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Lipase (EC 3.1.1.3) also known as glycerol hydrolase (acylglycerol hydrolases), its natural substrate for the natural oils in plants, animals and micro-organisms are prevalent in many, is one of the first to be studied enzymes. First discovered early twentieth century research abroad microbial lipases, microbial lipases are widely used, because it has the substrate specificity, chiral selectivity, temperature stability, and stability under alkaline conditions, is widely used in washing agents, food, oil processing, cosmetics and pharmaceuticals industrial production. In this paper, using fat assimilation tablet with tributyrin - Victoria blue tablet combining functional screening method, soil samples from the glacier isolated four producing lipase lipase producing fungi and three bacteria. Use 16S/18S sequence of molecular species identification method, the activity of a fungus with a higher initial identification of bacteria, belonging to the genus Penicillium and Acinetobacter named Penicillium sp.XMZ-9 and Acinetobacter sp.XMZ-26. Through the analysis of conserved sequence lipase designed for low-temperature lipase degenerate primers using PCR landed successfully been part of lipase genes related to DNA sequences. Based on the obtained partial gene sequence, using the TAIL-PCR method from Penicillium Penicillium sp.XMZ-9 obtained in two lipase genes, denoted LIPPA and LIPPB (GenBank database accession numbers are as FJ973462 and FJ973463). Lipase LIPPA structural gene LIPPA length 1014bp, no introns, encoding 337 amino acids and a stop codon. In the NCBI website homology with Blastp program compared the results showed that the highest similarity of 68%. The lipase LIPPB structural gene LIPPB length 1232bp, cDNA length 1122bp, contains two introns were located 538 ~ 598 for the 61bp intron sequences located 917 ~ 966 for the 49bp intron sequence, in its two ends of the intron typical sequence with 5'-GT-3 'and 5'-AG-3'. The gene encodes 373 amino acids and a stop codon, by homologous and analogous enzymes is compared to the amino acid sequence shows the highest similarity was 74%. These two genes in E. coli were heterologous expression, LIPPA majority expressed as inclusion bodies, refolded after denaturation with lipase activity was measured and expressed as low adaptability; LIPPB majority expressed as soluble proteins, Ni - affinity chromatography column purification, the determination of lipase which has a low activity. By conserved degenerate primers PCR amplified partial gene sequences obtained further use TAIL-PCR Acinetobacter Acinetobacter sp.XMZ-26 two full-length lipase gene sequence. Reuse genomic library prepared from Acinetobactersp.XMZ-26, he also won a lipase lipase gene and a gene-specific folding. That they will be denoted as lifA, lipA1, lipA2 with lipA3 (GenBank database accession number were GQ227699, GQ227701, GQ227702 and GQ227703); wherein, lifA downstream relationships with lipA1 above constitute operon structure. Folding lipase enzyme structural gene LifA LifA length 981bp, encoding 326 amino acids and a stop codon, by homologous and analogous enzymes is compared to the amino acid sequence shows the highest similarity was 43%. LipA1 lipase structural gene lipA1 length 984bp, encoding 327 amino acids and a stop codon, by homologous and analogous enzymes is compared to the display of the highest amino acid sequence similarity of 68%. LipA2 lipase structural gene lipA2 length 954bp, encoding 318 amino acids and a stop codon, by homologous and analogous enzymes is compared to the display of the highest amino acid sequence similarity of 69%. LipA3 lipase structural gene lipA3 length 1008bp, encoding 335 amino acids and a stop codon, by homologous and analogous enzymes is compared to the display of the highest amino acid sequence similarity of 37%. These four genes in E. coli of heterologous expression, expressed as inclusion bodies in addition to other lipA1, lifA, lipA2 with lipA3 were expressed as soluble protein form. Right LifA, LipA2 conducted with LipA3 Ni-affinity chromatography purification; inclusion on LipA1 preliminary purification, initially explored the folding enzymes LifA right refolding denatured LipA1 again, and found LifA can promote LipA1 folded enzymatic activity with the correct conformation, and improve its efficiency of in vitro refolding. On purified LipA2 with LipA3 enzymatic properties were measured. LipA2 The optimum reaction temperature of 15 ℃, the optimum reaction pH of 5.0, has good thermal stability, good pH stability, but, for most metal ions and chemical reagents are not sensitive, but the appropriate concentration of Ca 2 < / sup> can make the activity doubled, for most detergent is not sensitive to the more typical low lipase in the detergent industry has great potential. In contrast, LipA3 nature have large difference, and the optimum reaction temperature of 65 ℃, the optimum reaction pH of 5.0, has good thermal stability and pH stability, the majority of metal ions and chemical reagents are not sensitive and not sensitive to most detergent, typical of high lipase is more suitable for industrial processing.
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