Modeling and kinetic analysis of the reaction system using whole cells with separately and co-expressed D-hydantoinase and N-carbamoylase

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We developed a kinetic model that describes a heterogeneous reaction system for the production of D-p-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin using D-hydantoinase of Bacillus stearothermophilus SD1 and N-carbamoylase of Agrobacterium tumefaciens NRRL B11291. As a biocatalyst, whole cells with separately or co-expressed enzymes were used. The reaction system involves dissolution of substrate particles, enzymatic conversion, racemization of the L-form substrate, and transfer of the dissolved substrate, intermediate, and product through the cell membrane. Because the two enzymes have, different pH optimum, kinetic parameters were evaluated at different pH for the reaction systems. The model was simulated using the kinetic parameters and compared with experimental data, and it was found that the kinetic model-well describes the behavior of the reaction systems using whole cells with separately and co-expressed enzymes. Factors affecting the kinetics of the reaction systems were analyzed on the basis of the kinetic model. In the reaction system with separately expressed enzymes, racemization rate and transport of the reaction intermediate (N-carbamoyl-D-p-hydroxyphenylglycine) were revealed to be the limiting factors at neutral pH, resulting in accumulation of intermediate in the reaction medium. At alkaline condition, on the other hand, inhibition of N-carbamoylase by ammonia was severe and thereby the reaction rate significantly reduced. In the co-expressed enzyme system, accumulation of intermediate was negligible in the reaction medium, and the improved performance was observed compared to that with separately expressed enzymes. The present model might be applied for the optimization and development of the reaction system using two sequential enzymes. (C) 2002 Wiley Periodicals, Inc.
Publisher
JOHN WILEY & SONS INC
Issue Date
2002
Language
English
Article Type
Article
Keywords

D-AMINO ACIDS; THERMOSTABLE D-HYDANTOINASE; RECOMBINANT ESCHERICHIA-COLI; HETEROGENEOUS REACTION SYSTEM; D-P-HYDROXYPHENYLGLYCINE; MICROBIAL TRANSFORMATION; CONVERSION; AMIDOHYDROLASE; MICROORGANISMS; PURIFICATION

Citation

BIOTECHNOLOGY AND BIOENGINEERING, v.78, no.7, pp.779 - 793

ISSN
0006-3592
DOI
10.1002/bit.10259
URI
http://hdl.handle.net/10203/14102
Appears in Collection
BS-Journal Papers(저널논문)
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