Study On Microbial Enzymatic Sythesis Of D-p-hydroxyphenylglycine

D-p-hydroxyphenylglycine(D-p-HPG) is one of the important side chain in the semi-synthetic-lactam antibiotic molecules widely clinically used in the world. It is produced by enzymatichydrolysis of D-hydantoinase and D-decarbamoylase from D,L-p-hydroxyphenylhydantoin(D,L-HPH) .A strain MMR003, used for D-p-HPG production in industry was classified asBurkholderia pickettii by morphological observation and biochemical characterization. The geneencoding the D-hydantoinase enzyme was cloned, sequenced and expressed in Escherichia coli.The nucleotide sequence of the 5.0 kb insert of subclone pXZ-total was determined. One openreading frame of 1374 bp was found and predicted to encode a polypeptide consisting of 458amino acids in size. The amino acid sequence alignment of D-hydantoinase from Burkholderiapickettii shows the 85% homologous with the corresponding enzyme from Agrobacteriumradiobatcer NRRL B11291.The D-hydantoinase gene (dha) harboured in the plasmid pXZPH2 inE.coli BL21(DE3) was highly expressed by IPTG induction. The D-hydantoinase activity forD,L-p-hydroxyphenylhydantoin is 0.66U/ml broth, which is 2-fold increase compared to that of theparent strain Burkholderia pickettii. D-Amino acids, important intermediates in the production of semisynthetic penicillines andcephalosporins ,are currently prepared from the corresponding hydantoins using bacterial biomasscontaining two enzymes ,hydantoinase and carbamylase . These enzymes convert the hydantoinsfrist into N-carbamyl-D-p-hydroxyphenylglycine and then into corresponding D-amino acids.Inan attempt to select more efficient biocatalysts ,the hydantoinase and carbamylase genes fromBurkholderia pickettii were cloned in Escherichia coli, achieve a co-experssed strains. The geneswere assembled to give the carbamylase gene preceding the hydantoinase gene. The recombinantstrains stably and constitutively produced the two enzymes and efficiently converted thecorresponding hydantoins onto p-hydroxyphenylglycine and phenylglycine . The order of geneswithin the operon and the growth temperature of the strains turned out to be important for bothenzyme and D-amino acid production.The configuration with the carbamylase gene preceding thehydantoinase gene was the most efficient one when the biomass was grown at 22℃ rather than 37℃.This biomass produced D-amino acid twice as efficienty as the industrial strain of Burkholderiapickettii . The efficiency was found to be correlated with the level of carbamylaseproduced ,indicating that the concentration of this enzyme is the rate-limiting factor in D-aminoacid production under the conditions used on an industrial scale.