Chao-Hung Kao

Enantioselective synthesis of L-homophenylalanine by whole cells of recombinant Escherichia coli expressing L-aminoacylase and N-acylamino acid racemase genes from Deinococcus radiodurans BCRC12827

l‐Homophenylalanine (l‐HPA) is a chiral unnatural amino acid used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. To develop a bioconversion process with dynamic resolution of N‐acylamino acids for the l‐HPA production, N‐acylamino acid racemase (NAAAR) and l‐aminoacylase (LAA) genes were cloned from Deinococcus radiodurans BCRC12827 and expressed in Escherichia coli XLIBlue. The recombinant enzymes were purified by nickel‐chelate chromatography, and their biochemical properties were determined. The NAAAR had high racemization activity toward chiral N‐acetyl‐homophenylalanine (NAc‐HPA). The LAA exhibited strict l‐enantioselection to hydrolyze the NAc‐l‐HPA. A stirred glass vessel containing transformed E. coli cells expressing D. radiodurans NAAAR and LAA was used for the conversion of NAc‐d‐HPA to l‐HPA. Unbalance activities of LAA and NAAAR were found in E. coli cell coexpressing laa and naaar genes, which resulted in the accumulation of an intermediate, NAc‐l‐HPA, in the early stage of conversion and a low productivity of 0.83 mmol l‐HPA/L h. The results indicated that low activity of LAA present in the biomass is the rate‐limiting factor in l‐HPA production. In the case of two whole cells with separately expressed enzyme, the enzymatic activities of LAA and NAAAR could be balanced by changing the loading of individual cells. When the activities of two enzymes were fixed at 3600 U/L, 99.9% yield of l‐HPA could be reached in 1 h, with a productivity of 10 mmol l‐HPA/L h. The cells can be reused at least six cycles at a conversion yield of more than 96%. This is the first NAAAR/LAA process using NAc‐HPA as substrate and recombinant whole cells containing Deinococcus enzymes as catalysts for the production of l‐HPA to be reported.

A gene cluster involved in pyrimidine reductive catabolism from Brevibacillus agri NCHU1002.

Genes involved in pyrimidine reductive catabolism (pyd) were isolated from a moderate thermophile, Brevibacillus agri NCHU1002, and nine ORFs in an 8.2-kb DNA fragment were identified by DNA sequence analysis. The pyd gene cluster included three closely spaced ORFs, designated pydA, pydB, and pydC, transcribed in the same orientation. Based on their amino acid sequence identity and enzyme activity assay, the gene products were identified as dihydropyrimidine dehydrogenase (PydA), dihydropyrimidinase (PydB), and beta-alanine synthase (PydC). Northern blot and primer extension analyses revealed that the pydBC genes are induced by dihydrouracil and regulated under the control of sigma(54) recognized promoter at transcriptional level as a polycistronic operon. All results indicate that the pydABC genes participate in the pathway of the pyrimidine reductive catabolism. This is the first bacterial pyd gene cluster to be reported.

A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and l-N-carbamoylase activities as biocatalyst for the production of l-homophenylalanine

A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.0. This enzyme was completely thermostable at 50 degrees C for 20 days. A whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by coexpression of the pydB gene and a thermostable L-N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and L-N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-beta-D-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for L-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10mM D,L-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for L-HPA production.

Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus.

A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co(2+) and Mn(2+), and completely inhibited by Cu(2+). Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.