Molecular and enzymatic characterization of a subfamily I.4 lipase from an edible oil-degrader Bacillus sp. HH-01.
An edible-oil degrading bacterial strain HH-01 was isolated from oil plant gummy matter and was classified as a member of the genus Bacillus on the basis of the nucleotide sequence of the 16S rRNA gene. A putative lipase gene and its flanking regions were cloned from the strain based on its similarity to lipase genes from other Bacillus spp. The deduced product was composed of 214 amino acids and the putative mature protein, consisting of 182 amino acids, exhibited 82% amino acid sequence identity with the subfamily I.4 lipase LipA of Bacillus subtilis 168. The recombinant product was successfully overproduced as a soluble form in Escherichia coli and showed lipase activity. The gene was, therefore, designated as lipA of HH-01. HH-01 LipA was stable at pH 4-11 and up to 30°C, and its optimum pH and temperature were 8-9 and 30°C, respectively. The enzyme showed preferential hydrolysis of the 1(3)-position ester bond in trilinolein. The activity was, interestingly, enhanced by supplementing with 1 mM CoCl(2), in contrast to other Bacillus lipases. The lipA gene seemed to be constitutively transcribed during the exponential growth phase, regardless of the presence of edible oil.
Kamijo
Takashi
T
Graduate School of Science and Technology, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba-shi, Chiba, Japan.
Saito
Akihiro
A
Ema
Sadaharu
S
Yoh
Inchi
I
Hayashi
Hiroko
H
Nagata
Ryo
R
Nagata
Yoshiho
Y
Ando
Akikazu
A
D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D002122:Calcium Chloride / Q000378:metabolism; D003001:Cloning, Molecular; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005516:Food Microbiology; D015870:Gene Expression; D020869:Gene Expression Profiling; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009821:Oils / Q000378:metabolism*; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014280:Triglycerides / Q000378:metabolism
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D020536:Enzyme Activators; D009821:Oils; D012336:RNA, Ribosomal, 16S; D014280:Triglycerides; D008049:Lipase; D002122:Calcium Chloride; C009564:trilinolein