Characterization of two novel lipase genes isolated directly from environmental sample.
Two novel lipase genes (lipJ02, lipJ03) were isolated directly from environmental DNA via genome-walking method. Lipase gene lipJ02 contained an open reading frame (ORF) of 1,425 bp and encoded a 474-amino acids lipase protein, while lipase gene lipJ03 contained an ORF of 1,413 bp and encoded a 470-amino acids lipase protein. The lipase genes were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant P. pastoris were screened via a high-throughput method. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized. The optimum temperature for the purified lipase LipJ02 and LipJ03 was 30 and 35 degrees C, respectively, at pH 8.0. They exhibited similar thermostability, but LipJ02 exhibited better pH stability than LipJ03.
State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
Zhengbing
Z
Jiang
Hongping
H
Wang
Yushu
Y
Ma
Dongzhi
D
Wei
D016386:Chromosome Walking / Q000379:methods; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D004783:Environmental Microbiology; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D008049:Lipase