| Lipases can catalyze hydrolysis, as well as synthesis. Microbial lipases are usually more useful than those from plants or animals, because of the possible high yields, easiness of genetic manipulation, regular supply due to absence of seasonal fluctuations. In our study,16 bacteria and 16 fungi were isolated from five soil samples using rhodamine B-olive oil plates. All the isolates were rescreened by determination of activity and regioselectivity of zymotic fluid. And then SHERLOCK(?) Microbial Identification System, or sequences analysis of 26-28S rDNA or ITS were used to identify these strains. Finally,7 high producing strains were screened out. Pseudomonas sp. B1-1, Acinetobacter sp. B1-2, Acinetobacter sp. B5-1 and Trichosporon sp. F1-2 are sn-1(3) regioselective, while Staphylococcus sp. B2-1, Acinetobacter sp. B3-8 and Galactomyces candidum F1-1 have no positional selectivity. The catalytic activity of T. sp. F1-2 are the highest among all the isolates. Thus, it became the main study object. Phylogenetic analysis of T. sp. F1-2 showed that it has a close evolutionary relationship with T. cacaoliposimilis and T. laibachii.The specific activity of extracellular lipase of T. sp. F1-2 was found to be higher than intracellular one, so the former one is easier for purification. And it was successfully purified 3.96 fold via the process including ammonium sulfate precipitation (50% saturability), dialysis (8000-14000 Da) and DEAE-sepharose FF weak anion exchange column (pH 8.3). The specific activity of the purified lipase was 223.13 U/mg, and its molecular weight was 32.6 kDa according to SDS-PAGE. Comprehensive characterization of T. sp F1-2 lipase was studied. It could not keep long-time activity above 45℃, and its optimal reaction temperature was 50℃. It was stable at pH 7-9, and its optimal reaction pH was 8. It showed sn-1(3) regioselectivity, and also had significant substrate specificity. The octoate was its preference. It could keep stable with Na+、K+、Ca2+、Mg2+ and Mn2+, and Zn2+ was its most effective inhibitor. It was susceptible to different kinds of surfactants, but showed better tolerance to nonionic ones, compared with anion ones. It showed excellent organic solvent stability, diethyl ether, dichloromethane, methylbenzene and hexane can even promote its activity to some extent. The performance that it could keep good activity in strong polarity solvents, like glycerol, dimethyl sulfoxide and Methanol, is not common among lipases.Two inducing ways were studied. Adding inducing oil and lipids produced by itself were respectively regarded as inducer for lipase-producing fermentation. When inducing oil was added to the broth, T. sp F1-2 would transport it into inside of the strain, and formed intracellular lipid bodies. And emulsification of inducing oil was monitored before transportation. And tiny oil drops were acquired, which is thought to be beneficial to transportation. When fermentation was carried out with low concentration of glucose, the trasportaion of inducing oil could be very significant in 24 h, for the first carbon source soon ran out. When fermentation was carried out with high concentration of glucose, the strain would also transport the oil and formed a lot of lipid bodies, even though the glucose was still enough. And that probably is the reason that T. sp Fl-2 could still have a high yield of lipase in the fermentation with high content glucose. Analysis of fatty acid composition confirmed that intracellular fatty acids are the same with those of inducing oil, no matter what the concentration of glucose was. So the lipid body is really from inducing oil. On the other hand, the study proved that T. sp Fl-2 still can produce many lipid bodies on its own during fermentation without inducing oil. Since producing lipid needs lipases to take part in, and meanwhile the generated lipids could become internal inducing oil to further promote production of the lipase. As a result, the strain could also produce lipase without adding inducing oil. The lipids produced by T. sp Fl-2 mainly contained myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid. Lowering the nitrogen concentration was good for lipids accumulation, and meanwhile affect the content of each fatty acid. According to the comparison between two inducing ways, adding inducing oil was more efficient than the situation with own produced lipids. And initially adding inducing oil in seed media can produce more lipases.But relatively low yield limits T. sp F1-2 application in industry. To obtain high-producing mutants, the atmospheric and room temperature plasma was applied to this wild strain. And a high throughput screening method using 96-well plates was set up; in this protocol, enzyme activity of fermented broth was measured by p-nitrophenyl palmitate method. With this approach,60 mutants were successfully screened. The mutation rate and positive one were 51.7% and 28.3% respectively, when lipase activity was regarded as the screening factor. According to the results of shake-flask fermentation for 96 h, lipase yields of mutant A13 and mutant A5 were respectively 2.64 and 1.54 times higher than that of the wild strain. And both of them showed good genetic stability. Further study revealed that mutant A13 could reach the plateau of lipase output 24 h ahead of the wild strain, which is the most important advantage to this mutant.In addition to the content above, special concentration was focused on two crucial characters of lipases, regioselectivity and acyl migration. Regioselectivity of lipases plays a crucial role in structured lipids synthesis and oil modification. Because of differences between aqueous and non-aqueous conditions, regioselectivity analyzed by the conventional hydrolysis method might not always match that in synthesis reactions. In this paper, acidolysis of lauric acid and camellia oil was developed to directly evaluate regioselectivity in solvent-free media. Fatty acid composition and positional distribution of reaction products were detected by gas chromatography. Through this protocol, Lipozyme RMIM, L02, L03 and L04 were identified as sn-1(3) regioselective, L01 was partially selective, and Novozym 435 was nearly non-selective. Predictability of the model reaction has been verified by replacing substrates of lipases. According to the comparative analysis between the acidolysis method and traditional hydrolysis one, regioselectivity in two situations is generally the same, except selectivity of Novozym 435 which is susceptible to solvent systems. Therefore the present method avoids limits of hydrolysis evaluation systems in synthesis reactions.Besides acidolysis model reaction, an interesterification model reaction was also set up to study the acyl migration. The substrates of model reaction were equimolar mixture of trilaurin and 1,3-palmitin-2-olein, and three immobilized lipase preparations as catalysts were used to the interesterification. Analysis of triacylglycerol (TAG) content and fatty acid (FA) distribution monitored the lipase-catalyzed interesterification in sn-1,3 positions and FA exchange in the sn-2 position caused by acyl migration. Lipase from Rhizopus oryzae immobilized on polypropylene showed high sn-1(3) regioselectivity, and minimal exchange in the sn-2 position. With lipase from Thermomyces lanuginosus on silica (Lipozyme(?)TL IM), completely randomized FA distribution was obtained in 24 h. T. lanuginosus lipase on polypropylene caused a moderate rate of FA exchange in the sn-2 position. Thus, the T. lanuginosus lipase and silica promoted randomization of FA distribution, while the R. oryzae lipase and polypropylene did not. Higher water activity promoted hydrolysis and thereby increased concentrations of partial acylglycerols, but at the same time a decrease in the acyl migration rate of these intermediates was also observed. The net result was that at a certain degree of interesterification, there was no significant effect of water activity on the degree of exchange in the sn-2 position. On the other hand, a low water activity had the major advantage of giving a high yield of TAG. |
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