title: In vivo immobilization of D-hydantoinase in Escherichia coli.
abstract: D-P-Hydroxyphenylglycine (D-HPG) is a precursor required for the synthesis of semi-synthetic antibiotics. This unnatural amino acid can be produced by a transformation reaction mediated by D-hydantoinase (D-HDT) and d-amidohydrolase. In this study, a method was developed to integrate production and immobilization of recombinant D-HDT in vivo. This was approached by first fusion of the gene encoding D-HDT with phaP (encoding phasin) of Ralstonia eutropha H16. The fusion gene was then expressed in the Escherichia coli strain that carried a heterologous synthetic pathway for polyhydroxyalkanoate (PHA). As a result, d-HDT was found to associate with isolated PHA granules. Further characterization illustrated that D-HDT immobilized on PHA exhibited the maximum activity at pH 9 and 60°C and had a half-life of 95 h at 40°C. Moreover, PHA-bound d-HDT could be reused for 8 times with the conversion yield exceeding 90%. Overall, it illustrates the feasibility of this approach to facilitate in vivo immobilization of enzymes in heterologous E. coli strain, which may open a new avenue of enzyme application in industry.
authors: Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan 32003, Taiwan. Electronic address: chensy@saturn.yzu.edu.tw.
authors: Shan-Yu
authors: SY
authors: Chen
authors: Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Taoyuan 32003, Taiwan.
authors: Yi-Wen
authors: YW
authors: Chien
authors: Department of Chemical Engineering, Feng Chia University, 100 Wen-Hwa Road, Taichung 40724, Taiwan; Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan; Department of Medical Research, China Medical University Hospital, Taichung 40447, Taiwan.
authors: Yun-Peng
authors: YP
authors: Chao
mesh_terms: D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism*; D020118:Cupriavidus necator / Q000235:genetics; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D002479:Inclusion Bodies; D037121:Plant Lectins / Q000235:genetics; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis
chemical_list: D004800:Enzymes, Immobilized; D037121:Plant Lectins; D054813:Polyhydroxyalkanoates; C008825:phasin; C023010:4-hydroxyphenylglycine; D000581:Amidohydrolases; C020047:dihydropyrimidinase; D005998:Glycine
keywords: In vivo immobilization; Phasin; Polyhydroxyalkanoate; d-Hydantoinase; d-p-Hydroxyphenylglycine