Screening Of Lipase Producing Strains And Cloning Of Lipase Genes

Lipases(carboxylesterases,EC 3.1.1.3) can catalyze both the hydrolysis and synthesis of long-chain acylglycerols.Lipases exist widely in animals,plants,and microorganisms.Lipases from microorganisms have been utilized widely in industrial fields including detergents,food,oil processing, cosmetic and medicine due to substrate specificity,enantiomeric selectivity,thermostability and alkaline stability.Seven lipase-yield strains were isolated from soil samples near glaciers in Xinjiang province.Two lipolytic strains XMZ-9 and XMZ-26 with higher lipase activity were selected and purified.Based on morphological characteristics and phylogenetic comparisons of their 16S/18S rDNA,these two strains were identified as Penicillium sp.and Acinetobacter sp.respectively.They were named as Penicillium sp.XMZ-9 and Acinetobacter sp.XMZ-26.Two lipase genes LIPPA and LIPPB from Penicillium sp.XMZ-9 were cloned and expressed in Escherichia coli respectively.The partial nucleotide sequences of two genes were obtained by touchdown PCR using the degenerate primers designed according to the conservative domains of lipase. The full-length sequences of two genes were obtained by future TAIL-PCR using specific primers designed according to the partial nucleotide sequences.The LIPPA(FJ973462) gene is 1014bp long, comprising one open reading frame encoding a polypeptide of 337 amino acids.It was identified as lipase due to its conserved GXSXG motif.Its deduced amino acid sequence showed 68%identity to the reported lipase genes using Blastp program in NCBI.The LIPPB(FJ973463) gene is 1232bp long, comprising two introns(61bps and 49bps) and a coding region sequence of 1122bps encoding a polypeptide of 373 amino acids.Its deduced amino acid sequence showed 74%identity to the reported lipase genes.Both LIPPA and LIPPB were cloned and expressed under control of the incurable lacZ promoter,and the recombinant proteins LIPPA and LIPPB were purified by Ni-Column.They both showed high activity at low temperature and were unstable at high temperature.These results indicated that they were cold-adapted lipases.One lipase-specific foldase gene LifA and three lipase genes LipA1,LipA2 and LipA3 were cloned from Acinetobacter sp.XMZ-26 and expressed in Escherichia coli respectively.The partial nucleotide sequences of these genes were obtained by touchdown PCR using the degenerate primers designed according to the conservative domains of lipase-specific foldase and lipase respectively.A genomic library of Acinetobacter sp.XMZ-26 was constructed,and a recombinant plasmid,pUC-lip1,was selected by colony PCR using specific primers designed according to the partial nucleotide sequences. The insert sequence of the pUC-lip1 was 2435bp long containing two complete open reading frames. The two ORFs were predicted to encode a complete lipase-specific foldase LifA and a lipase LipA1. The lifA(GQ227699) gene is 981bp long,comprising one open reading frame encoding a polypeptide of 326aa.Its deduced amino acid sequence showed 43%identity to the reported lipase-specific foldase genes using Blastp program in NCBI.The lipA1(GQ227701) gene is 981bp long,comprising one open reading frame encoding a polypeptide of 327aa.Its deduced amino acid sequence showed 68%identity to the reported lipase genes.Both lifA and lipA1 were expressed in Escherichia coli respectively,and the recombinant proteins LifA was purified by Ni-Column,but LipA1 formed inclusion body after induced by IPTG.Through the denaturation and renaturation experiments,it was found that LifA could help LipA1 to form the active conformation.Besides,the full-length sequences of other two lipase genes lipA2 and lipA3 were obtained by future TAIL-PCR using specific primers designed according to the partial nucleotide sequences.The lipA2(GQ227702) gene is 954bp long,comprising one open reading frame encoding a polypeptide of 317aa.Its deduced amino acid sequence showed 69%identity to the reported lipase genes.The lipA3(GQ227703) gene is 1008bp long,comprising one open reading frame encoding a polypeptide of 335aa.Its deduced amino acid sequence showed 37%identity to the reported lipase genes.Both lipA2 and lipA3 were expressed in Escherichia coli respectively,and the recombinant proteins LipA2 and LipA3 were purified by Ni-Colurnn and characterized.Both of them behaved highest activity at pH10.0 and were remarkably stable at pH 7 to 12.However,temperature optima and stabilities of lipA2 and lipA3 were extremely different.The lipA2 and lipA3 showed highest activity at 15℃and 65℃respectively.While LipA2 was unstable at high temperature,LipA3 had strong stability at higher temperature.These results indicated that LipA2 was typical cold-adapted lipases,whereas LipA2 was thermophilic lipases.Their reaction kinetics and the effects of metal ions and chemicals were also detected.In conclusion,they had the valuable application potentialities in detergent and food industry.