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Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression and biochemical characterization

Paper ID Volume ID Publish Year Pages File Format Full-Text
34600 45035 2013 7 PDF Available
Title
Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression and biochemical characterization
Abstract

•A lipase gene from Bacillus subtilis DR8806 was cloned and functionally expressed.•The 26 kD recombinant enzyme was optimally active at 70 °C and pH 8.0.•The enzyme demonstrated high compatibility toward commercial detergents.•Effect of organic solvents and ionic liquids on enzyme activity was evaluated.•The lipase can potentially be applied in biotransformation and detergent industry.

A thermo-alkaliphilic lipase from Bacillus subtilis DR8806 was functionally expressed as an N-terminal 6xHis-tagged recombinant enzyme in Escherichia coli BL21 using pET-28a(+) expression vector. Sequence analysis revealed an open reading frame of 639 bp encoding a 212-amino acid protein containing the well-conserved Ala-His-Ser-Met-Gly motif. One-step purification of the His-tagged recombinant lipase was achieved using Ni-NTA affinity chromatography with a specific activity of 1364 U/mg. The purified enzyme with an apparent molecular mass of 26.8 kDa demonstrated the maximum activity at 70 °C and pH 8.0 for hydrolysis of p-nitrophenylbutyrate as substrate. The enzyme activity was strongly inhibited by divalent ions of heavy metals such as Hg2+ and Cu2+, while retained over 90% of the original activity in the presence of several reagents including DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)), SDS (sodium dodecyl sulfate), urea, DMF (dimethylformamide), DTT (dithiothreitol), glycerol and Triton X-100. While being considerably stable in organic solvents, imidazolium-based ionic liquids (ILs) had stimulatory effects on the activity of purified lipase. Remarkable stabilization of enzyme at alkaline pH and in ionic liquids as well as its thermostability/thermoactivity are among the most fundamental characteristics which offer great potential for various biotechnological applications including detergent formulation, bioremediation processes and biotransformation in non-aqueous media.

Keywords
Cloning; Lipase; Bacillus subtilis DR8806; Biochemical characterization; Ionic liquids
First Page Preview
Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression and biochemical characterization
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 48, Issue 11, November 2013, Pages 1679–1685
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering