Characterization of two soil metagenome-derived lipases with high specificity for p-nitrophenyl palmitate.
Two novel genes (pwtsB and pwtsC) encoding lipases were isolated by screening the soil metagenomic library. Sequence analysis revealed that pwtsB encodes a protein of 301 amino acids with a predicted molecular weight of 33 kDa, and pwtsC encodes a protein of 323 amino acids with a predicted molecular weight of 35 kDa. Furthermore, both genes were cloned and expressed in Escherichia coli BL21 (DE3) using pET expression system. The expressed recombinant enzymes were purified by Ni-nitrilotriacetic acid affinity chromatography and characterized by spectrophotometric with different p-nitrophenyl esters. The results showed that PWTSB displayed a high degree of activity and stability at 20 degrees C with an optimal pH of around 8.0, and PWTSC at 40 degrees C with an optimal pH of around 7.0. P-nitrophenyl palmitate (p-NPP) was identified as the best substrate of PWTSB and PWTSC. The specific activities of PWTSB and PWTSC were 150 and 166 U/mg, respectively toward p-NPP at 30 degrees C, about 20-fold higher than that toward p-nitrophenyl butyrate (C4) and caprylate (C8). In conclusion, our results suggest that PWTSB is a cold adapt lipase and PWTSC is a thermostable lipase to long-chain p-nitrophenyl esters.
Wei
Ping
P
Department of Basic Medicine, Taishan Medical University, Taian, Shandong, China. wp360@yahoo.cn
Bai
Liping
L
Song
Wengang
W
Hao
Gangping
G
D000595:Amino Acid Sequence; D004269:DNA, Bacterial / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D016680:Genome, Bacterial; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010168:Palmitates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012988:Soil Microbiology; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D010168:Palmitates; D011994:Recombinant Proteins; C050015:4-nitrophenyl palmitate; D008049:Lipase