himachal Pradesh, India.

Gagandeep Kaur,Pushpender K Sharma,Rohit Sharma,Vinay Sharma,Swati Verma,Amninder Singh

doi:10.1007/s13205-016-0369-y

Abstract

In the present investigation, a gene encoding extracellular lipase was cloned from a Bacillus licheniformis. The recombinant protein containing His-tag was expressed as inclusion bodies in Esherichia coli BL21DE3 cells, using pET-23a as expression vector. Expressed protein purified from the inclusion bodies demonstrated ~22 kDa protein band on 12 % SDS-PAGE. It exhibited specific activity of 0.49 U mg−1 and % yield of 8.58. Interestingly, the lipase displayed activity at wide range of pH and temperature, i.e., 9.0–14.0 pH and 30–80 °C, respectively. It further demonstrated ~100 % enzyme activity in presence of various organic solvents. Enzyme activity was strongly inhibited in the presence of β-ME. Additionally, the serine and histidine modifiers also inhibited the enzyme activities strongly at all concentrations that suggest their role in the catalytic center. Enzyme could retain its activity in presence of various detergents (Triton X-100, Tween 20, Tween 40, SDS). Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel β-sheet in the core of the 3D structure. The function of these conserve sequences have not fully understood.

Highlights

Lipases are triacylglycerol hydrolases (E.C. 3.1.1.3) that catalyzes variety of conversion reactions, ranging from interesterification, esterification, alcoholysis, acidolysis and aminolysis (Gupta et al 2004; Hayes 2004; Kim et al 2002; Nagao et al 2001; Saxena et al 2003)
Sequence and structural analysis employing in silico tools revealed that the lipase contained two highly conserved sequences consisting of ITITGCGNDL and NLYNP, arranged as parallel b-sheet in the core of the Gagandeep Kaur and Amninder Singh have contributed to this work
Screening of hot spring soil for the lipase-producing strain resulted in isolation of bacterial strain that showed a clear zone of hydrolysis around the colonies (Fig. 1)

Summary

Introduction

Lipases are triacylglycerol hydrolases (E.C. 3.1.1.3) that catalyzes variety of conversion reactions, ranging from interesterification, esterification, alcoholysis, acidolysis and aminolysis (Gupta et al 2004; Hayes 2004; Kim et al 2002; Nagao et al 2001; Saxena et al 2003) They are frequently used in various industries including detergents, leather, dairy, baking and pharmaceutical (Jaeger and Eggert 2002). A lipase was characterized from Bacillus licheniformis strain that was isolated from the mangrove sediments This enzyme showed stability up to 70 °C and pH 12.0 (Annamalai et al 2011).

Methods

Results

Conclusion