Takayuki Uwajima

Crystallization and some properties of acetylpolyamine amidohydrolase from Mycoplana bullata.

During the course of investigations on the catabolism of acetylpolyamines by microorganisms, we found that acetylpolyamine amidohydrolase was produced by Mycoplana bullata FERM BP-1845 and isolated the enzyme from the cell-free extract in crystalline form. The enzyme had an apparent molecular weight of 67 kDa and was composed of two identical subunits. The enzyme activity was inhibited by o-oxyquinoline and the crystalline enzyme contained one zinc atom per each subunit. The enzyme had an optimal pH around 8.0 with acetylputrescine as substrate and showed broad substrate specificity and high affinity towards various acetylpolyamines, such as acetylputrescine, acetylcadaverine, acetylspermidine, and acetylspermine.

Production of aldehyde oxidases by microorganisms and their enzymatic properties

In order to establish an efficient process to decompose environmentally toxic aldehydes, dioxygen-dependent aldehyde oxidase (ALOD) from microorganisms was first sought, and some bacteria and actinomycetes were found to produce the enzyme in their cells. Methylobacillus sp., Pseudomonas sp. and Streptomyces moderates were selected as the representative ALOD-producing strains and their enzymes were partially purified and characterized. The three ALODs could oxidize a wide range of aldehydes including formaldehyde, aliphatic aldehydes, and aromatic aldehydes, though their preferences differ depending on their producing strains. The other enzymatic properties were also determined with regard to their producing strains. Methylobacillus sp. ALOD had the most acidic optimum pH for its activity and stability and Pseudomonas sp. ALOD had the highest stability against heat treatment. Three native ALODs had molecular weights ranging from 140 to 148 kDa and were composed of three subunits of different sizes: large (85 to 88 kDa), medium-sized (37 to 39 kDa) and small (18 to 23 kDa).

Isolation and identification of the gene of cholesterol oxidase from Brevibacterium sterolicum ATCC 21387, a widely used enzyme in clinical analysis

The gene coding cholesterol oxidase (CHOD) from Brevibacterium sterolicum, which is widely used in clinical analysis, has been selected from pUC19-based gene bank in E. coli MM294 by colony-hybridization using synthetic DNA as probe. The gene was identified to encode the protein having the same amino acid sequence as that determined from amino-acid sequence analysis. The expression of the CHOD gene in E. coli was not observed, probably due to the transcription failure. Attempts are being made to express it in various hosts including Streptomyces lividans, Corynebacterium glutamicum, and B. sterolicum itself.

Chemo-enzymatic synthesis of optically pure l-leucovorin, an augmentor of 5-fluorouracil cytotoxicity against cancer

Optically pure l-leucovorin was synthesized on a large scale by the combination of chemical and enzymatic processes. After reduction of folate with zinc, dihydrofolate was reduced asymmetrically to (6)-tetra-hydrofolate by use of dihydrofolate reductase from E. coli C600/pTP600, with simultaneous NADPH cofactor recycling using glucose dehydrogenase from Gluconobacter scleroideus KY3613. Calcium l-leucovorin.4H2O (113 g) was obtained from (6S)-tetrahydrofolate via 5,10-methyenyltetrahydrofolate by formylation, reflux, addition of calcium ions and floricil column chromatography, with an overall yield of 50% based on folate. The l-leucovorin showed optical purity of 99.9% de as (6S)-form.

Oxidation of 3β-hydroxyandrostenes by the 3β-hydroxysteroid oxidase (cholesterol oxidase) from brevibacterium sterolicum prior to their analysis by gas—liquid chromatography—mass spectrometry

Abstract The 3β-hydroxysteroid oxidase from Brevibacterium sterolicum has been applied to the oxidation of a number of 3β-hydroxyandrostenes, including polar steroids containing up to three other hydroxylic groups. The substrates, products, and derivatives thereof have been examined by gas—liquid chromatography. Retention index increments for these conversions, and for parallel transformations of other steroids, show considerable regularities, and together with mass spectrometric data afford characteristic structural information.

Hyperproduction of N-acetylneuraminate lyase by the gene-cloned strain ofEscherichia coli

SummaryOptimal culture medium for production ofN-acetylneuraminate lyase (NANA lyase) by the gene-cloned strain ofEscherichia coli, E. coli(pNALl), was screened to develop the process for the industrial production of NANA lyase. Out of nutrients tested, corn steep liquor (CSL) and sugarcane molasses were superior nutrient sources for the enzyme production. Hyperproduction (6–8 units/ml-broth) was achieved on the medium consisted of CSL and molasses.

Purification and properties of an esterase from Aspergillus nomius HS-1 degrading ethylene glycol dibenzoate

We isolated a fungal strain HS-1 utilizing either ethylene glycol dibenzoate or ethyl benzoate as sole source of carbon and energy, and identified it as Aspergillus nomius, based on morphological and rDNA analyses. An enzyme hydrolyzing the esters was purified from the culture supernatant of the strain to an electrophoretically homogeneous state. The enzyme was a carboxyl esterase with a monomeric structure, of which the molecular mass was about 60000, and inhibited by phenylmethylsulfonyl fluoride. The enzyme hydrolyzed various benzoate esters and p-nitrophenyl esters, and its hydrolysis rates for the most favorable substrates, n-butyl benzoate and p-nitrophenyl valerate, were comparable.

Optimum culture conditions for the epoxidation of cis-propenylphosphonate to fosfomycin by Cellvibrio gilvus

SummaryApproximately 470 strains of various microorganisms were tested for their ability to epoxidize cis-propenylphosphonate (PPOH) to (−)-cis-1,2-epoxypropylphosphonate (fosfomycin, FOM). Cellvibrio gilvus KY 3412 was selected as the best strain. To obtain higher activity, FOM-resistant stains were derived by N-methyl-N′-nitro-N-nitroguanidine mutagenesis. Mutant KY 3413, showing ten times higher FOM resistance, was selected. The conditions for the conversion of PPOH to FOM during the cultivation of the mutant were optimized. The addition of both cobalt and vanadium ions to the culture medium greatly stimulated the conversion. Furthermore, when the pH was maintained at pH 8.0 during cultivation, the highest conversion was attained. The molar conversion yield of FOM was inversely dependent on the initial concentration of PPOH, that is, conversions of 100% at less than 0.05% PPOH and of 40% at 0.5% wPOH were attained after 5 days cultivation.

[236] Tyramine oxidase (Sarcina lutea)

Publisher Summary This chapter focuses on tyramine oxidase (Sarcina lutea). Tyramine oxidase catalyzes the oxidation of tyramine and dopamine with the consumption of 1 mole of oxygen, and the production of 1 mole of aldehyde, 1 mole of ammonia, and 1 mole of hydrogen peroxide. In the presence of the catalase, hydrogen peroxide is converted to water and 0.5 mole of oxygen. The assay described in the chapter is based on the oxygen consumption obtained with tyramine as the substrate in the presence of added catalase. The chapter also discusses purification procedure and properties.

Large-scale chemoenzymic synthesis of calcium (6S)-5-formyl-5,6,7,8-tetrahydrofolate [(–)-leucovorin] using the NADPH recycling method

Chemoenzymic large-scale synthesis of the calcium salt of (6S)-5-formyltetrahydrofolic acid [(–)-leucovorin, (6S)-5] was achieved from folic acid 1via(6S)-tetrahydrofolic acid [(6S)-3] by using dihydrofolate reductase (DHFR) produced by Escherichia coil, harbouring a high-expression plasmid, pTP64–1. On the other hand, for the diastereoselective reduction of 7,8-dihydrofolic acid 2 to tetrahydrofolate (6S)-3, a new NADPH recycling system was constructed by coupling with glucose dehydrogenase from Gluconobacter scieroides. Having these enzymic systems to hand, compound 1 was reduced by zinc powder in alkaline solution to give compound 2 which, without isolation, was reduced enzymatically to afford tetrahydrofolate (6S)-3(94% de). The pH adjustment of the reaction mixture containing dihydrofolate 2 was done with phosphoric acid in order to remove zinc ion which inhibited the following enzymic reduction. The formed tetrahydrofolate (6S)-3 was converted into entirely optically pure N-formyl compound (6S)-5 on a large scale. The specific rotation value of (–)-leucovorin was [α]D20–13.3 (c 1, water). For the comparison of pharmacological effects, a completely optically pure form of (+)-leucovorin [(6R)-5] was also prepared on a preparative scale. Compound (6S)-5 was 300-fold more active compared with the (6R)-diastereoisomer.