Diversity Of Cold-related Lipases In Glacier Soil And Cloning Of Some Lipase Genes

Lipase (EC 3.1.1.3) hydrolyzes triacylglycerols and catalyzes the reactions of ester synthesis and transesterification. It is a versatile biocatalyst with good stability in organic solvents, excellent substrate specificity and high stereo-selectivity. Due to the characteristics of gentle reaction conditions, fewer by-products and less contamination to environment, lipases have been extensively applied in industries. Cold-related lipases are a group of lipases with high catalytic efficacy at low temperatures and sensitivity to high temperature, and have potential applications in fields of detergents, food industry and biodegradation of oil-polluted soil.The purpose of this study was to investigate lipase diversity in the microorganisms from glacier soil based on culture-independent metagenomic DNA method. The validity of this method was confirmed by lipase gene cloning and heterogeneous expression based on the cell culture approach. The lipase fragments obtained in diversity study will be used for further lipase gene cloning from glacier soil.Based on the amino acid sequence alignment of low temperature lipases in Hormone-sensitive Lipase (HSL) family obtained by the Entrez search at NCBI, two conserved motifs [V/L]-[F/Y/D]-[F/I]-H-G-G-[G/A] and G-[D/V]-S-[A/V]-G-G-[N/C]- [L/M] were found around the oxyanion hole and the active site, respectively. A set of degenerate primers (CLF and CLR) was designed based on these two conserved motifs.Microbial metagenomic DNA was isolated using the direct lysis method from No. 1 glacier soil in China. Primers CLF and CLR were used to directly amplify fragments from the metagenomic DNA via PCR reaction. The PCR products were used to construct a cold-related lipase gene fragment clone library (GCLP). Among the 300 clones sequenced for analysis, 201 clones encoding partial lipases shared 33–82% identity to the known lipases in GenBank based on BLAST analysis and were subject to phylogenetic analysis. The results indicated that the lipases fragments in GCLP library were diverse and had high similarity with cold-adapted lipases in GenBank. A distinct cluster far from any other known lipases may represent an unidentified HSL subfamily.A total of twelve lipase-producing microorganisms were obtained from glacier soil by selective isolation. Among them, the genus Brachybacterium and Mrakia was first reported to produce lipase. Using the degenerate primers CLF and CLR, eight lipase gene fragments were isolated and shared 27–87% identity in the amino acid sequence. These lipase fragments were aligned with the sequences from GCLP library. Four lipase fragments were identical to the clones in GCLP library, wherease the others had no counterparts in the library. The full-length genes of Lip3A, LipXD, LipDB5 and LipDB8 corresponding to the clones in GCLP library and LipM from Mrakia sp. were obtained using TAIL-PCR technology. LipM, Lip3A,LipXD,LipDB5 and LipDB8 encoded a protein of 836, 320, 318, 310 and 313 amino acids, respectively. The highest amino acid sequence identity of these five deduced proteins to the lipases in GenBank was 43, 52, 54, 70 and 71%, respectively; suggesting the novelty of these lipase genes. Structure prediction of Lip3A,LipXD and LipDB8 were carried out using homologous modeling. Compared to the thermostable lipases, these lipases were more flexible and incompact.LipDB5 were expressed in Escherichia coli BL21(DE3). And Lip3A and LipXD were expressed in Pichia pastoris GS115. Lipase activity was detected after induction of the recombinant cells, indicating the successful expression of these lipase genes. The crude enzyme of recombinant LipDB5,Lip3A and LipXD were characterized: the optimum temperature on lipase activity were 30, 40 and 37°C, respectively; at 10°C, they retained 47, 17 and 33% relative activity with reference to the activity at optimum temperature; in thermostability test, LipDB5 retained approximately 34% of the activity after incubation at 60°C for 30 min, Lip3A and LipXD retained approximately 16 and 26% of the activity after incubation at 50°C for 30 min, respectively. The thermodynamic properties indicated that LipDB5, lip3A and LipXD had cold-related enzyme properties.Our study demonstrates that degerate PCR technology is a rapid and efficient screening method to evaluate the diversity of lipase genes of complex environmental samples. The lipase fragments obtained in diversity study established research foundation for high throughput screening of novel lipase genes from environmental metagenomic library.