Kimiyasu Isobe

Thin-film polyamine biosensor: substrate specificity and application to fish freshness determination

Abstract A polyamine biosensor was developed by immobilizing putrescine oxidase on micro planar thin-film hydrogen peroxide electrodes. The sensor showed a rapid response to putrescine (90% response: 40 s) and the relationship between putrescine concentration and output current was linear between 0.03 and 3 μM under the optimum reaction conditions (pH 8.0, 30°C). The substrate specificity of the polyamine sensor was determined using a flow-injection analysis system. In addition to the main substrate putrescine, the sensor also converted cadaverine (46%), spermidine (45%), agmatine (13%), 1,6-diaminohexane (3%), 1,7-diaminoheptane (2%) and tyramine (13%, non-enzymatic response). These conversion rates were high compared with those of soluble putrescine oxidase. This is probably due to broadening of the enzymes active centre during the immobilization procedure. The sensor was applied to fish freshness determination during a fish storage experiment. The sensor response obtained in fish extracts increased with increasing storage time and agreed with liquid chromatographic amine determinations.

Enzymatic Production of Glyoxal from Ethylene Glycol Using Alcohol Oxidase from Methanol Yeast

A new oxidative reaction of ethylene glycol was found with two alcohol oxidases from methanol yeast, Candida sp. and Pichia pastoris. Both alcohol oxidases oxidized ethylene glycol to glyoxal via glycolaldehyde. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Candida alcohol oxidase were around 8.5 and 5.5, respectively, and their apparent Kms were 2.96 m and 28.6 mm, respectively. The optimum temperature was 40°C at pH 7.0. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Pichia alcohol oxidase were around 8.0 and 6.0, respectively, and their optimum temperatures were 50 and 45°C, respectively, at pH 7.0. The apparent Km for glycolaldehyde was found to be 83.3 mm. For the accumulation of glyoxal, addition of catalase was effective, and a higher amount of glyoxal was obtained at a much lower temperature than the optimum for the alcohol oxidase. When 0.1 m ethylene glycol and glycolaldehyde were incubated with 80 units of the Pichia enzyme at 10°C, both substrates were almost completely converted to glyoxal after 10 and 3h of incubation, respectively.

Purification and some properties of cholesterol oxidase stable in detergents from γ-proteobacterium Y-134

Cholesterol oxidase (CHO) with high stability in detergents was found from an isolated strain, Y-134, belonging to the gamma-subclass of Proteobacteria. CHO production reached its maximum by incubation at 30 degrees C for 12 d. It was purified from cell-free extract prepared by mixing the cells with 0.4% Triton X-100. The absorption spectrum of the purified enzyme exhibited maxima at 274 and 410 nm, and a shoulder at 330 nm. The molecular mass was 115 kDa with two identical subunits of 58 kDa. The enzyme oxidized cholest-5-en-3beta-ol (cholesterol) and 5alpha-cholestan-3beta-ol (dihydrocholesterol) at a high reaction rate, and the K(m) value for cholesterol was 65 microM. The stability of the enzyme was higher than other CHOs in nonionic detergents with high values of hydrophilelipophile balance (HLB) such as Triton X-450 and sodium cholate. NH2-terminal sequence analysis showed a high similarity to CHO from Burkholderia cepacia, but not to CHOs from Streptomyces or Brevibacterium.

Primary structure determination of mono- and diacylglycerol lipase from Penicillium camembertii

The complete amino acid sequence of mono‐ and diacylglycerol lipase from Penicillium camembertii was determined. This lipase has a single polypeptide chain consisting of 276 amino acid residues with two disulfide linkages. The primary structure was revealed by sequencing the digests of the intact and S‐pyridylethylated proteins by trypsin, endoproteinase Lys‐C and V8 protease. The two‐dimensional electrophoresis was also carried out to confirm the internal sequence. The catalytic triad of this lipase was Ser, Asp and His, and one potential N‐glycosylation site was also revealed.

Purification and characterization of an L-amino acid oxidase from Pseudomonas sp. AIU 813.

An L-amino acid oxidase was found from a newly isolated strain, Pseudomonas sp. AIU 813. This enzyme was remarkably induced by incubation with L-lysine as a nitrogen source, and efficiently purified using an affinity chromatography with L-lysine as ligand. The enzyme oxidized L-lysine, L-ornithine and L-arginine, but not other L-amino acids and d-amino acids. The oxidase activity for L-lysine was detected in a wide pH range, and its optimal was pH 7.0. In contrast, the oxidase activity for L-ornithine and L-arginine was not shown in acidic region from pH 6.5, and optimal pH for both substrates was 9.0. The enzyme was a flavoprotein and composed of two identical subunits with molecular mass of 54.5 kDa. The N-terminal amino acid sequence was similar to that of putative flavin-containing amine oxidase and putative tryptophan 2-monooxygenase, but not to that of L-amino acid oxidases.

A Simple Enzymatic Method for Production of a Wide Variety of D-Amino Acids Using L-Amino Acid Oxidase from Rhodococcus sp. AIU Z-35-1

A simple enzymatic method for production of a wide variety of D-amino acids was developed by kinetic resolution of DL-amino acids using L-amino acid oxidase (L-AAO) with broad substrate specificity from Rhodococcus sp. AIU Z-35-1. The optimum pH of the L-AAO reaction was classified into three groups depending on the L-amino acids as substrate, and their respective activities between pH 5.5 and 8.5 accounted for more than 60% of the optimum activity. The enzyme was stable in the range from pH 6.0 to 8.0, and approximately 80% of the enzyme activity remained after incubation at 40°C for 60 min at pH 7.0. D-Amino acids such as D-citrulline, D-glutamine, D-homoserine or D-arginine, which are not produced by D-aminoacylases or D-hydantoinases, were produced from the racemic mixture within a 24-hr reaction at 30°C and pH 7.0. Thus, the present method using L-AAO was versatile for production of a wide variety of D-amino acids.

A new enzymatic method for glycolaldehyde production from ethylene glycol

Abstract A new enzymatic method for glycolaldehyde production from ethylene glycol was investigated using alcohol oxidase from Pichia pastoris or glycerol oxidase from Aspergillus japonicus . Both alcohol and glycerol oxidases oxidize ethylene glycol to glyoxal via glycolaldehyde, but glycolaldehyde was remarkably accumulated using a high concentration of ethylene glycol. The glycolaldehyde formation was also affected by buffer species and reaction pH. Under the optimum conditions, 0.92 or 0.97 M glycolaldehyde was formed from 1.0 M ethylene glycol using alcohol oxidase or glycerol oxidase. This enzymatic method was superior to the chemical method in terms of conversion yields and selectivity of glycolaldehyde.

A method for glyoxylic acid production using cells of Alcaligenes sp. GOX373

Abstract A microorganism capable of producing glyoxylic acid from glycolic acid was obtained by enrichment culture with medium containing 1,2-propanediol. It was identified as a member of the genus Alcaligenes . The maximal activity of the enzyme responsible for glyoxylic acid production by this strain was obtained by culturing at pH 5–6 for 3 days. The optimum conditions of cell-reaction for accumulating high concentration of glyoxylic acid were investigated using lyophilized cells of 3 day cultivates: Approximately 0.65 M glyoxylic acid was produced from 1.0 M glycolic acid by incubating at 20°C for 15 days in 1.0 M TES-NaOH buffer, pH 7.0. In addition, the production of glyoxylic acid was improved to 0.95 M by keeping the reaction pH at neutral region. The method proposed here is more advantageous for glyoxylic acid production than enzymatic methods.

A rapid enzymatic assay for total blood polyamines

We have designed a rapid, reliable enzymatic assay for total blood polyamines, as based on the combination of substrate specificity of polyamine oxidase (PAO) from Aspergillus terreus and putrescine oxidase (PUO) from Micrococcus rubens. Quinone dye, derived from hydrogen peroxide generated in the oxidation reaction, is measured spectrophotometrically at 555 nm, and total amounts of putrescine (Put), cadaverine (Cad), spermidine (Spd), and spermine (Spm) can be readily determined. The minimum detection limit is about 0.4 mumol/L whole blood. Bilirubin, hemoglobin, reducing substances, and anticoagulants show no apparent interference. Analytical recovery averaged 98.5%. Within-run and between-day precisions ranged from 1.02% (61.04 mumol/L) to 2.84% (13.07) and 1.54% (50.67) to 3.27% (14.74), respectively. Results obtained with this method and those by high-performance liquid chromatography (HPLC) (r = 0.955) or the enzymatic differential method (r = 0.944) correlated well. In our opinion, this method is superior to other assays being used to determine blood polyamines.

An enzymatic differential assay for urinary diamines, spermidine, and spermine

SummarySubsequent to the hydrolysis of urinary conjugated amines by heating with hydrochloric acid, free amines were isolated by cation-exchange chromatography. SPD and SPM in an aliquot of amine extract were first oxidized by PAO from Penicillium chrysogenum, producing PUT and hydrogen peroxide. DIAs, which consist of the initially present DIAs plus PUT produced by PAO, were subsequently oxidized by PUO from Micrococcus rubens, producing hydrogen peroxide. In an another aliquot of the amine extract DIAs and SPD were oxidized by PUO, producing hydrogen peroxide. Quinone dye, derived from hydrogen peroxide generated in each end-point reaction, was measured spectrophotometrically at 555 nm, and the amounts of the respective amines in urine were calculated. Significantly elevated levels of DIA, SPD, SPM, and an elevated DIA to SPD ratio were found in urine from 46 cancer patients, as compared to 34 normal control subjects. An increase in DIA and the ratio of DIA to SPD was found at clinical tumor stage I of the alimentary tract. The levels of DIA remained fairly constant and the ratio of DIA to SPD was consistently decreased with advancing clinical tumor stages. In patients who had undergone curative resection, there were greater decreasing rates (80% of cases for DIA and 80% for SPD) than in patients who had undergone noncurative resection (45.5% for DIA and 36.4% for SPD).