Gene cloning, purification, and characterization of a cold-adapted lipase produced by Acinetobacter baumannii BD5.
Acinetobacter baumannii BD5 was isolated from waters of Baek-du mountain, and the lipase gene was cloned using a PCR technique. The deduced amino acid sequence of the lipase and lipase chaperone were found to encode proteins of 325 aa and 344 aa with a molecular mass of 35 kDa and 37 kDa, respectively. The lipase gene was cloned and expressed in Escherichia coli BL21 (trxB) as an inclusion body, which was subsequently solubilized by urea, and then purified using Ni-affinity chromatography. After being purified, the lipase was refolded by incubation at 4oC in the presence of a 1:10 molar ratio of lipase:chaperone. The maximal activity of the refolded lipase was observed at a temperature of 35 degrees and pH 8.3 when p-NP caprate (C10) was used as a substrate; however, 28% of the activity observed at 35 degrees was still remaining at 0 degrees . The stability of the purified enzyme at low temperatures indicates that it is a cold-adapted enzyme. The refolded lipase was activated by Ca2+, Mg2+, and Mn2+, whereas Zn2+ and Cu2+ inhibited it. Additionally, 0.1% Tween 20 increased the lipase activity by 33%, but SDS and Triton X-100 inhibited the lipase activity by 40% and 70%, respectively.
Park
In-Hye
IH
Department of Biotechnology, Faculty of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea.
Kim
Sun-Hee
SH
Lee
Yong-Seok
YS
Lee
Sang-Cheol
SC
Zhou
Yi
Y
Kim
Cheol-Min
CM
Ahn
Soon-Cheol
SC
Choi
Yong-Lark
YL
D040981:Acinetobacter baumannii / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003080:Cold Temperature; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D008049:Lipase