Characterization and a point mutational approach of a psychrophilic lipase from an arctic bacterium, Bacillus pumilus

Abstract

A bacterium with lipolytic activity was isolated from the Chukchi Sea within the Arctic Ocean. The lipase BpL5 from the isolate, Bacillus pumilus ArcL5, belongs to subfamily 4 of lipase family I. The optimum pH and temperature of the recombinant enzyme BpL5, as expressed in Escherichia coli, were 9.0 and 20 °C, respectively. The enzyme retained 85 % of its activity at 5 °C. There was a significant difference between temperatures for maximal activity (20 °C) and for protein denaturation (approx. 45 °C). The enzyme preferred middle-chain (C8) p-nitrophenyl substrates. Two mutants, S139A and S139Y, were rationally designed based on the 3D-structure model, and their activities were compared with that of the wild type. The both mutants showed significantly improved activity against tricaprylin.

Cold adaptation of tRNA nucleotidyltransferases: A tradeoff in activity, stability and fidelity

“…CD spectroscopy at 222 nm is frequently used to follow the temperature-dependent denaturation of proteins, monitoring the unfolding of alpha-helical elements. [48][49][50][51] As tertiary interactions in the 3D structure are usually destroyed before the secondary structures, [52][53][54][55] the unfolding of alpha-helical elements definitely indicates denaturation and functional loss of a protein. Hence, these measurements give a conservative estimation of a proteins melting temperature, allowing to discriminate psychrophilic CCA-adding enzymes from their thermostable counterparts based on the unfolding of secondary structures.…”

Recent advances on sources and industrial applications of lipases

“…A recent study by Gupta et al reports a detailed comparative analysis of 3D structures of different lipases with respect to their structural determinants. Some of the latest studies on 3D structures of recombinant lipases include lipase isolated from arctic sea microorganism with improved activity, thermally stable mutated lipase from C. antarctica, thermo, alkali tolerant cloned lipase from Bacillus (B.) licheniformis, etc.…”

“…The study reported by Clementz et al, includes immobilization of recombinant R. oryzae lipase on bovine bone support which has shown high porosity, large surface area, and suitable porous structure, thereby resulting in improved biocatalytic activity of lipase. Further, two novel mutants of a psychrophilic lipase isolated from an arctic bacterium, B. pumilus were rationally designed based on the 3D‐structure model and resulted in significant improvement in their activities as compared with that of the wild type . It was noted from the study of Raftari et al, that the production was increased to 34‐fold higher than the native enzyme through metabolic engineering of lipase from Burkholderia cepacia which was cloned and highly expressed in Lactococcus lactis .…”

“…Further, two novel mutants of a psychrophilic lipase isolated from an arctic bacterium, B. pumilus were rationally designed based on the 3D-structure model and resulted in significant improvement in their activities as compared with that of the wild type. 54 It was noted from the study of Raftari et al, 289 that the production was increased to 34-fold higher than the native enzyme through metabolic engineering of lipase from Burkholderia cepacia which was cloned and highly expressed in Lactococcus lactis.…”

Integrative computational approach for genome-based study of microbial lipid-degrading enzymes

“…MIS38 showed that this enzyme was predicted to be most stable at low temperature, particularly in the catalytic domain (Mohamad Ali et al 2013). In another study, mutant enzymes were rationally designed based on the 3D model of wildtype enzyme, a psychrophilic lipase, from an arctic bacterium, producing an engineered lipase with improved activity of some substrates (Wi et al 2014).…”