A novel method of producing the key intermediate ASI-2 of ranirestat using a porcine liver esterase (PLE) substitute enzyme.

(R)-2-amino-2-ethoxycarbonylsuccinimide (ASI-2) is a key intermediate used in the pharmaceutical industry and is valuable for the industrial synthesis of ranirestat, which is a potent aldose reductase inhibitor. ASI-2 was synthesized in a process combining chemical synthesis and bioconversion. Bioconversion in this study is a key reaction, since optically active carboxylic acid derivative ((R)-1-ethyl hydrogen 3-benzyloxycarbonylamino-3-ethoxycarbonylsuccinate, Z-MME-AE) is synthesized from a prochiral ester, diethyl 2-benzyloxycarbonylamino-2-ethoxycarbonylsuccinate, Z-MDE-AE, at a theoretical yield of 100%. Upon screening for microorganisms that asymmetrically hydrolyze Z-MDE-AE, Bacillus thuringiensis NBRC13866 was found. A novel esterase EstBT that produces Z-MME-AE was purified from Bacillus thuringiensis NBRC13866 and was stably produced in Escherichia coli JM109 cells. Using EstBT rather than porcine liver esterase (PLE), ASI-2 was synthesized with a 17% higher total yield by a novel method, suggesting that the esterase EstBT is a PLE substitute enzyme and therefore, may be of interest for future industrial applications.

A novel method of producing the pharmaceutical intermediate (R)-2-chloromandelic acid by bioconversion.

(R)-2-Chloromandelic acid (R-CM) is one of the chiral building blocks used in the pharmaceutical industry. As a result of screening for microorganisms that asymmetrically hydrolyze racemic 2-chloromandelic acid methyl ester (CMM), Exophiala dermatitidis NBRC6857 was found to produce R-CM at optical purity of 97% ee. The esterase that produces R-CM, EstE, was purified from E. dermatitidis NBRC6857, and the optimal temperature and pH of EstE were 30°C and 7.0, respectively. The estE gene that encodes EstE was isolated and overexpressed in Escherichia coli JM109. The activity of recombinant E. coli JM109 cells overexpressing estE was 553 times higher than that of E. dermatitidis NBRC6857. R-CM was produced at conversion rate of 49% and at optical purity of 97% ee from 10% CMM with 0.45 mg-dry-cell/L recombinant E. coli JM109 cells. Based on these findings, R-CM production by bioconversion of CMM may be of interest for future industrial applications.

Genetic analysis around aminoalcohol dehydrogenase gene of Rhodococcus erythropolis MAK154: a putative GntR transcription factor in transcriptional regulation.

NADP(+)-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 catalyzes the reduction of (S)-1-phenyl-1-keto-2-methylaminopropane ((S)-MAK) to d-pseudoephedrine, which is used as a pharmaceutical. AADH is suggested to participate in aminoalcohol or aminoketone metabolism in this organism because it is induced by the addition of several aminoalcohols, such as 1-amino-2-propanol. Genetic analysis of around the aadh gene showed that some open reading frames (ORFs) are involved in this metabolic pathway. Four of these ORFs might form a carboxysome-like polyhedral organelle, and others are predicted to encode aminotransferase, aldehyde dehydrogenase, phosphotransferase, and regulator protein. OrfE, a homologous ORF of the FadR subfamily of GntR transcriptional regulators, lies downstream from aadh. To investigate whether or not orfE plays a role in the regulation of aadh expression, the gene disruption mutant of R. erythropolis MAK154 was constructed. The ΔorfE strain showed higher AADH activity than wild-type strain. In addition, a transformed strain, which harbored multi-orfE, showed no AADH activity even in the induced condition with 1-amino-2-propanol. These results suggest that OrfE is a negative regulator that represses aadh expression in the absence of 1-amino-2-propanol.

Directed evolution of an aminoalcohol dehydrogenase for efficient production of double chiral aminoalcohols.

The aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154, which can be used as a catalyst for the stereoselective reduction of (S)-1-phenyl-1-keto-2-methylaminopropane to d-pseudoephedrine (dPE), is inhibited by the accumulation of dPE in the reaction mixture, limiting the yield of dPE. To improve this weak point of the enzyme, random mutations were introduced into aadh, and a mutant enzyme library was constructed. The mutant library was screened with a color detectable high-throughput screening method to obtain the evolved enzymes showing the activity in the presence of a high concentration of dPE. Two mutant enzymes showed higher tolerability to dPE than the wild type enzyme. Each of these enzymes had a single amino acid substitution in a different position (G73S and S214R), and a third mutant enzyme carrying both of these amino acid substitutions was constructed. Escherichia coli transformant cells, which express mutant AADHs, showed activity in the presence of 100mg/ml dPE. A kinetic parameter analysis of the wild type and mutant enzymes was carried out. As compared with the wild type enzyme, the mutant enzymes carrying the S214R amino acid substitution or both the S214R and G73S substitutions showed higher k(cat) values, and the mutant enzymes carrying the G73S amino acid substitution or both the G73S and S214R substitutions showed higher K(m) values. These results suggest that the Ser214 residue plays an important role in enzyme activity, and that the Gly73 residue participates in enzyme-substrate binding.

Practical resolution system for DL-pantoyl lactone using the lactonase from Fusarium oxysporum.

We developed an enzymatic resolution system for DL-pantoyl lactone that uses immobilized mycelia of Fusarium oxysporum, which produce a lactone-hydrolyzing enzyme (lactonase). The lactonase catalyzes the stereospecific hydrolysis of D-pantoyl lactone. One hundred eighty repeated batch reactions (total reaction time, 3780 h) were made with mycelia entrapped in calcium alginate gels as the catalyst, in the presence of 90 mM CaCl2. With a 300 gl(-1)DL-pantoyl lactone solution as the substrate, the hydrolysis rate for DL-pantoyl lactone was > 40% and the optical purity of D-pantoic acid was 90% enantiomer excess. Immobilized mycelia retained 70% of their initial lactonase activity, even after 180 batch reactions. The estimated half-life of the lactonase activity of the immobilized mycelia was 6000 h, which is 35 times higher than that of the free mycelia. The process has been exploited commercially since 1999.

Biocatalytic deprotection of a cetraxate ester by Microbacterium sp. strain 7-1W cells.

Enzymatic deprotection of the terminal ester bond of a cetraxate methyl ester was done with resting cells of Microbacterium sp. strain 7-1W, which produces an esterase catalyzing a regioselective hydrolysis reaction, as the catalyst. When 20 g of cetraxate methyl ester in 50 ml of a reaction mixture was incubated with 5 g of wet cells for 17 h, 96% of the substrate was converted to the desired product, cetraxate, quantitatively.

Purification and characterization of a novel esterase promising for the production of useful compounds from Microbacterium sp. 7-1W.

A novel esterase catalyzing regioselective hydrolysis was purified from the membrane fraction of Microbacterium sp. 7-1W, and characterized. The enzyme was solubilized with Brij 58 and purified 13.8-fold to apparent homogeneity with 2.58% overall recovery. The relative molecular mass of the native enzyme as estimated by gel filtration was more than 600,000 Da, and the subunit molecular mass was 62,000 Da. The enzyme catalyzed cleavage of the terminal ester bonds of cetraxate esters and pantothenate esters. The K(m) and V(max) values for methyl cetraxate were 0.380 mM and 7.76 micromole min(-1) mg(-1) protein, respectively. The enzyme was inhibited by serine hydrolase inhibitors.