title: Molecular cloning and heterologous expression of a true lipase in Pichia pastoris isolated via a metagenomic approach.
abstract: Lipases are important enzymes for various biotechnological applications. By using functional expression screening, lipZ03, a novel lipase gene, was isolated from a soil-derived metagenomic library. The gene was supposed to encode a protein of 617 amino acids with a C-terminal targeting signal region and four potential N-linked glycosylation sites. The protein sequence shared a conserved GXSXG motif (X represents any amino acid residue) with other microbial lipases. Gene lipZ03 was expressed in Pichia pastoris and the molecular weight was estimated to be approximately 65 kDa by electrophoresis. The optimum reaction temperature and pH value for LipZ03 was 50°C and 9.0, respectively. The enzyme was highly stable in the temperature range of 40-60°C and under alkaline conditions (pH 8-10). Lipolytic activity was significantly enhanced by Ca(2+) and Mg(2+) ions, but dramatically inhibited by Cu(2+), Ni(2+) and Hg(2+) ions and EDTA. The purified enzyme preferentially hydrolyzed relatively long-chain triacylglycerols and was a true lipase rather than an esterase. Using a multi-stepwise methanol supply, the purified LipZ03 achieved a conversion yield of biodiesel production up to 74% after 36 h. Some interesting characteristics described here showed that the recombinant lipase may have potential to be a useful enzyme in industrial applications.
authors.affiliation: MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, PR China. huahero@ipbcams.ac.cn
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zheng
authors.forename: Liguo
authors.initials: L
authors.lastname: Liu
authors.forename: Cuina
authors.initials: C
authors.lastname: Liu
authors.forename: Qi
authors.initials: Q
authors.lastname: Jin
mesh_terms: D020816:Amino Acid Motifs; D001665:Binding Sites; D056804:Biofuels; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D015698:Genomic Library; D006031:Glycosylation; D008049:Lipase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D008274:Magnesium / Q000378:metabolism; D056186:Metagenomics / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012988:Soil Microbiology; D013379:Substrate Specificity; D013997:Time Factors; D014280:Triglycerides / Q000378:metabolism
chemical_list: D056804:Biofuels; D004791:Enzyme Inhibitors; D014280:Triglycerides; D008049:Lipase; D008274:Magnesium; D002118:Calcium