Screening,Cloning,Expressing,and Characterization Of Lipase From Burkholderia Gladioli

Lipase has tremendous prospects for its industrial application. In order to obtain the application value of the Lipase, the current studies mainly focus on its development and reform. The purpose of this study is to screen the strain of producing Lipase, to build efficient recombinants, and to probe the application of lipase gene.In this study, the samples were obtained from oil soaked soil, and a strain that produces Lipase efficiently was screened out,designated strain BG-01. Identification of strain BG-01 was carried out with molecular biology method. Sequence of its 16S rDNA blasting results show that strain BG-Olwas most related to Burkholderia gladioli By designing primers and cloning, the lipase gene of BG-01 was obtained, named lipL.The lipL open reading frame was composed of 1278 nucleotides, which encoding a polypeptide of 425 amino acids. Sequence blasting results show 98.6% identity with lipase gene of Burkholderia gladioli BSR3.Then lipL sequence information was submited to GenBank database and a GenBank accession number of KR023991 was provided.The result of bioinformatics analysis shows that LipaseL belongs to the Lipase_GDSL₂ family.4 recombinants were constructed with plasmid pET-22b and pET-32a and expressed in E.coli BL21（DE3）, and the target proteins formed in inclusion bodies after IPTG induction. The inclusion bodies were successfully refolded by large numbers of dilution methods, and then were purified to a single band in SDS-PAGE analysis by immobilized metal affinity chromatography（IMAC）, resulting a specific activity of 118.2 U/mg.The enzymatic properties of refolded JDLipaseL was analyzed, The optimum pH of JDLipaseL is 8.5, it still shows a high enzyme activity when pH is between 7 and 9,and it is capable of alkali-stable in this pH range. The optimum temperature of JDLipaseL is 55℃, and kept fairly stable between 35℃ and 75℃,and remained more than 50 percent of the maximium activity. So JDLipaseL belongs to the mesophilic enzyme. Using the optimum substrate pNPP as reaction substrate, some parameters were obtained according to Lineweaver-Burk plot, the Km of JDLipaseL is 6.2×10-4 M, Vmax is 161.4 μmol/（L·min）.5 mM potassium, lithium, magnesium, manganese ions play weak roles in promoting enzyme activity, and the calcium ion has a most significant promotion to enzyme activity and the relative enzyme activity ratio reaches 1.3 times. Copper ion, cobalt ion and ferric ion are factors with obvious inhibitory effect on the enzyme activity. To investigate tolerance to a variety of short-chain alcohols, JDLipaseL was disposed with 10% of the organic solvent, the result shows propanol plays a slightly possitive role on the enzyme activity. Methanol, ethanol, isopropanol, butanol, glycerol, acetonitrile have little effect on the enzyme activity respectively, and residual activity remained about 90%. While inhibitory effect of acetone and chloroform were obvious.