Koichi Mitsukura

Purification and Characterization of a Novel (R)-Imine Reductase from Streptomyces sp. GF3587

The (R)-imine reductase (RIR) of Streptomyces sp. GF3587 was purified and characterized. It was found to be a NADPH-dependent enzyme, and was found to be a homodimer consisting of 32 kDa subunits. Enzymatic reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) resulted in the formation of 9.8 mM (R)-2-methylpyrrolidine ((R)-2-MP) with 99% e.e. The enzyme showed not only reduction activity for 2-MPN at neutral pH (6.5–8.0), but also oxidation activity for (R)-2-MP under alkaline pH (10–11.5) conditions. It appeared to be a sulfhydryl enzyme based on the sensitivity to sulfhydryl specific inhibitors. It was very specific to 2-MPN as substrate.

Oxidation of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids by Acetobacter and Serratia strains

Conversion of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids was carried out using Acetobacter rancens IFO3297, A. pasteurianus IFO13753 and Serratia liquefaciens LF14. IFO3297 produced 110g 2-furoic acid l-1 from furfural with a 95% molar yield. 5-Hydroxymethyl-2-furancarboxylic acid was produced from the corresponding aldehyde by using whole cells LF14. IFO13753 and LF14 both converted isophthalaldehyde, 2,5-furandicarbaldehyde, 2,5-thiophenedicarbaldehyde and 2,2’ biphenyldicarbaldehyde to the corresponding formylcarboxylic acid with 86--91% molar yields.

Molecular and Catalytic Properties of 2,4′-Dihydroxyacetophenone Dioxygenase from Burkholderia sp. AZ11

The gene dad encoding 2,4′-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M r=90 kDa) was a homotetramer with a subunit M r of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K m for DHAP and the apparent V max were estimated to be 1.60 μM and 6.28 μmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl2 and HgCl2 strongly inhibited the enzyme, while FeSO4 weakly activated it.

Synthesis of (R)-2-phenylpropanoic acid from its racemate through an isomerase-involving reaction by Nocardia diaphanozonaria

Abstract(R)-2-Phenylpropanoic acid was synthesized from the racemic acid through an isomerization reaction involving resting cells of Nocardia diaphanozonaria JCM3208. The isomerization activity of the cells was enhanced 25-fold by adding 5.5 mM racemic 2-phenylpropanoic acid to the culture medium. When 5 mM racemic 2-phenylpropanoic acid was included in the reaction mixture (4 ml) containing resting cells (100 mg dry cell wt) in 25 mM K2HPO4/KH2PO4 buffer (pH 7.0) at 30 °C for 8 h, 4.56 mM (R)-2-phenylpropanoic acid (95.8% e.e.) was formed with a 91% molar conversion yield.

Bioconversion of 1-adamantanol to 1,3-adamantanediol using Streptomyces sp. SA8 oxidation system

To efficiently produce 1,3-adamantanediol (1,3-ad(OH)(2)) from 1-adamantanol (1-adOH), our stocks of culture strains and soil microorganisms were surveyed for hydroxylation activity towards 1-adOH. Among them, the soil actinomycete SA8 showing the highest hydroxylation activity was identified as Streptomyces sp. based on 16S ribosomal DNA sequence analysis. The reaction products were purified by silica gel column chromatography, and from NMR and MS analyses, they were identified as 1,3-ad(OH)(2) and 1,4-ad(OH)(2). Streptomyces sp. SA8 produced 5.9 g l(-1) 1,3-ad(OH)(2)from 6.2 g l(-1) 1-adOH in culture broth after 120 h at 25 degrees C. Using resting cells, 2.3 g l(-1) 1,3-ad(OH)(2) was produced after 96 h of incubation at a 69% conversion rate. In both cases, 1,4-ad(OH)(2) was formed as a byproduct at a rate of about 15%. Strain SA8 also hydroxylated 2-adamantanol and 2-methyl-2-adamantanol.

Imine Reductases for Chiral Amine Synthesis

Abstract The discovery of imine reductases catalyzing the enantioselective reduction of unnatural imines leads to an effective method for the synthesis of chiral amine. The characterization and application of imine-reducing enzymes have been reviewed. Among them, Δ1-piperidine-2-carboxylate/Δ1-pyrroline-2-carboxylate reductases can be useful for the synthesis not only of cyclic amino acids but also of N-methyl- l -amino acids. Enantioselective imine reductases acting on unnatural imines afforded various five-, six-, and seven-membered ring cyclic amines with high optical purity. Furthermore, dihydroisoquinoline, dihydro-β-carboline, and indoles are acceptable as substrates, and the enantioselectivities of imine reductases for their substrates are very high. From X-ray crystal structure analyses, several imine reductases have been revealed to be NADPH-dependent homodimeric proteins with N-terminal Rossmann-fold motif, and the reaction mechanisms were proposed.

Oxidative Degradation of 4-Hydroxyacetophenone in Arthrobacter sp. TGJ4

The 4-hydroxyacetophenone assimilating bacterium Arthrobacter sp. TGJ4 was isolated from a soil sample. The resting cell reaction suggested that the strain cleaved 4-hydroxyacetophenone and its 3-methoxy derivative to the corresponding carboxylic acids and formaldehyde. Some properties of the enzyme catalyzing the cleavage reaction were examined.

Characteristics and function of Alcaligenes sp. NBRC 14130 esterase catalysing the stereo-selective hydrolysis of ethyl chrysanthemate

Aims:  Alcaligenes sp. NBRC 14130 was found as a strain hydrolysing a mixture of (±)‐trans‐ and (±)‐cis ethyl chrysanthemates to (1R,3R)‐(+)‐trans‐chrysanthemic acid. The Alcaligenes cells also have hydrolytic activity for 6‐aminohexanoate‐cyclic dimer (6‐AHCD, 1,8‐diazacyclotetradecane‐2,9‐dione). The correlation of function on the enzyme from the Alcaligenes strain with hydrolysis activities for both ethyl chrysanthemate and 6‐AHCD was demonstrated.