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Journal of agricultural and food chemistry

Facile immobilization of evolved agrobacterium radiobacter carbamoylase with high thermal and oxidative stability.


PMID 18636689

Abstract

Optically pure amino acids have been widely used as intermediates in the synthesis of antibiotics, antifungal agents, pesticides, and sweeteners. Of particular importance, d- p-hydroxyphenylglycine (D-HPG) can be produced from d, l-hydroxyphenly hydantoin (D,L-HPH) in a two-step reaction mediated by d-hydantoinase and N-carbamoyl- d-amino acid amidohydrolase (or carbamoylase). To make this production more industrially appealing, the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 cloned in an Escherichia coli strain was intensively mutated to improve the thermal stability of carbamoylase by three rounds of DNA shuffling. After an extensive screening of the mutant library, the mutant E. coli strain M303 was obtained to produce variant carbamoylase, CBL303, with three critical mutated residues, including V40A, G75S, and V237A. Further characterization showed that in comparison with the wild-type counterpart the evolved carbamoylase exhibited more than 20-fold tolerance to heat and, in addition, hydrogen peroxide as a result of the synergistic effect caused by the three mutations. Moreover, with the fusion of the chitin-binding domain (ChBD) of Chitinase A1, the evolved carbamoylase CBL303 was specifically adsorbed on chitin beads. Subsequent analysis indicated that the linkage between the enzyme and the affinity matrix was substantially stable. The half-life of the immobilized carbamolyase CBL303 could reach 210 h at 45 degrees C, whereas its free form had that of 17 h. In particular, when applied to D-HPG production, the immobilized enzyme could be recycled 16 times with the achievement of 100% conversion yield. Along with the previous illustration of d-hydantoinase immobilization, the success achieved by immobilization of the evolved carbamoylase in this work apparently offers a promising way for the efficient production of D-HPG from D,L-HPH.

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