Noriyuki Doukyu

Characteristics and biotechnological applications of microbial cholesterol oxidases

Microbial cholesterol oxidase is an enzyme of great commercial value, widely employed by laboratories routinely devoted to the determination of cholesterol concentrations in serum, other clinical samples, and food. In addition, the enzyme has potential applications as a biocatalyst which can be used as an insecticide and for the bioconversion of a number of sterols and non-steroidal alcohols. The enzyme has several biological roles, which are implicated in the cholesterol metabolism, the bacterial pathogenesis, and the biosynthesis of macrolide antifungal antibiotics. Cholesterol oxidase has been reported from a variety of microorganisms, mostly from actinomycetes. We recently reported cholesterol oxidases from gram-negative bacteria such as Burkholderia and Chromobacterium. These enzymes possess thermal, detergent, and organic solvent tolerance. There are two forms of cholesterol oxidase, one containing a flavin adenine dinucleotide cofactor non-covalently bound to the enzyme (class I) and the other containing the cofactor covalently linked to the enzyme (class II). These two enzymes have no significant sequence homology. The phylogenetic tree analyses show that both class I and class II enzymes can be further divided into at least two groups.

Isolation of Paenibacillus illinoisensis That Produces Cyclodextrin Glucanotransferase Resistant to Organic Solvents

A bacterium that secreted cyclodextrin glucanotransferase (CGTase) in a medium overlaid with n-hexane was isolated and identified as Paenibacillus illinoisensis strain ST-12 K. The CGTase of the strain was purified from the culture supernatant. The molecular mass was 70 kDa. The enzyme was stable at pH 6 to 10 and active at pH 5.0 to 8.0. The optimum temperature at pH 7.0 was 65°C in the presence of 5 mM CaCl2. The enzyme produced mainly β-cyclodextrin. The total yield of α-, β-, and γ- cyclodextrins was increased 1.4-fold by the addition of ethanol. In particular, the yield of β-cyclodextrins in the presence of 10% (vol/vol) ethanol was 1.6-fold that without ethanol. The CGTase was stable and active in the presence of large amounts of various organic solvents.

Discovery of glpC, an organic solvent tolerance-related gene in Escherichia coli, using gene expression profiles from DNA microarrays.

ABSTRACT Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance.

Stabilities and Conformational Transitions of Various Proteases in the Presence of an Organic Solvent

The half‐life of the activity of the PST‐01 protease that was secreted by organic solvent‐tolerant Pseudomonas aeruginosa PST‐01 was very long in the presence of methanol as compared to that in the absence of methanol. The conformational transitions of the PST‐01 protease, α‐chymotrypsin, thermolysin, and subtilisin in the presence and absence of methanol were monitored by measuring the CD spectra. The conformational stabilities of the PST‐01 protease and subtilisin in the presence of methanol were higher than those in the absence of methanol. This resulted in high stability of these proteases in the presence of methanol. Furthermore, it was suggested that the organic solvent stabilities of enzymes were closely related to the secondary structure by monitoring the conformational transitions of polyamino acids, which form the particular conformations, in the presence and absence of methanol.

Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli

Aims:  To investigate the involvement of osmoprotectant transporters in organic solvent tolerance in Escherichia coli and to construct an E. coli strain with high organic solvent tolerance.

Cloning, sequence analysis, and expression of a gene encoding Chromobacterium sp. DS-1 cholesterol oxidase

Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53–62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. Km and Vmax values of the cholesterol oxidase were estimated from Lineweaver–Burk plots. The Vmax/Km ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.

Contributions of mutations in acrR and marR genes to organic solvent tolerance in Escherichia coli

The AcrAB-TolC efflux pump is involved in maintaining intrinsic organic solvent tolerance in Escherichia coli. Mutations in regulatory genes such as marR, soxR, and acrR are known to increase the expression level of the AcrAB-TolC pump. To identify these mutations in organic solvent tolerant E. coli, eight cyclohexane-tolerant E. coli JA300 mutants were isolated and examined by DNA sequencing for mutations in marR, soxR, and acrR. Every mutant carried a mutation in either marR or acrR. Among all mutants, strain CH7 carrying a nonsense mutation in marR (named marR109) and an insertion of IS5 in acrR, exhibited the highest organic solvent-tolerance levels. To clarify the involvement of these mutations in improving organic solvent tolerance, they were introduced into the E. coli JA300 chromosome by site-directed mutagenesis using λ red-mediated homologous recombination. Consequently, JA300 mutants carrying acrR::IS5, marR109, or both were constructed and named JA300 acrR IS, JA300 marR, or JA300 acrR IS marR, respectively. The organic solvent tolerance levels of these mutants were increased in the following order: JA300 < JA300 acrR IS < JA300 marR < JA300 acrR IS marR. JA300 acrR IS marR formed colonies on an agar plate overlaid with cyclohexane and p-xylene (6:4 vol/vol mixture). The organic solvent-tolerance level and AcrAB-TolC efflux pump-expression level in JA300 acrR IS marR were similar to those in CH7. Thus, it was shown that the synergistic effects of mutations in only two regulatory genes, acrR and marR, can significantly increase organic solvent tolerance in E. coli.

Peptide Synthesis of Aspartame Precursor Using Organic‐Solvent‐Stable PST‐01 Protease in Monophasic Aqueous‐Organic Solvent Systems

The PST‐01 protease is a metalloprotease that has zinc ion at the active center and is very stable in the presence of water‐soluble organic solvents. The reaction rates and the equilibrium yields of the aspartame precursor N‐carbobenzoxy‐l‐aspartyl‐l‐phenylalanine methyl ester (Cbz‐Asp‐Phe‐OMe) synthesis from N‐carbobenzoxy‐l‐aspartic acid (Cbz‐Asp) and l‐phenylalanine methyl ester (Phe‐OMe) in the presence of water‐soluble organic solvents were investigated under various conditions. Higher reaction rate and yield of Cbz‐Asp‐Phe‐OMe were attained by the PST‐01 protease when 30 mM Cbz‐Asp and 500 mM Phe‐OMe were used. The maximum reaction rate was obtained pH 8.0 and 37 °C. In the presence of dimethylsulfoxide (DMSO), glycerol, methanol, and ethylene glycol, higher reaction rates were obtained. The equilibrium yield was the highest in the presence of DMSO. The equilibrium yield of Cbz‐Asp‐Phe‐OMe using the PST‐01 protease attained 83% in the presence of 50% (v/v) DMSO.

Enhancement of the aspartame precursor synthetic activity of an organic solvent-stable protease

The PST-01 protease is highly stable and catalyzes the synthesis of the aspartame precursor with high reaction yields in the presence of organic solvents. However, the synthesis rate using the PST-01 protease was slower than that observed when thermolysin was used. Structural comparison of both enzymes showed particular amino acid differences near the active center. These few residue differences in the PST-01 protease were mutated to match those amino acid types found in thermolysin. The mutated PST-01 proteases at the 114th residue from tyrosine to phenylalanine showed enhancement of synthetic activity. This activity was found to be similar to thermolysin. In addition, mutating the residue in the PST-01 protease with arginine and serine showed more improvement of the activity. The mutant PST-01 protease should be more useful than thermolysin for the synthesis of the aspartame precursor, because this enzyme has higher stability and activity in the presence of organic solvents. The results show the potential of organic solvent-stable enzymes as industrial catalysts.

Stereospecific Oxidation of 3β-Hydroxysteroids by Persolvent Fermentation with Pseudomonas sp. ST-200

Pseudomonas sp. strain ST-200 isolated from a humus soil effectively oxidizes cholesterol dissolved in organic solvents but not that suspended in the growth medium. The organism does not assimilate cholesterol. This organism oxidized a variety of 5α- or 5-ene-sterols dissolved in organic solvent. First, the 3β-OH group was oxidized to a ketone group. The 3α-OH group was scarcely oxidized. Successively, C-6 position of 5-ene-steroids was hydroxylated, and a double bond of 5-ene-steroids was transferred from Δ(5) to Δ (4). Then, the 6-OH group was oxidized to a ketone group. Persolvent fermentation with ST-200 would provide an effective, convenient, and stereospecific method to oxidize the C-3 and C-6 positions of steroids.