Surface display of transglucosidase on Escherichia coli by using the ice nucleation protein of Xanthomonas campestris and its application in glucosylation of hydroquinone.
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.
Wu
Po-Hung
PH
Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan.
Giridhar
R
R
Wu
Wen-Teng
WT
D020816:Amino Acid Motifs; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D001709:Biotechnology / Q000379:methods*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D005947:Glucose / Q000737:chemistry; D005959:Glucosidases / Q000737:chemistry* / Q000378:metabolism; D006873:Hydroquinones / Q000737:chemistry*; D008858:Microscopy, Phase-Contrast; D008956:Models, Chemical; D008957:Models, Genetic; D010447:Peptide Hydrolases / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D013347:Subcellular Fractions; D016959:Xanthomonas campestris / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D006873:Hydroquinones; C063113:ice nucleation protein; D005959:Glucosidases; D010447:Peptide Hydrolases; D005947:Glucose; C031927:hydroquinone