Surface display of transglucosidase onEscherichia coli by using the ice nucleation protein ofXanthomonas campestris and its application in glucosylation of hydroquinone

Abstract

A surface anchoring motif using the ice nucleation protein (INP) of Xanthomonas campestris pv. campestris BCRC 12,846 for display of transglucosidase has been developed. The transglucosidase gene from Xanthomonas campestris pv. campestris BCRC 12,608 was fused to the truncated ina gene. This truncated INP consisting of N- and C-terminal domains (INPNC) was able to direct the expressed transglucosidase fusion protein to the cell surface of E. coli with apparent high enzymatic activity. The 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 degrees C 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.

High cell density cultivation of Escherichia coli with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

“…In our previous work on the construction of recombinant E. coli displaying transglucosidase, we focused on the enzymatic characteristics of the whole cells in hydroquinone glucosylation [26]. We wish to extend this work to establish a high cell density culture for the production of E. coli cells displaying transglucosidase for use as biocatalysts, for large-scale production of -arbutin by glucosylation of hydroquinone.…”

“…Sam7 nin5 lacUV5-T7 gene 1)] was transformed with plasmid pInaXNC1-aglA for the expression of INPNC-transglucosidase fusion protein [26]. The growing E. coli cells were induced with IPTG or lactose to express the fusion protein.…”

“…For example, using Pseudomonas syringae ice nucleation protein (INP) as a carrier protein, enzymes such as levansucrase [10], carboxymethylcellulase [11], and organophosphorus hydrolase [23] have been successfully displayed on Escherichia coli surface for use as whole-cell biocatalysts in the related applications. In the previous study [26], we have successfully constructed engineered E. coli displaying transglucosidase on the cell surface using INP anchoring motif. The whole cells expressing INPNC-transglucosidase at surface with very high transglucosylating activity were applied as biocatalysts in the glucosylation of hydroquinone with maltose as the sugar donor, and a high yield of arbutin was obtained [26].…”

“…In the previous study [26], we have successfully constructed engineered E. coli displaying transglucosidase on the cell surface using INP anchoring motif. The whole cells expressing INPNC-transglucosidase at surface with very high transglucosylating activity were applied as biocatalysts in the glucosylation of hydroquinone with maltose as the sugar donor, and a high yield of arbutin was obtained [26].…”

Production of recombinant EGFP via surface display of ice nucleation protein and self-cleavage intein

“…By fusing various target proteins to the C-terminus of INP, it was found that the engineered host cell had the surface-localized activities of the target proteins [10][11][12][13][14][15].…”