| Lipase (EC3.1.1.3) is ubiquitous in nature, which is used in a wide range of field such as fine chemical, detergent, pharmaceutical, food, leather processing, paper manufacture, textile, feed industry. In the term of catalytic feature, lipases have shown many advantages:high regiospecificity and stereoselectivity; no requirement of coenzyme; mild reaction condition and little by-product. Another important property of lipases is that they play a catalytic role in heterogeneous system or organic phase. Usually, numerous lipases catalyze the hydrolysis of esters to the corresponding acids and alcohols in water, while in organic phase esterification, transesterification, aminolysis, acidlysis and alcoholysis can be catalyzed by lipases. However, organic solvents often give rise to denature of lipase and decreased activity. Therefore, screening of organic solvent-tolerant lipases with high activity in the presence of organic solvents has become a focus of researches on lipase. In the present paper, a novel strain producing an organic solvent-tolerant lipase was successfully screened, and the fermentation condition was optimized. Furthermore, the lipase was purified and characterized. The lipase and it cognate foldase genes were cloned and co-expressed. Finally, the use of the immobilized recombinant lipase for the synthesis of butyl acetate was attempted.The paper was divided into five parts.The screening and identification of a strain producing an organic solvent-tolerant lipase were included in the first part. A novel strain producing an organic solvent-tolerant lipase was obtained after enrichment cultivation, initial screening and the second screening. The activity of lipase in the culture was 1.63 U/ml, with the residual activity of 95.7% after incubating at 30℃for 30 min in the presence of benzene (25%v/v).The colony of strain CS-2 was of round shape, bright, milky yellow. Strain CS-2 was rod shaped, Gram negative and it had capsule, single-end flagella and no spore. It can grow at 25℃,37℃and 45℃and can not produce urease and sulfide hydrogen. It can utilize citrate, D-lactose, D-fructose, D-glucose, mannitol and glycerol, but not D-maltose and sucrose. It can hydrolyze gelatin, casein and tween-80, but not starch. The length of 16S rDNA sequence of strain CS-2 was 1499bp, and the sequence data were submitted to Genbank with the accession number GQ254065. According to the morphological trait, biochemical feature and 16S rDNA sequence, strain CS-2 was name after Pseudomonas aeruginosa CS-2. The crude lipase from Pseudomonas aeruginosa CS-2 showed stability in some organic solvents.The optimization of fermentation condition was included in the second part. The maximum growth was achieved at 24 h, and then the growth was decreased slowly. Substantial lipase production started at 18 h and reached to maximum at 48 h. Organic nitrogen source was preferred over inorganic nitrogen resource. Peptone and tryptone was determined to be best organic nitrogen source for CS-2 lipase production. Gum arabic, Tween-20, Tween-80 and Polyvinyl alcohol 124 enhanced the lipase production, whereas CTAB and Triton X-100 played a negative role in lipase production. The optimal temperature, rotary shaking speed initial pH for lipase production was 37℃,200r/min and 7.5, respectively.The purification and characterization of the extra-cellular lipase secreted by Pseudomonas aeruginosa CS-2 were included in the third part. The purified lipase was obtained by ultra-filtration, acetone precipitation and ion exchange chromatography. CS-2 lipase was purified about 25.5-fold with an overall yield of 45.5%. Molecular mass of the native lipase was about 33.9 kDa and 36 kDa, as determined by SDS-PAGE and size chromatography. Therefore, this suggested the lipase was a monomer. The C terminal of the lipase was KNASLWDPGRG. No protein was identical with the C-terminal sequence of CS-2 lipase using blastp. The temperature optimum of the CS-2 lipase was 50℃, and was stable in a temperature range of 40-70℃with more than 80% of the maximum activity. CS-2 lipase showed good thermostability. It remained 62.7% residual activity after incubated at 80℃for 30min. CS-2 lipase showed an optimum activity at pH 8.0, and was stable over a pH range of 7.0-9.0. It kept over 80% residual activity after incubated in a pH range of 4.0-10.0, which showed the lipase exhibited pH stability. These results confirmed the lipase was an alkaline lipase. Ca2+ increased the lipase activity, while other metal ions inhibited the activity. The activation and inhibition of activity were related to the concentration of metal ions. CTAB, tween 20, tween 80 and triton X-100 inhibited the lipase activity. On the contrary, gum Arabic and polyvinyl alcohol 124 enhanced lipase activity. Among the ester of different fatty acids with longer chain,p-nitrophenyl palmitate was the best substrate as compared to other esters. The lipase was activated in the presence of acetonitrile, and it showed stability in DMSO and some immiscible organic solvents to some extent.The clone and expression of the genes of CS-2 lipase and foldase in E.coli were included in the fourth part. The length of CS-2 lipase and foldase gene was 2025bp, and there was no stop codon in the CS-2 lipase gene. The genes of CS-2 lipase and foldase were co-expressed in E. coli BL21 (DE3). The CS-2 lipase was soluble in recombinant cell.To our knowledge, there was no report that a gene without a stop codon can be expressed as an active form. The activity of 203.8 U/mg was achieved after affinity chromatography, in which purification fold was 10.2 and yield rate was 40.9%. The purified enzymes were stained in SDS-PAGE with two bands and their molecular mass was estimated to be 35.7 kDa and 38.3 kDa, respectively. The expression of CS-2 lipase gene in soluble state should be aided by the co-expression of its foldase gene.The synthesis of butyl acetate in heptane by the immobilized recombinant CS-2 lipase was included in the fifth part. The optimum reaction condition was on the following:reaction time (10h); water activity (0.02); reaction temperature (55℃); the concentration of acetic acid and n-butanol (0.1mol/L and 0.2mol/L); the addition of 4A molecular sieve (0.5g) at 8h of reaction time. The conversion of 98.2% was achieved in the optimum reaction condition. The conversion of substrate decreased only from 98.2% to 87.4% after five cycles in use of the immobilized lipase.Therefore, the immobilized lipase showed good catalytic capability and repeated use for the synthesis of butyl acetate.
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