Keiko Kita

Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes

Abstract The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7% enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13 500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.

Enzymatic production of ethyl (R )-4-chloro-3-hydroxybutanoate: asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by an Escherichia coli transformant expressing the aldehyde reductase gene from yeast

Abstract The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the aldehyde reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP+, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP+, based on the amounts of NADP+ added and CHBE formed, was about 5100 mol/mol.

Degradation of the metal-cyano complex tetracyanonickelate (II) by Fusarium oxysporum N-10

Abstract A fungus with the ability to utilize a metal-cyano compound, tetracyanonickelate (II) {K2[Ni (CN)4]; TCN}, as its sole source of nitrogen was isolated from soil and identified as Fusarium oxysporum N-10. Both intact mycelia and cell-free extract of the strain catalyzed hydrolysis of TCN to formate and ammonia and produced formamide as an intermediate, thereby indicating that a hydratase and an amidase sequentially participated in the degradation of TCN. The enzyme catalyzing the hydration of TCN was purified approximately ten-fold from the cell-free extract of strain N-10 with a yield of 29%. The molecular mass of the active enzyme was estimated to be 160 kDa. The enzyme appears to exist as a homotetramer, each subunit having a molecular mass of 40 kDa. The enzyme also catalyzed the hydration of KCN, with a cyanide-hydrating activity 2 × 104 times greater than for TCN. The kinetic parameters for TCN and KCN indicated that hydratase isolated from F. oxysporum was a cyanide hydratase able to utilize a broad range of cyano compounds and nitriles as substrates.

Chiral alcohol synthesis with yeast carbonyl reductases

Abstract Synthesis of chiral alcohols, ( R )- and ( S )-4-chloro-3-hydroxybutanoate ethyl esters (CHBE), was performed through the enzymatic asymmetric reduction of 4-chloroacetoacetate ethyl ester (CAAE). The enzymes reducing CAAE to ( R )- and ( S )-CHBE were found to be produced by Sporobolomyces salmonicolor and Candida magnoliae , respectively. The enzyme of S. salmonicolor is a novel NADPH-dependent aldehyde reductase (AR) belonging to the aldo-keto reductase superfamily. The C. magnoliae enzyme also seems to be a novel NADPH-dependent carbonyl reductase. When AR-overproducing Escherichia coli transformant cells or C. magnoliae cells were incubated in an organic solvent–water two-phase system, 300 or 90 mg/ml of CAAE was almost stoichiometrically converted to ( R )- or ( S )-CHBE (>92% e.e.), respectively.

Cloning of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122, and characterization of its gene product

A DNA fragment that carries the gene coding for poly(3-hydroxybutyrate) (PHB) depolymerase was cloned from the chromosomal DNA of Alcaligenes faecalis AE122 isolated from seawater. The open reading frame encoding the precursor of the PHB depolymerase was 1905 base pairs (bp) long, corresponding to a protein of 635 amino acid residues (M(r) = 65,208). The promoter site, which could be recognized by Escherichia coli RNA polymerase, was upstream from the gene, and the sequence adhering to the ribosome-binding sequence was found in front of the gene. The deduced amino acid sequence agreed with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 28 onwards. Analysis of the deduced amino acid sequence revealed the domain structure of the protein; a signal peptide of 27 amino acids long was followed by a catalytic domain of about 400 amino acids, a fibronectin type III module sequence, and a putative substrate binding domain. The molecular mass (62,526) of the mature protein deduced from the nucleotide sequence was significantly lower than the value (95 kDa) estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but coincided well with the value (62,426) estimated from matrix-assisted laser desorption ionization mass spectra. By comparison of the primary structure with those of other PHB depolymerases, the substrate binding domain was found to consist of two domains, PHB-specific and poly(3-hydroxyvalerate)-specific ones, connected by a linker region. The PHB depolymerase gene was expressed in Escherichia coli under the control of the tac promoter. The enzyme expressed in E. coli was purified from culture broth and showed the same catalytic properties as the enzyme from A. faecalis.

Purification and characterization of new aldehyde reductases from Sporobolomyces salmonicolor AKU4429

Abstract New aldehyde reductases (AR), ARII and ARIII, which reduce ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl 4-chloro-3-hydroxybutanoate (CHBE), with NADPH as a cofactor, were purified from Sporobolomyces salmonicolor AKU4429. The two enzymes were different from another aldehyde reductase (ARI) which had already been purified and characterized [Yamada et al., FEMS Microbiol. Lett., 70 (1990) 45; Kataoka et al., Biochim. Biophys. Acta, 1122 (1992) 57]. ARII catalyzed the stereospecific reduction of 4-COBE to (S)-CHBE (92.7% enantiomeric excess (e.e.)). In contrast, ARIII reduced 4-COBE to (R)-CHBE (38.4% e.e.). ARII was characterized further, and reduced aliphatic and aromatic aldehydes, as well as carbonyl compounds, such as camphorquinone, but did not accept aldose as a substrate. The enzyme is a monomer protein with a relative molecular mass of 34,000. Its isoelectric point is 5.0. The NH2-terminal amino acid sequence of ARII is different from that of ARI, which catalyzes the stereospecific reduction of 4-COBE to (R)-CHBE (100% e.e.).

Chiral alcohol synthesis with microbial carbonyl reductases in a water-organic solvent-two-phase system.

Abstract: Production of chiral 4‐chloro‐3‐hydroxybutanoate ethyl esters (CHBE) was performed through microbial asymmetric reduction of 4‐chloroacetoacetate ethyl ester (CAAE). The enzymes reducing CAAE to (R)‐ and (S)‐CHBE were found to be produced by Sporobolomyces salmonicolor and Candida magnoliae, respectively. The enzyme of S. salmonicolor is a novel NADPH‐dependent aldehyde reductase (AR) belonging to the aldo‐keto reductase superfamily. When AR‐overproducing Escherichia coli transformant cells or C. magnoliae cells were incubated in an organic solvent‐water two‐phase system, 300 or 90 mg/mL of CAAE was almost stoichiometrically converted to (R)‐ or (S)‐CHBE (>92% ee), respectively.

Purification, crystallization, and characterization of the extracellular invertase from Zymomonas mobilis

Abstract Zymomonas mobilis IFO 13756 (ATCC 29191) produces three kinds of sucrose-hydrolyzing enzymes, E1, E2, and E3. Extracellular enzymes E2 and E3 bound to the cell surface were released from cells by suspension in 20 mM potassium phosphate buffer (pH 7.0) and incubation at 30°C for 10 min with gentle shaking. After centrifugation of the cell suspension, E3 was isolated from the supernatant as crystals in a 52-fold purification. The enzyme consisted of a monomer subunit having a molecular mass of 58 kDa and its isoelectric point was pH 3.2. The N-terminal amino acid sequence was MFNFNASRWTRAQAMKVNKFDL. The enzyme catalyzed the hydrolysis of sucrose, and was identified as an invertase that had a strict substrate specificity for sucrose. The optimum pH and temperature were pH 5.5 and 50°C, respectively. Thiol reagents inhibited the enzyme activity markedly.

Enzymatic preparation of [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol and hydroxypyruvate using the methanol-assimilating system of methylotrophic yeasts

Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C1-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO2. When [13C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, 13C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the 13C content of each carbon (atoms/100 atoms) was estimated to be 50%. [13C]Methanol was also useful for the enrichment of dihydroxyacetone with 13C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of 13C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol; the molar yield of the ester relative to methanol added was 92.5%

Restriction endonuclease AfaI from Acidiphilium facilis, a new isoschizomer of RsaI: purification and properties

Abstract We have purified Afa I endonuclease, an isoschizomer of Rsa I, from Acidiphilium facilis strain 28H. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis, and composed of a single polypeptide chain with a molecular weight of 30 000. Afa I endonuclease, like Rsa I, recognizes the tetranucleotide sequence 5′-G-T-A-C-3′, and cleaves between the T and A to produce blunt-ended fragments. The yield of the enzyme is 50–100-times that of the Rsa I, which is form a phototrophic bacterium, Rhodospseudomonas sphaeroides strain 28/5.