Screening For Bacteria Producing Positionally Non-Specific Lipase And Expression Of Lipase Genes

Lipases (EC 3.1.1.3), a series of enzymes that catalyze the hydrolysis of various fatty esters are widely distributed in animals, plants and microorganisms. Lipases also catalyze ester synthesis, transesterification and interesterification without cofactors. The lipases from bacteria have the advantages of great variety, broad pH and temperature for catalysis, which are convenient for industrial produce and easy to get pure enzyme. So microbial lipases are a class of useful enzymes that have practical and potential applications in the detergent, food processing, bioenergy, organic synthesis, and pharmaceutical industries. Lipases from various bacteria have different characteristics, such as pH and temperature stability, organic solvent tolerance, and selectivity of substrate and hydrolysis position. Researchers aim at finding the lipases with various desirable traits, and put them in use at different fields. A lipase that does not exhibit any positional specificity may markedly improve the efficiency of hydrolysis and transesterification. In addition, high activity and high productivity are two most desirable properties of lipases in line with industrial applications. Screening of strains from nature that produce lipases with these desirable traits, and expression of lipase genes with high efficiency in heterologous system, are two important approaches to accelerate the study and application of lipases. Main results of this study are as following:1. A strain was isolated from soil which could produce positionally non-specific lipase with high production. The olive oil was used as the sole carbon source in the first-round screening plates, while Rhodamine B plates and Tween-80 plates were used as second-round screening plates.40 strains with high productivity of lipase were isolated from second-round screening plates and cultured in liquid media to examine the lipase activity. Positional selectivity of lipase was determined according to the thin-layer chromatography analysis of the hydrolysis products of triolein. A strain, identified as Burkholderia cepacia S31, produced positionally non-specific lipase with high activity (76 U/mL) in the presence of olive oil in the medium. 2. The culture media components and fermentation conditions were optimized in order to improve the productivity of S31. Based on the results of single-factor experiments and orthogonal experiments of media components and fermentation parameters, the optimum culture conditions for S31 was as follows:the culture medium composed of 2% bran,1% peptone,0.05% MgSO4,4% Tween-80 and 0.2% K2HPO4,with the initial pH of 7.0; The overnight culture was inoculated to 40 mL medium in 250 mL shaking flask to a concentration of 3%(v/v), and cultured at 30℃with 180 rpm shaking for 66 h. The maximum activity reached 283.6 U/mL.3. The catalysis characteristics of S31 lipase were examined after purification from the culture supernatant. After a combination of purification steps including ammonium sulfate fractionation, DEAE ion-exchange chromatography and two rounds of gel filtration, we obtained a single chromatographic peak with high lipase activity. The molecular weight of the purified protein was estimated to be about 35 kDa by SDS-PAGE. S31 lipase was purified with the purification fold of 19.8 and the yield of 13.3%。The S31 lipase appeared to be a thermophilic enzyme, with the optimal temperature of 65-70℃. The lipase retained 80% activity after incubation at 70℃for 1 h, and retained 96% and 85% activity after incubation for 12 h at 50℃and 55℃, respectively, which showed that it was a thermostable enzyme. The optimal pH value of S31 lipase was 9.0. S31 lipase retained high activity after incubation at different pH values for 12 h, especially the residual activity retained larger than 80% after treated in buffers of pH 5-9. The lipase was inhibited by Fe2+, Cu2+ and was activated by Ca2+, Mn2+, K+, Na+ and Mg2+. S31 lipase is tolerant to several organic solvents, such as methanol, n-hexane, n-butanol, toluene and ethyl acetate, some of which even enhanced the activity. S31 lipase could catalyze various fatty acid esters with different chain length, with the best affinity to middle-chain fatty acid.4. S31 lipase gene was cloned and expressed in Bacillus subtilis. The lipase gene lip A comprises of 1095 nucleotides that encoded 364 amino acids, followed by its chaperone gene lip B comprising of 1035 nucleotides. LipB is necessary for folding and secretion of LipA. The sequence was deposited to GenBank with the accession number FJ638612.We investigated the secretive expression of the lipase gene in Bacillus subtilis, which is a non-toxic and safe host. The fragment containing lip A and lip B was inserted into vector pHT43, and then transformed into B. subtilis DB104. The lipase was secretively expressed from DB104 under IPTG induction, and the maximum activity reached 135 U/mL after fermentation for 54 h in the MSR medium in addition of 0.5%Triton X-100. The electro-transformation method was imporoved by adding 0.5 M trehalose to electroporation medium, and bacteria cells were harvested at OD600 nm of 1.0-1.2. The transformation efficiency was enhanced by 10 folds.5. Proteus vulgaris lipase gene was cloned and heterologous expressed. P. vulgaris T6 was another lipase-producing strain which had been isolated and kept in our lab. T6 lipase (PVL) could cleave all the three ester bonds and had the best affinity to long chain fatty acid. PVL was ligated into vector pET-DsbA and transformed into Escherichia coli BL21 (DE3). BL21 [pET-DsbA-PVL] was cultured in LB medium (pH 8.5) supplemented with 15 mg/mL glucose and 200μg/mL amplicilin to OD600 of 1.2, and then induced with 100μg/mL IPTG for 15 h at 15℃. In this optimal condition, the maximum lipase activity reached 192.2 U/mL. The protein products of recombinant BL21 were purified by affinity chromatography, and a single band showing lipase activity appeared in SDS-PAGE, which was about 31 kDa. The traits of PVL from recombinant BL21 cells were similar to those of the native enzyme.6. B. subtilis expression vector pMMP43 was constructed. We reconstructed pMM1525 by substituting its xylose promoter with B. subtilis high-efficient promoter P43 to generate pMMP43. PVL gene was inserted into pMMP43 and expressed in B. subtilis WB800, which secreted PVL with activity of 60 U/mL. Another pHPQ vector (a vector constructed in our lab) was used to harbour PVL gene. The maximum activity of WB800 [pHPQ-PVL] was 200 U/mL after culture for 72 h.