One-Step Production of D-p-Hydroxyphenylglycine by Recombinant Escherichia coli Strains

Abstract

The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one-step conversion of DL-HPH to D-HPG. In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.

Recombinant Polycistronic Structure of Hydantoinase Process Genes in Escherichia coli for the Production of Optically Pure d -Amino Acids

“…The conversion percentages increased as temperatures increased up to 55°C, but at higher temperatures the percentage of D-methionine conversion decreased sharply. Similar results have been reported for recombinant systems with D-hydantoinase and D-carbamoylase enzymes whose thermostability was analyzed (4,19) and for the system also including the hydantoin racemase enzyme expressed separately (16).…”

“…For this reason, both parameters were studied with both multienzymatic systems. Previous studies have described different optimal pHs for D-hydantoinase and D-carbamoylase enzymes (4,10,19). The optimal pH described for both hydantoin racemases is 7.5 (12,15).…”

“…This method is both cheaper and less contaminating than the chemoenzymatic process (10). In this cascade of reactions, named the hydantoin process (1) (4,8,19), only 50% of the remaining hydantoins are converted to the corresponding amino acid, while the other 50% correspond to L-hydantoin, which is not hydrolyzed by D-hydantoinase. For these hydantoins, faster racemization is possible via an enzymatic reaction incorporating a third enzyme named hydantoin racemase together with D-hydantoinase and D-carbamoylase enzymes.…”

“…High velocities of chemical racemization have been observed only for D,L-5-phenyl-and D,L-5-p-hydroxy-phenylhydantoin because of the resonance stabilization by the 5-substituent, while all other hydantoins take many hours to racemize (13). Consequently, although the hydantoinase process has been successfully used to produce 100% D-phenyl-and D-p-hydroxy-phenylglycine (4,8,19), only 50% of the remaining hydantoins are converted to the corresponding amino acid, while the other 50% correspond to L-hydantoin, which is not hydrolyzed by D-hydantoinase. For these hydantoins, faster racemization is possible via an enzymatic reaction incorporating a third enzyme named hydantoin racemase together with D-hydantoinase and D-carbamoylase enzymes.…”

Carrageenan: a review

“…Recombinant E. coli cells expressing both dihydropyrimidinase and carbamoylase were immobilised in κ-carrageenan and were able to convert d/l-hydroxyphenylhydantoin to d-p-hydroxyphenylglycine. In a singlestep reaction a conversion of 93% was obtained, while a 20% value was observed with the strain of Agrobacterium radiobacter, which contained the original dihydropyrimidinase gene cloned into E. coli (Chao et al 1999). …”