[Cloning and overexpression of lipase gene from Geotrichum candidum Y162].
By means of bioinformatics, we aligned nucleotide sequence of reported lipase gene from Geotrichum. Primers were designed based on the conservative nucleotide sequence, and the lipase gene of G. candidum Y162 was cloned for the first time in China. Nucleotide sequencing revealed that the open reading frame has 1692 nucleotides without any introns, encoding 563 amino acid residues including a signal sequence of 19 amino acid residues, which is 86% identical to lipase I of G. fermentans. Subsequently, we cloned the lipase gene into expression vector pPIC9K, and then transformed into Pichia pastoris GS115. Cultures of recombined P. pastoris accumulated active enzyme in the supernatant to levels of 55 U/mL after induction for 96 hours in shake flasks. The purified lipase exhibited maximum activity at 50 degrees C and pH 8.0, and was stable between pH 6.0 and 10.0 and below 60 degrees C. Lipase activity was compatible with the presence of organic solvents such as methanol, n-heptane, hexane, cyclohexane, glycerol, benzene and diethyl ether. Lipase showed hydrolysis preference for triacylglycerol substrates containing cis-9 unsaturated fatty acid. The results suggest that the lipase could be a candidate for industrial applications.
Yan
Jinyong
J
College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China. yjiny@126.com
Yang
Jiangke
J
Xu
Li
L
Yan
Yunjun
Y
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005848:Geotrichum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D005656:Fungal Proteins; D008049:Lipase