A new process for (6S)-tetrahydrofolate production from dihydrofolate was designed that used dihydrofolate reductase and an NADPH regeneration system. Glucose dehydrogenase from Gluconobacter scleroides KY3613 was used for recycling of the cofactor. The reaction mixture contained 200 mM dihydrofolate, 220 mM glucose, 2 mM NADP, 14.4 U/ml dihydrofolate reductase, and 14.4 U/ml Glucose dehydrogenase, and the reaction was complete after incubation at pH 8.0, and 40 degrees C for 2.5 hr. With (6S)-tetrahydrofolate as the starting material, l-leucovorin was synthesized via a methenyl derivative. The purity of the l-leucovorin was 100%, and its diastereomeric purity was greater than 99.5% d.e. as the (6S)-form.

Poly(ethyleneglyco1)-bound NAD (PEG-NAD) was covalently linked to Thermus thermophilus malate dehydrogendse with a bifunctional reagent, 3,3'-(1,6-dioxo-1,6-hexanediyl)bis-2-thiazolidinethione. The covalently linked malate-dehydrogenase -PEG -NAD complex (MDH-PEG-NAD) was purified by DEAE-Sephadex column chromatography to remove unbound PEG-NAD, and fractionated by blue-Sepharose column chromatography into four preparations: MDH-PEG-NAD I, MDH-PEG-NAD 11, MDH-PEG-NAD 111 and MDH-PEG-NAD IV. The average numbers of NAD moieties covalently bound per subunit of MDH-PEG-NAD I, MDH-PEG-NAD 11, MDH-PEG-NAD I11 and MDH-PEG-NAD IV were 1.2, 1.2, 0.8 and 0.5, respectively, and the values were confirmed by sodium dodecyl sulfate/polydcrylamide gel electrophoresis. 60 -80% bound NAD moiety of these preparations of MDH-PEG-NAD was reduced by the enzyme moiety in the presence of Lmalate, and the specific activity of the enzyme moiety of the preparations was more than 80% that of the native enzyme.MDH-PEG-NAD I has the following properties. The K , value for exogeneous NAD is three times that of the native enzyme. The coenzyme activity of its NAD moiety is 20-40% that of native NAD for alcohol and lactate dehydrogenases. The complex catalyzes the oxidation of L-malate in the presence of the redox system of 5-ethylphenazinium ethyl sulfate and a tetrazolium salt with a rate constant of 0.11 s-l. The coenzyme moiety of the complex can also be recycled by coupled reactions of the active site of the same complex and alcohol dehydrogenase. These results indicate that MDH-PEG-NAD works as an NAD(H)-regeneration unit for coupled reactions.A covalently linked dehydrogenase-NAD complex is an attractive catalytic unit if the bound NAD can be used by other enzymes as well as by itself. Such a complex serves as an NAD(H)-regeneration unit in enzyme reactors and also provides many interesting questions about the effects of fixing a readily dissociable coenzyme in the vicinity of an enzyme. Several efforts have been made for this purpose [l -31 but only the alcohol-dehydrogenase -NAD complex, reported by MAnsson et al. [2], has been clearly demonstrated to have covalently bound NAD. As for the complex, recycling of the bound NAD by lactate or malate dehydrogenase [4] and kinetic properties of the alcohol dehydrogenase activity of the complex [5] have also been investigated.We have prepared poly(ethyleneglyco1)-bound NAD (PEG-NAD) as a macromolecular NAD derivative for enzyme reactors [6, 71. PEG-NAD has a unique structure in that one NAD molecule and one amino group are linked with Correspondence to I. Urabe,

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Effects of aliphatic esters, aldehydes and some impurities, as monovinyl and divinyl acetylene which exist normally in vinyl acetate, on the polymerization of vinyl acetate were studied. Esters and saturated aldehydes such as methyl acetate, ethyl acetate, isopropyl acetate and dimethyl oxalate etc., and acetaldehyde and butyraldehyde act only as transfer agent and the values of the transfer constants on these substances were determined. Unsaturated aldehyde such as crotonaldehyde and also benzaldehyde, and monovinyl and divinyl acetylene act as retarder. The several reaction constants relating the retarding effect were obtained. ZUSAMMENFASSUNG:Es wurde der EinfluB von aliphatischen Estern, Aldehyden und Verunreinigungen, die imvinylacetat iiblicherweise vorhanden sindwie Monovinylacetylen und Divinylacetylen a d die Polymerisation von Vinylacetat untersucht. Ester und gesattigte aliphatische Aldehyde, wie z. B. Methylacetat, Athylacetat, Isopropylacetat, Dimethyloxalat usw., Acetaldehyd und Butyraldehyd, wirken nur als ubertrager. Ihre tfbertragungskonstanten wurden bestimmt. Ungesattigte Aldehyde, wie Crotonaldehyd, aber auch Benzaldehyd, ferner Mono-und Divinylacetylen wirken als Verzogerer. Die Polymerisationsgeschwindigkeit von Vinylacetat in Gegenwdrt solcher Verzogerer wurde nach der Gleichung von KICE analysicrt, und die verschiedenen Geschwindigkeitskonstanten wprden bestimmt. *) This report is a summarization of several of the author' papers which appear in the 1) I. SAKURADA and R. INOUE, Chem. High Polymers (Japan) 7 (1950) 211. Industrially pure vinyl acetate, made by the KURASAIKI RAYON COMPANY, was used in all tests. The monomer was washed with aqueous NaHSO, and water. It was then dried and rectified. The fraction boiling at 72.5"C. was partially polymerized, and the unpoly-5) A. J. BUSELLI, M. K. LINDEMANN, and C. E. BLADES, J. Polymer Sci. 28 (1958) 485. 6) T. OHSUGI, Chem. High Polymers [Japan] 6 (1949) 460. 7) 0. L. WHEELER, S. L. ERNST, and R. N. CROZIER, J. Polymer Sci. 8 (1952) 409. 8 ) 0. L. WHEELER, E. LAVIN, and R. N. CROZIER, J. Polymer Sci. 9 (1952) 157. 9) M. YANO and M. MATSUMOTO, J. chem. SOC. Japan; Ind. Chem. Sect. (Kogyo Kagaku

The intrinsic viscosity of the sulfate of aminoacetalized polyvinyl alcohol (Am‐PVA) in N/10 K2SO4 solution has been studied to disclose the influence of the number of ionic substitutents on the intrinsic viscosity of the polymer. The number of ionic substitutents is easily changed by controlling the degree of aminoacetalization, which is determined by a selection of the conditions of aminoacetalization. β‐Cyclohexylamino n‐butyraldehyde dimethylacetal, CH3(C6H11NH)CH·CH2·CH(OCH3)2, and β‐cyclohexylamino‐n‐propylaldehyde dimethylacetal, (C6H11NH)CH2·CH2·CH(OCH3)2, were used as aminoacetals. The aminoacetalization reaction was carried out in a system of aminoacetal, polyvinyl alcohol (PVA), sulfuric acid, and water. The reaction product was purified by dialysis, and the degree of aminoacetalization was estimated from the nitrogen content as given by the semimicro Kjeldahl method. The PVA was used unfractionated and its degree of polymerization, as determined by the viscosity method, was found to be between 900 and 2400. The solution viscosity was measured in an Ostwald viscometer designed to reduce the kinetic energy term for which a correction was made. It was first ascertained that the viscosity in N/10 K2SO4 solution was not influenced by the rate of shear in the range of our experiment. The intrinsic viscosity was found from linear extrapolation, a linear relation between ηsp and c, being well realized in the concentration range from 1.5 to 9 g./l. The intrinsic viscosity increased with the degree of aminoacetalization in an S‐shaped fashion. Compared to the same degree of aminoacetalization, Sakurada‐Houwink's equation [η] = KPa was found to hold well at each degree of aminoacetalization, and the index a was found to increase with the degree of aminoacetalization. The volume expansion α3 of the polymer in the solution was estimated from [η]M/[η]θM0 according to Flory's theory, in which M is the molecular weight of the sulfate of Am‐PVA, M0 the molecular weight of each original PVA, [η] the intrinsic viscosity of the sulfate of Am‐PVA, and [η]θ is the mean intrinsic viscosity of each original PVA in Flory's unperturbated state. The viscosity in a mixture of acetone (35.2%) and water (64.8%) was used for [η]θ, its value being calculated from [η]θ = 1.06 P1/2, having reference to Sakurada's report. Then (α5 − α2/M1/2) was calculated and plotted against the degree of aminoacetalization x. It was found that these points fall on one curve, independently of the degree of polymerization of the original PVA. When [(α5 − α3)/M1/2]x − [(α5 − α3)/M1/2]0 was plotted against x in a log‐log scale (we are subtracting the value [((α5 − α3)/M1/2]0 for PVA) it was found that a linear relation between them holds well independently of the degree of polymerization of PVA. The empirical formula [(α5 − α3)/M1/2]x − [(α5 − α3)/M1/2]0 = Axn was thus obtained. In both Am‐PVA's n is 1.45, but A differs slightly. When the ionic strength is constant, {[(α5 − α3)/M1/2]x − [(α5 − α3)/M1/2]0} should be proportional to x2/(M/r 20)3/2...

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Chemoenzymic large-scale synthesis of the calcium salt of (6s) -5-formyltetrahydrofolic acid [ (-)leucovorin, (6S)-5] was achieved from folic acid 1 via (6s)-tetrahydrofolic acid [(6S)-3] by using dihydrofolate reductase (DH FR) produced by Zscherichia coli, 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 scleroides. 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) -