| Summary: | 博士 === 國立清華大學 === 化學工程學系 === 96 === A surface anchoring motif using the ice nucleation protein (INP) of Xanthomonas campestris pv. campestris BCRC 12846 for displaying transglucosidase was developed. The transglucosidase gene from Xanthomonas campestris pv. campestris BCRC 12608 was fused to the truncated ina gene. This truncated INP consisting of N- and C-terminal domains (INPNC) was able to direct the expression of transglucosidase fusion protein to the cell surface of E. coli.
Localization of the truncated INPNC-transglucosidase fusion protein was examined by Western blot analysis and immunofluorescence labeling, and by whole-cell enzyme activity in the glucosylation of hydroquinone. The glucosylation reaction was carried out at 40℃ for 1 h, which gave 23 g/L of alpha-arbutin, and the molar conversion based on the amount of hydroquinone reached 83 %. The use of whole-cells of the wild type strain resulted in an alpha-arbutin concentration of 4 g/L and a molar conversion of 16 % only under the same conditions. The results suggested that E. coli displaying transglucosidase using truncated INPNC as an anchoring motif can be employed as a whole-cell biocatalyst in glucosylation.
Recombinant E. coli displaying transglucosidase on the surface was used as whole-cell biocatalyst in hydroquinone glucosylation, and its enzymatic characteristics were also studied. The enzymatic activity of recombinant E. coli was seven to twelve-fold higher than that of X. campestris when using the same amount of cells. In the enzymatic characterization experiments, the optimal temperature was found to be 40℃. The optimum pH for the glucosylation of hydroquinone by E. coli displaying transglucosidase was 7.2. In the study of the effect of hydroquinone on the conversion of alpha-arbutin, it was found that the inhibitory effect of hydroquinone was profound at high concentrations of hydroquinone. In addition, conversion of hydroquinone to arbutin was inhibited slightly by high initial concentration of arbutin in the reaction mixture. Taken together, the results demonstrated that the enzyme was inhibited by both substrate and product.
A fed-batch culture strategy for the high-cell density cultivation of recombinant E. coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for alpha-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as a feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of alpha-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76 % molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.
We have successfully demonstrated the application of fed-batch culture strategy for the production of recombinant E. coli cells anchoring surface-displayed transglucosidase, for use as a biocatalyst in alpha-arbutin synthesis. Although the transglucosylating activity of recombinant cells using lactose as an inducer was slightly lower than that of the biocatalyst produced by IPTG induction, the use of fed-batch culture by lactose induction resulted in a higher productivity of whole cell biocatalysts. Therefore, fed-batch cultivation coupled with lactose induction offers an attractive strategy for the mass production of recombinant E. coli cells for use as whole-cell biocatalysts in biotransformations such as alpha-arbutin synthesis.
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