Open AccessCloning, purification, and characterization of a cold‐adapted esterase produced by P sychrobacter cryohalolentis K 5 T from S iberian cryopeg
Author(s) -
NovototskayaVlasova Ksenia,
Petrovskaya Lada,
Yakimov Sergey,
Gilichinsky David
Publication year - 2012
Publication title -
fems microbiology ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.377
H-Index - 155
eISSN - 1574-6941
pISSN - 0168-6496
DOI - 10.1111/j.1574-6941.2012.01385.x
Subject(s) - biology , thermostability , biochemistry , escherichia coli , enzyme , esterase , recombinant dna , thermophile , histidine , bacteria , affinity chromatography , amino acid , microbiology and biotechnology , gene , genetics
A psychrotrophic gram‐negative bacterium P sychrobacter cryohalolentis K 5 T was previously isolated from a cryopeg within S iberian permafrost and its genome has been completely sequenced. To clone and characterize potential cold‐active lipases/esterases produced by P . cryohalolentis K 5 T , we have identified their potential genes by alignment with amino acid sequences of lipases/esterases from related bacteria. One of the targets, EstPc , was cloned and overexpressed in E scherichia coli B L21 ( DE 3) cells. The recombinant protein was produced with a 6x histidine tag at its C ‐terminus and purified by nickel affinity chromatography. Purified recombinant protein displayed maximum esterolytic activity with p ‐nitrophenyl butyrate ( C 4) as a substrate at 35 °C and pH 8.5. Activity assay conducted at different temperatures revealed that EstPc is a cold‐adapted esterase which displayed more than 90% of its maximum activity at 0–5 °C. In contrast to many known cold‐active enzymes, it possesses relatively high thermostability, preserving more than 60% of activity after incubation for 1 h at 80 °C. It was activated by Ca 2+ , Mn 2+ , and EDTA whereas Zn +2 , Cu +2 , Co +2 , Ni +2 , and Mg +2 inhibited it. Various organic solvents (ethanol, methanol and others) inhibited the enzyme. Most non‐ionic detergents, such as Triton X ‐100 and Tween 20 increased the lipase activity while SDS completely inhibited it.