Screening And Heterologous Expression Of Stenotrophomonas Maltophilia Lipase, And Its Application In Enzymatic Preparation Of L-menthol

Lipases (triacylglycerol lipases, EC 3.1.1.3) are ubiquitous enzymes which act on ester bonds, either synthesizing or hydrolyzing, in organic media or aqueous. Although lipases could be obtained from animals and plants, microbial lipases own useful characteristics such as low production cost and high yield, amenability to genetic manipulation, broad substrate specificity diversity in catalytic activities, and stability in organic solvents. The wide berth for employment in a variety of industries, such as food, pharmaceuticals, cosmetics and chiral chemicals, endowed by the features listed above. However, if lipases are naturally stable and exhibit high activity in organic solvents, they can be conveniently employed in industrial manufactory.A novel cold-active and organic solvent-tolerant lipase from Stenotrophomonas maltophilia CGMCC 4254 was isolated, purified and characterized. The high stereoselective lipase was sought to preferentially transesterification l-menthol in hydrophobic solvent, extremely simplifying separation of l-menthol from the diasteromeric mixture. These results imply that the lipase is an excellent biocatalyst for the enzymatic preparation of l-menthol in industrial scale.The first part of this dissertation is the screening of novel OST lipase in resolution of menthol diasteromeric mixture. With the aim of screening for potential OST lipases, we used an established fluorescence-based microplate assay to screen of OST lipase. Using this approach, we have identified 21 bacterial lipases that show stable in the presence of toluene. The 21 lipases from these microorganisms were secondary screened by detecting their stereoselectivity. Using this method, the lipase from S. maltophilia shwoing excellent optical purity of product and higher conversions of substrate were selected.Optimization of media component and fermentation conditions allowed a lipolytic activity in liquid of 757.1U/L,10-fold high than original medium. The lipase was purified to give a specific activity of 38.9 U/mg by ammonium sulfate precipitation and hydrophobic interaction chromatography. SML displayed remarkable stability in the presence of high content of hydrophobic solvents and pure hydrophilic solvents for 7 days. SML retained 57% maximum activity at 5℃ and over an alkaline pH range. The lipase exhibited a conversion of 95.9% l-menthol (100mM) toward 4-pairs-menthol mixture, and enantiomeric excess (eep) of higher than 98% and diastereomeric excess (de) of higher than 85% was achieved.The third part of the dissertation is cloning, amino acid sequence analysis and heterologous expression of LipSM54. A genomic DNA library derived from S. maltophilia contained approximately 64,500 genomic clones. Sequence analysis revealed that one 1518-bp ORF codes a hydrolase, named as LipSM54. On the basis of phylogenetic tree, conserved motif alignment and catalytic mechanism, we proposed that LipSM54 and its homologs belong to a novel bacterial lipase family. The expression of recombinant lipase in E. coli BL21(DE3) reached 4235U/L, 3.3-times higher than lipolytic activity in S. maltophilia. The enzymatic transesterification of menthol mixture could be effectively achived in high enantioselectivity (E>200) by LipSM54.The fourth part of the dissertation is the design of protein structure in increasing the thermostability of LipSM54. Confirmation of catalytic triad (115S-225D-253H) and oxyanion hole residues (44W-116Y) was conducted by analysis of 3D homologs model and site-directed mutagenesis, as well as the catalytic mechanism involving the hydrogen atom from Trp44. The integrated computational forecast -experimental has yielded a LipSM54 variant (F73R-G165D) with about 1000-fold increase in half-life at 50℃ and remarkable stability in 50%(v/v) solvent. The novel hydrogen bond and salt bridge generated newly contributed to the increase of structural rigidity, which was confirmed by the molecular dynamics and thermodynamics.The fifth part of the dissertation is the optimization of reaction catalyzed by recombinant LipSM54. Hexane and vinyl acetate were chosen as an appropriate organic solvent and acyl donor for transesterification. The ratio of vinyl acetate to l-menthol was chosen at 2:1, and enantiomeric excess (eep) of higher than 99% (E>200) was achievable at a conversion of 28.5% dl-menthol (100 mM) at 30℃. LipSM54 exhibited diastereoselectivity of higher 40 at 10%(m/v) concentration during 24h toward 4-pairs-menthol mixture. The space-time yield 1.1 g l-menthyl acetate/L/h were achieved by LipSM54 when production accumulated to 132 mM in 24 h-transesterification of 10%(w/t) mixture.