| The development of green chemistry encourages the design of products and processes that minimize the use and generation of hazardous substances with high efficiency, low energy consumption and low emission. Bioconversion, also known as biotransformation refers to the use of microorganisms or enzymes to carry out a chemical reaction, is an important part of green chemistry. Lipases perform essential roles in bioconversion, because of their various catalytic reactions and substrates. Currently, the main types of lipase applied in the industry are free and immobilized enzymes. Whole-cell biocatalyst is a novel immobilization technology to improve cost efficiency of enzymes, has caused the attention of researchers. Yeast surface display technique has been often utilized to construct whole-cell biocatalysts, making enzymes displayed on the cell surface by gene modification, with better performance in enzyme activity, stability, cost and recycle.Here, based on the yeast surface display system with a-agglutinin as the anchor,4recombinant yeast whole-cell biocatalysts have been successfully constructed, displaying codon optimized ROL and CALB, respectively. One of them, ROL-displayed S.cerevisiae, was chosen as a whole-cell biocatalyst to study in this paper. Enzyme activities and properties, oil hydrolysis in aqueous phase, biodiesel synthesis in non aqueous phase, and influence of bioimprinting have been studied. The main results are summarized as follows.The results of halo assay by tributyrin plate medium and no detection of lipase activity in supernatant of liquid medium have indicated that the yeast whole-cell ROL biocatalyst was constructed successfully. To our knowledge, this was the first attempt to combine the techniques of yeast surface display with a-agglutinin as the anchor and codon optimization for whole-cell biocatalyst construction. Consequently, the hydrolysis activity was25±0.89U/g dried cells, the esterification activity was5.12U/g dried cells and the optimum pH and temperature was pH7.0and40℃.The application of the whole-cell biocatalyst in aqueous phase was studied in the test of tributyrin hydrolysis. The conversion of butyric acid reached96.91%after144h. The whole-cell biocatalyst could be applied in hydrolysis of various substrates with ester bond in the future. Then, the bioimprinting technique was applied to whole-cell biocatalyst to improve the activity and thermostability in organic phase for the first time. Bioimprinting of lipases with fatty acid was shown to be a feasible and effective method for obtaining highly active enzymes in organic solvents. The whole-cell biocatalyst was bioimprinted with oleic acid, gaining2-fold (96.30%to50.32%) increase in the bioconversion of oleic acid methyl ester. However, the influence of bioimprinting on activity and thermostability of the whole-cell biocatalyst in aqueous phase was not obvious.Furthermore, the bioimprinted whole-cell lipase biocatalyst was applied to biodiesel bioconversion, gaining5-fold (77.71%to15.46%) increase in the alcoholysis of soybean oil to biodiesel. Moreover, the conversion of biodiesel was up to95.45℃2.73%after a27h reaction at60℃. Surprisingly, the yeast cells were still alive after the reaction, showing outstanding thermostability and tolerance of organic solvent.These results indicate that the combination of bioimprinting with yeast surface display technique to prepare whole-cell biocatalyst has been prove to be a powerful strategy for obtaining an even more active and stable biocatalyst preparation for industrial bioconversion. |
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