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A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.

a-Ionone, a-methylionone, and a-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from a-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-a-ionone, trans-3-hydroxy-a-ionone, and 3-oxo-a-ionone. 2,3-Dehydro-a-ionone, 3,4-dehydro-o-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from a-methylionone and a-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for a-ionones and elucidation of stereochemical features of the bioconversion. lonones and their derivatives are widely distributed in nature. They are important constituents of many kinds of essential oils and are presumably generated from carot-* Corresponding author.

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Wehave been studying the bioconversion of terpenes by fungi. Terpenes containing a cyclohexane ring in their molecules, such as ionones,1 2) isophorone3) and oxoisophorone,4) were found to be converted into oxidative or reductive compounds by Aspergillus niger JTS 191. It was apparent that the main conversion reaction in the furigus was regio-and stereospecific hydroxylation. There have been reports5~7) on the microbial conversion of acyclic monoterpenes with one or more double bonds. However, there have been very few on the microbial conversion of /?-myrcene, an acyclic monoterpene.As /?-myrcene has conjugated double bonds and a gemdimethyl terminal in its molecule and it is a basic material for synthetic terpenes,8} we were interested in the conversion of this compound by the fungus. A. niger JTS 1911} was cultured in a 3-1 Erlenmeyer flask containing one liter of medium as previously described.4) To the resulting culture broth, one ml of /?-myrcene was added as the substrate. The culture was further incubated at 28°C with shaking. Ten ml of the broth was removed each day for analysis of the substrate and conversion products. Gas liquid chromatography (GLC) was performed as previously described,4) except that the oven temperature was programmed from 60 to 160°C (2°C/min).The total content of conversion products in the neutral fraction corresponded to less than 10% of the initially added substrate, for the incubation.As the contents of substrate and conversion products in the mediumwere low, those in the mycelia were analyzed.Mycelial pellets were separated from the culture broth by suction filtration and then weighed. The substrate in the mycelia was extracted twice with 100mlof ethyl acetate. The pooled extract was analyzed by GLCwithout concentration, to prevent loss due to vaporization of the substrate.As shown in Fig.

Manystudies have been reported on the preparation of chiral compounds from achiral ones with enzymes or microorganisms. Wehave studied the conversion of terpenes by Aspergillus niger and reported that the main reaction was regio-and stereoselective hydroxylation.1'2* It has been reported that 2,6,6-trimethyl-2-cyclohexene-1 ,4-dione (4-oxoisophorone) is asymmetrically reduced to (6jR)-2,2,6-trimethylcyclohexane-l ,4-dione by yeasts3) and actinomycetes.4) We have reported that the conversion of isophorone and ionones by Aspergillus niger afforded chiral oxidation products.5) Therefore, it is interesting as to whether the main reaction is reduction or oxidation on conversion of 4-oxoisophorone by the fungus. This paper deals with identification of the conversion products of 4-oxoisophorone with A. niger, the conversion pathway and stereochemical aspects of the conversion. Spores of Aspergillus niger JTS 191 (4x lO7) were inoculated into a 3-1 Erlenmeyer flask containing one liter of medium consisting of 3% sucrose, 0.2% NaNO3, 0.1% K2HPO4, 0.05% KC1, 0.05% MgSO4à"7H2O, 0.1% yeast extract and distilled water (pH 7.2). Cultivation was carried out at 28°C for 48hr under gyratory shaking at 200rpm. To the resulting culture broth, oneml of 4

Thermostable purine nucleoside phosphorylases, PUNPI and PUNPII, have been purified from Bacillus stearothermophilus JTS 859. The characterization of PUNPIwas reported previously. [Hori et al, Agric. Biol Chem. 53, 2205] PUNPII had a molecular weight of 113,000, consisting of 4 identical subunits (Mw28,000). The isoelectric point was 5.3. The Michaelis constants for inosine, guanosine, and adenosine were 0.22, 0.34, and 0.075 mM, respectively. The optimal temperature of the reaction was 70°C. The enzyme was stable at 70°C. Although other reported purine nucleoside phosphorylases were SH-enzymes, PUNPIIwas not a SH-enzymebecause the enzyme reaction was not inhibited by PCMBand iodoacetic acid, the optimal pH of the enzyme reaction was from 7.0 to ll.0, and the enzyme did not contain cysteine.PUNPII and PUNPI were different in several points. Not PUNPI but PUNPII could catalyze the phosphorolysis of adenosine. Specific activity of PUNPI and II for inosine were 405 and 50.6|imol/min/mg protein at 60°C, respectively.PUNPI was stable at 80°C. PUNPII was stable at 70 C, but was denatured at 80 C.Purine nucleoside phosphorylase (PUNP) (EC. 2.4.2. 1) catalyzes the reversible phosphorylation of ribonucleoside and deoxyribonucleoside derivatives of hypoxanthine, guanine, and xanthine as follows: purine nucleoside + phosphate <± purine base+pentose-1 -phosphate Since the substrate specificity of PUNPis not strict, the enzymehas been used to prepare purine nucleoside analogues, such as hypoxanthine arabinoside, virazole, and 6-mercaptopurine nucleoside.1~4)Wehave studied the practical application of ribosyl transfer reaction by thermostable PUNP and pyrimidine nucleoside phosphorylase (PYNP) (EC 2.4.2.2) at high temperatures.5) Wereported the purification and the characterization of a thermostable PUNP, PUNPI from Bacillus stearothermophilus JTS 859, and also the existence of another PUNP, PUNPII, in our previous paper.6) This paper 3219 describes the purification and the characterization of PUNPII. These two enzymes were different in their substrate specificity, specific activity, and thermal stability. Not PUNPI but PUNPII could catalyze the phosphorolysis of adenosine. The specific activities of PUNPI and II for insoine were 405 and 50.6 /xmol/min/mg protein at 60°C, respectively. PUNPI was stable at 80°C. PUNPII was stable at 70°C but was denatured at 80°C. Materials and MethodsMicroorganism. Bacillus stearothermophilus JTS 859, which produced thermostable purine nucleoside phosphorylases, was used as the enzyme source.Chemicals. Inosine was purchased from Yamasa Chemicals (Tokyo).Peptone A and yeast extract were purchased from Kyokuto Co. (Tokyo).Culture conditions. Medium,containing (g/1 of water) Peptone A 20, yeast extract 10, glucose 3, inosine 1, and

Manythermophiles, which can grow at 65°C, were examined as to their ability to produce 5-methyluridine from inosine and thymine (5-methyluracil) in the presence of phosphate and cells as enzymesources. Bacillus stearothermophilus JTS 859 was selected as a strain that synthesized 5-methyluridine efficiently. The reaction is supposed to be carried out by a combination of a thermostable purine nucleoside phosphorylase and a thermostable pyrimidine nucleoside phosphorylase. Their halflives were 7200hr and 400hr at 63 C, and 148hr and 14hr at 70 C, respectively.Nucleosides such as inosine, adenosine, uridine and cytidine are produced by a fermentation method, but thymidine has not been produced by such a method. Thymidine is practically produced by hydrolysis and separation from deoxyribonucleic acid derived from natural sources such as milt. As an alternative to this method, we developed a method for the practical production of thymidine from non-natural sources. Wetried to synthesize 5-methyluridine (5MU) from thymine (5-methyluracil) and inosine enzymatically at first by means of the ribosyl transfer reaction, as shown in the following chemical equations, and to chemically reduce the hydroxy group at the 2'-position on the ribose ring of 5MUto yield thymidine.Inosine + Phosphate-åº Ribose-1 -phosphate + Hypoxanthine (1)Ribose-1 -phosphate + Thymine-åº 5-Methyluridine + PhosphateWeselected inosine as a ribose donor because it it produced abundantly in industrial fermentations. This paper describes the synthesis of 5-methyluridine using thermostable enzymes produced by a thermophile. 197The ribosyl transfer reaction has been studied for a long time.1~4) This reaction is carried out by purine nucleoside phosphorylase, pyrimidine nucleoside phosphorylase, uridine phosphorylase, thymidine phosphorylase and a combination of these enzymes. The substrate specificities of these enzymes are not strict, therefore, the ribosyl transfer reaction has been applied for the synthesis of nucleoside analogues, 5-fluorouridine, 5-bromouridine and so on.5~7) It is necessary that the enzymes used for practical application should be stable for a long time. Enzymes derived from mesophiles have been used for practical application.As for thermostable enzymes derived from thermophiles, only pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus NCA10 has been reported.8)The application of thermostable enzymes has several advantages.9) The reaction can be carried out at high temperature for a long time and the reaction rate is also high. The higher solubility of the reactants allows operation with higher concentrations of reactants. The solubility of 5MUis 83g/1 at 25°C and above 500 g/1 at 63°C. Thermostable enzyme reactors are resistant to bacterial contamination due to the high operation temperature. This is impor-