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KMID : 1094720140190010076
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Biotechnology and Bioprocess Engineering
2014 Volume.19 No. 1 p.76 ~ p.82
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Efficient and cost-effective production of D-p-hydroxyphenylglycine by whole-cell bioconversion
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Zhang Junli
Cai Zhen
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Abstract
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D-p-hydroxyphenylglycine (D-HPG) is a widely used intermediate for the synthesis of semi-synthetic antibiotics. It can be produced from DL-5-p-hydroxyphenylhydantoin through two sequential enzymatic reactions catalyzed by D-hydantoinase and N-carbamoylase. However, the low productivity and high production cost of the current process significantly limit its industrial application. To set up an efficient and cost-effective whole-cell bioconversion process for D-HPG production, a recombinant E. coli strain was constructed by co-expressing D-hydantoinase and N-carbamoylase from Agrobacterium sp. Then a cheap medium formulation, which uses glycerol and corn steep liquor (CSL) as carbon and nitrogen sources and without addition of any foreign inducer, was developed for high level of enzyme expression. Galactose, melibiose, and raffinose in CSL were found to be capable of inducing T7 promoter. Moreover, this CSL-containing cheap medium exhibited higher expression levels than the traditional LB+IPTG medium for several different enzymes tested, indicating that this medium might be a better alternative to the commonly used LB+IPTG medium for enzyme expression under the control of a T7 promoter. Further optimization revealed that low expression temperature not only increased enzyme expression, but also stabilized the enzyme-expressing cells and the plasmids therein. Wholecell bioconversion was carried out in 55 t water containing 1.8 t substrate and the resting cells expressed in 40 t medium. The specific D-HPG productivity reached 1.68 g/h/g dry cell weight, with a molar yield of 97.8%. To the best of our knowledge, this is the highest productivity reported to date and the first description of this process using large-scale production.
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KEYWORD
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D-p-hydroxyphenylglycine, whole-cell bioconversion, D-hydantoinase, N-carbamoylase, large-scale
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