Dextrin dextranase (EC 2.4.1.2), which synthesizes dextran from maltooligosaccharides, was purified from Acetobacter capsulatus ATCC11894 cells by n-butanol extraction, Phenyl-Toyopearl chromatography, and Toyopearl HW-65S gel filtration. The molecular weight of this enzyme was 300, 000 by SDS-gradient-PAGE. The optimum temperature and pH were 37-45°C and 4.0-4.2. The enzyme was stable at below 45°C and at pH 3.5-5.2 for 30 min treatment. This enzyme synthesized dextran from maltooligosaccharides except for maltose, from which a little glucose and panose were produced. On the reaction with G4, the enzyme synthesized dextran and also maltose, panose, and a small amount of glucose as byproducts. Acetobacter dextran synthesized from G4 was compared with commercial Leuconostoc dextran by dextranase digestion, and it was suggested that these structures were different from each other.

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α-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORE) consisted of 499 amino acids with a molecular weight of 55, 000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3' non-coding regions, respectively, so far determined. The α-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active α-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.

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Glucose isomerase from Streptomyces phaeochromogenes was purified from a commerical preparation, Swetase, by DEAE-cellulose, Bio-Gel A-0.5m, and hydroxyapatite column chromatographies. It was found to be 2 fractions; F-A, not adsorbed on hydroxyapatite and F-B, adsorbed on hydroxyapatite. They were homogeneous in ordinary and SDS-PAGE and had similarities in some enzymatic and physico-chemical properties. The differences, however, were found in the N-terminal amino acid, which was only serine for F-A while it was serine and alanine for F-B, and also in their peptide mapping patterns of digests with trypsin, Achromobacter protease I, and cyanogen bromide. The results suggest that glucose isomerase from S.phaeochromogenes was composed of the two kinds of isozymes and that each of isozymes was a tetramer constituted of non-identical subunits.

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Fluorescent labeling of uronic acid with water-soluble carbodiimide, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), was examined. The optimum concentration of EDC was 100-200 mM and pH 4.75 was essential for the EDC activation of glucuronic acid. To form the amide linkage between the activated intermediate forms and a nucleophilic amino compound, a fluorescent reagent EDAN (N-1-ethylene-diaminonaphthalene) was used. Column chromatography with Bio-Gel P-2 was effective to remove excess EDAN from the fluorescent EDAN derivative of glucuronic acid. About 0.62-4.3μg/ml of glucuronic acid derivative gave a linear correlation with the relative fluorescence intensity. Although this method could be applicable to various lactones, mutual separation of these fluorescent derivatives by high performance liquid chromatography requires further examination.

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A host-vector system was established in Lactobacillus helveticus with β-galactosidase activity as a selection marker. Plasmid pBG10 was constructed by joining the β-galactosidase gene from L. bulgaricus, the promoter region of the erythromycin resistance gene from pAMβ1, the replication region of pBR329, and the replication region the L. helveticus cryptic plasmid pLJ1. L. helveticus SBT2195 (Lacmutant), transformed with pBG10, was selected on skim milk plates. The structural gene of α-amylase (1536bp) from Bacillus licheniformis, inserted downstream of the promoter region of the erythromycin resistance gene of pBG10, was expressed in L. helveticus SBT2195. Plasmid pBG10 is a food-grade and expression vector in L. helveticus.

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Inhibition from binding of Cholera toxin (CT) to Chinese hamster ovary (CHO) -k1 cells and ganglioside G_M1 by lactoferrin (Lf) and κ-casein glycomacropeptide (GMP) from cow's milk was examined. Both Lf and GMP effectively reduced the CT-derived morphological changes in CHO-K1 cells. The competitive binding assay demonstrated that both Lf and GMP inhibited the binding of CT to G_M1, although their affinity for CT was lower than that of G_M1. The inhibitory effect of Lf and GMP seemed to be attributed to their terminal sialic acid, although the sugar chain sequence only partially fitted to the CT-receptor.

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1, 2-Diaminobenzimidazoles 2 were synthesized by N-amination of 2-aminobenzimidazoles 1 with hydroxylamine-O-sulphonic acid. Substituted 1-alkyl and 1-alkylarylbenzimidazoles 3 were prepared from various benzimidazoles by alkylating with the corresponding alkyl halides. As an example, 1-(4-chlorobenzyl)-5, 6-dimethyl-5, 6-dimethylbenzimidazole (3₄) was N-aminated with O-(mesitylenesulphonyl)-hydroxyl-amine to give 5, 6-dimethyl-1-(4-chlorobenzyl)-3-aminobenzimidazolium mesitylenesulphonate (6). Compounds 4, 7 and 5 are derivatives of 1, 2-(5-nitro-2-furfurylideneamino)-benzimidazoles and were synthesized by the carbonyl-amine condensation of 5-nitro-2-furaldehyde with appropriate 1, 2-diaminobenzimidazoles 2 and 2-aminobenzimidazoles 1 and 3, respectively. An attempt to prepare the derivative of 3-(5-nitro-2-furfurylidenamino)-benzimidazolium mesitylenesulphonate (8) from compounds 6 was unsuccessful. The antimicrobial activitites of the above compounds were screened against different strains of bacteria and fungi. The general structure-activity relationships of tested benzimidazoles 3-7 were determined.

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The cDNA encoding Taka-amylase A (EC.3.2.1.1, TAA) was isolated to identify functional amino acid residues of TAA by protein engineering. The putative catalytic active-site residues and the substrate binding residue of TAA were altered by site-directed mutagenesis : aspartic acid-206, glutamic acid-230, aspartic acid-297, and lysine-209 were replaced with asparagine or glutamic acid, glutamine or aspartic acid, asparagine or glutamic acid, and phenylalanine or arginine, respectively. Saccharomyces cerevisiae strain YPH 250 was transformed with the expression plasmids containing the altered cDNA of the TAA gene. All the transformants with an expression vector containing the altered cDNA produced mutant TAAs that cross-reacted with the TAA antibody. The mutant TAA with alteration of Asp206, Glu230, or Asp297 in the putative catalytic site had no α-amylase activity, while that with alteration of Lys209 in the putative binding site to Arg or Phe had reduced activity.

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A cDNA sequence coding for feline interferon has been cloned for the first time by screening a cDNA library constructed using Okayama-Berg vector and mRNA derived from the feline cells (LAS-I) induced by TPA (12-o-tetradecanoylphorbor 13-acetate) for the ability of transient expression to produce feline interferon in COS1 monkey cells. The amino acid sequence, deduced from the nucleotide sequence by comparing it with the sequences of other mammalian IFNs, consists of 171 amino acids with 6 cysteins and an N-glycosylation site at amino acid position 79, and has about 60% homology to human IFN alpha 1. The interferon was partially purified through Blue Sepharose, and its molecular weight was estimated to be 2.4×10⁴. The antiviral activity was acid stable, and glycosylation was suggested.

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The structures of sugar chains of the glycoproteins from the microsomal fraction of developing castor bean endosperms have been analyze. The structural analyses were done by a fluoresecence method combined with component analysis, exoglycosidase digestions, partial acetolysis, Smith degradation, and 1^H-NMR spectroscopy. The estimated structures fell into three categories; the first was oligomannose-type, the second xylomannose-type, the third complex-type. Among these oligosaccharides, Man₃Fuc₁Xyl₁GlcNAc₂ (M3FX) and Man₆GlcNAc₂ (M6B) were the major structures. The structures of Man₄GlcNAc₂ (M4C) and Man₄Xyl₁GlcNAc₂ (M4X) have also been found in the microsomal glycoproteins of the developing bean endosperms. These results could indicate that the structures of M4C, M4X, and M3FX are formed in the stage of sugar chain processing in the mirosomal fraction, in which oligomannose-type sugar chains are modified into complex-type ones by several kinds of processing enzymes.

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The structure of a 1.6-kb SphI-HindIII DNA sequence necessary and sufficient for the replication of a 8.6-kb plasmid pLS11 of Bacillus subtilis IFO 3022, which is responsible for γ-polyglutamate production, has been characterized by using a trimethoprim (Tmp)-resistance gene derived form B. subtilis TTK24 chromosomal DNA as a selective marker. The 1.6-kb DNA sequence contains a rep gene encoding the protein (333 amino acids) essential for initiation of replication and a possible origin of replication. The predicted REP protein of pLS11 has an overall homology with the REP proteins of pUH1 (74.8% identity), pBAA1 (92.8%), and pFTB14 (78.7%) in Bacillus spp., pLP1 (42.1%) and pLAB1000 (36.3%) in Lactobacillus spp., and pUB110 (35.3%) and pC194 (37.4) in Staphylococcus aureus, but has not any similarity with the REP protein of the staphylococcal plasmid pT181.

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We have developed a genetic transformation system for a freeze-tolerant yeast Kluyveromyces thermotolerans. K. thermotolerans spheroplasts could be transformed with a YPp-type vector containing an autonomously replicating (ARS) of Saccharomyces cerevisiae. However, transformation with a Yep-type vector containing a replication origin of S. cerevisiae 2μM DNA was not successful. The cycloheximide resistance gene (RIM-C) of Candida maltosa and the URA3 gene of S. cerevisiae were successfully used to transform a prototrophic strain of K. thermotolerans and an Ura-mutant of this yeast isolated in this study, respectively. Transformation was also possible by using intact cells treated with lithiukm salts or thiol compounds. The YRp-type vectors were maintained as plasmids in the transformants under selective conditions. This is the first report of successful transformation of K. thermotolerans.

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An Actinomycete, strain K-128, which was isolated from soil, formed a transfer product when the strain was cultured in a medium containing both stevioside and curdlan. The transfer product in culture broth was separated by DIAION HP-20 column and TOYOPEARL HW-40F column chromatographies. From structural studies, the transfer product was identified as C13-O-β-6²-β-glucosylsophorosyl-C19-O-β-glucopyranosyl steviol. This product was the first to be obtained by a culture of an Actinomycete.

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A thermostable carboxyl esterase of Bacillus stearothermophilus excreted by recombinant B. brevis was purified to homogeneity using column chromatographies on DEAE Bio-Gel A, Sephacryl S-200HR, and Mono Q. The purified enzyme had a molecular weight of 29, 000. The optimum pH at 37°C was 7.5. The esterase had higher activity toward triglycerides with short-chain fatty acids than with long-chain ones. The activity of esterase was completely or significantly inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, iodoacetamide, and p-chloromercuribenzoic acid, while it was slightly stimulated in the presence of 2-mercaptoethanol. This observation suggests that that the enzyme is a serine enzyme having a sulfhydryl group for expressing the enzyme activity. The activation energy for inactivation of the enzyme was estimated to be 131 kcal·mol^-1. The kinetic parameters of this enzyme with p-nitrophenyl butyrate were measured : K_m was 0.132 mM, and V_max was 4.8 μmol·(mg protein)^-1·min<-1>.

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The galactosyl transfer reaction to branched cyclodextrins (CDs) was investigated using lactose as a donor substrate and branched CDs as acceptors by various β-galactosidases. Bacillus circulans β-galactosidase synthesized galactosyl transfer products to branched CDs, of which the galactose residues were linked at side chains of branched CDs, not directly at CD rings. Aspergillus oryzae and Penicillium multicolor β-galactosidases also produced derivatives galactosylated at side chains of branched CDs. The structures of main transgalactosylation products of branched CDs by these β-galactosidases seem to be different from those by B. circulans β-galactosidase, judging from the retention times on high performance liquid chromatography.

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Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0. Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DEP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodyecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5-13.0 at 37°C the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4-12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of microbial serine protease, although alanine of the NH₂-terminal amino acid was deleted.

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Pyrophosphate : D-fructose 6-phosphate 1-phosphotransferase was purified to homogeneity from rice seedlings. The enzyme had an apparent molecular weight of 100, 000 and consisted of a single protein. The pH optimum for the forward and reverse reactions was 8.1 and 7.9, respectively. Fructose-2, 6-bisphosphate (Fru-2, 6-P₂) shifted the pH optimu for both the forward and reverse reactions to slightly acidic. Half-maximum activation of the enzyme for Fru-2, 6-P₂ in the forward and reverse reactions was observed at 16 and 520nM, respectively. The enzyme had normal Michaelis-Menten kinetics in both the presence and absence of Fru-2, 6-P₂. Fru-2, 6-P₂-stimulated the enzyme activity by increasing V_max for Fru-6-P and PPi, and by raising the affinity for Fru-6-P, Fru-1, 6-P₂, and PPi. At a saturated concentration of Fru-2, 6-P₂, Pi strongly inhibited the enzyme in the forward reaction. The Dixon plots with Pi indicated that the inhibition behavior was of the mixed type with respect to both Fru-6-P and PPi with K_i of 1.46 and 1.53 mM, respectively.

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Twenty-four benzaldehyde O-alkyloximes having a carboxylic moiety a the α-position of the oxime-oxygen atom were prepared as abscisic acid (ABA) mimics. Their effects on some physiological and biochemical processes of selected plant species were investigated in comparison to those of ABA. The mimics exhibited ABA-like activity by inhibiting germination and α-amylase induction. They also induced stomatal closure coupled with a reduction in transpiration.

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The oxidation pathway of sulfite to sulfate in Thiobacillus thiooxidans JCM 7814 was investigated. Most of the activities of sulfite oxidation were in the membrane fractions of the cells. The addition of AMP (adenosine 5'-monophosphate) did not affect the activity. Two other reference strains of T. thiooxidans also gave the same results. Sulfite oxidation by T. denitrificans with an APS (adenosine 5'-phosphosulfate) pathway was evidently stimulated by the addition of AMP, while none of the effects occured upon the addition to T. novellus, which has an AMP-independent pathway. Although T. denitrificans produced APS from sulfite and AMP, T. thiooxidans JCM 7814 neither consumed AMP nor produced APS. T. thiooxidans JCM 7814 had sulfite dehydrogenase activity. These results showed that T. thiooxidans JCM 7814 has only the AMP-independent pathway. The presence of cytochromes a, b, and c was also shown from the difference spectrum of the membrane fraction prepared from T. thiooxidans JCM 7814 cells.

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Sexual aggregation involved in conjugative transfer of the Enterococcus faecalis plasmids pPD1 and pAD1 is enhanced by sex pheromones cPD2 and cAD1, respectively, which are excreted from recipient cells. PD78 (78kDa) were detectable on the surface of donors harboring pPD1 and pAD1, respectively, at the time of cell aggregation. The proteins PD78 and AD74 were purified and used to raise anti-PD78 and anti-AD74 antibodies. The antibodies blocked the sexual aggregation and the plasmid transfer, Anti-AD74 antibody reacted to both 153kDa proteins extracted from cPD1 and cAD1-induced donor cells after lysozyme digestion of cell wall. Pheromone-induced synthesis of PD78 and AD74, when both plasmids were present in the same cell, was independent.

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Molecular stability of chicken egg yolk immunoglobulin G (IgY) and that of rabbit IgG were compared by measuring antibody activities and conformational changes. Stability of rabbit IgG to acid denaturation was much higher than that of IgY. Conformation of the IgY molecule was readily changed in acidic conditions, resulting in a rapid loss of antibody activity. Much less stable natures of IgY to heat-treatment and guanidine-HCl denaturation than rabbit IgG were also observed. Differences in the structure between the two immunoglobulins that might participate in their different stability were inferred from their amino acid sequence data. Importance of the intramoleular disulfide linkage in the rabbit light chain and some other structural differences were suggested.

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Gourd seed inhibitors were purified in the following manner : gourd seeds were ground and extracted with 10 mM ammonium carbonate, pH 7.8. The extract was precipitated with 65-90% acetone and the acetone precipitates were gel filtered in a Cellulofine GCL-90-m column. Fractions of 3000 Da showing trypsin inhibitory activity were combined and purified further by ion exchange and reversed phase chromatographies. Three inhibitors, LLTI-I, II, and III were thus purified to homogeneity and the amino acid sequences of these inhibitors were : [table] The exact sequences are unique but very similar to proteinase inhibitors belonging to the squash family. Based on the sequence, it is assumed that the peptide bond (Arg-Ile) found in the three inhibitors is the reactive site for trypsin. The Ki values estimated for complexes of LLTI-I, II. and III with bovine trypsin were 3.6×10^-10M, and 3.0×10^-11M, respectively.

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Chitinase involved in plant self-defense was investigated by inoculation of yam (Dioscorea opposita Thunb) callus with autoclaved mycelia of Fusarium oxysporum Schl. f. sp. raphani Kend. et Snyd., ethylene, and chitin and chitosan oligosaccharides. The induction of chitinase was followed by measuring the enzyme activity, and the isozyme pattern was also analyzed by activity staining and immunoblotting after disc- and SDS-PAGE. Autoclaved Fusarium oxysporum induced acidic chitinases named E1 and E3 with MW 33, 500 in the callus, while ethylene and oligosaccharides of chitin and chitosan induced some chitinases more basic than E1 and E3, in addition to E1 and E3. These included large chitinases with MW 38, 000 and immunologically different chitinases from E1 and E3. In total, nine chitinase were induced. Isozyme patterns of chitinase induced were different among the elicitors used, although E1 and E3 were commonly induced in all cases. Oligosaccharides of chitin and chitosan showed different chain-length dependences on the induction of chitinase activity. In a series of chitin oligosaccharides, the induction time was more shortened by longer chain-length saccharide, although the highest levels of the activity were almost the same. In a series of chitosan oligosaccharides, the activity level was most induced by mono-saccharide, followed by hexa-, di-, tri-, tetra-, and hepta-saccharides, although the induction times were almost the same. In all cases, as the concentration of the elicitor increased, the induction time was shorter. The results of this study suggest that the plant cells response to various elicitors, and then produce different chitinase isozymes in a specific induction pattern.

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We studied the simplification of the usual colorimetric measurement of secondary amines, using nitrosation of secondary amines with KNO₂, degradation of KNO₂ with sulfamic acid, extraction of nitrosamines with CH₂Cl₂, and coloring of the nitrosamines. To skip the most time-consuming CH₂Cl₂ extraction step, the KNO₂ concentration for nitrosation was reduced from 60% to 400 mM. This modification enabled us not only to measure secondary amines more quickly and easily, but also to measure secondary amines yielding nirosamines hardly extractable with CH₂Cl₂.

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For efficient meiosis and sporulation, yeast cells must be starved for nitrogen, and only nonfermentable carbon sources may be present. To find how these nutritional signals regulate entry into meiosis, transcript levels from meiotic inducer genes (IME1 and IME2) were examined under various culture conditions. Our results suggest (1) that IME1 RNA levels are negatively regulated by cell divison arrest at G1 phase, (2) that the G1 arrest is not sufficient to induce IME2 transcription, and (3) that IME1 and IME2 transcriptions are inhibited by either nitrogen or glucose. Acetate was required for full expressions of IME1 and IME2 RNAs, and this induction required de novo protein synthesis. We have cloned SME2 and SME3 by screening genes which, when present in increased dosage, bypass the nitrogen repression of sporulation. RNA blot analysis suggests that SME2 and SME3 are positive transcriptional regulators for SGA1 (a late meiotic gene encoding glucoamylase) and IME1, respectively, and that transcript levels from SME2 and SME3 were also negatively regulated by cell division in a manner similar to that found for IME1. However, diploids homozygous for sme2 or sme3 disruption mutation sporulated normally like isogenic wild-type strains, indicating that SME2 and SME3 are dispensable for meiosis.

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A benzyl alcohol oxidase (BAO) was purified to homogeneity from Botrytis cinerea. The enzyme was found to have a molecular mass of 214kD with a trimeric structure, and optimal pH and temperature of 5.0 and 30°C, respectively. The enzyme activity was not sensitive to metal ions or to metal ion chelators, while thiol blocking reagents strongly inhibited BAO activity. Sulfur dioxide irreversibly inhibited the enzyme activity and the inhibitory effect of ethanol was weak and reversible. Benzyl alcohol was the most effective alcohol substrate for BAO. Para or meta monosubstituted benzyl alcohol with methyl or methoxy groups were good substrates. BAO also oxidized cinnamyl alcohol, furfuryl alcohol, and some terpenic alcohols with an alkenyl group near the reactive carbinol. Secondary alcohol, methanol and phenol were not substrates. Product inhibition studies suggested that benzaldehyde and benzyl alcohol were bound at different places to the active site. O₂ was the only electron acceptor identified and Botrytis cinerea benzyl alcohol oxidase was classified as EC 1.1.3.7. according to stoichiometrical studies. We discuss the metabolic role of BAO in the Botrytis cinerea-grape host-parasite relationship.

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Membrane-bound, pyrroloquinoline quinone-dependent, alcohol dehydrogenase functions as the primary dehydrogenase in the respiratory chain of acetic acid bacteria. In this study, an ability of the enzyme to directly react with ubiquinone was investigated in alcohol dehydrogenases purified from both Acetobacter aceti and Gluconobacter suboxydans by two different approaches. First, it was shown that the enzymes are able to reduce natural ubiquinones, ubiquinone-9or -10, in a detergent solution as well as a soluble short-chain homologue, ubiquinone-1. In order to show the reactivity of the enzyme with natural ubiquinone in a native membrane environment, futhermore, alcohol dehydrogenase was reconstituted into proteoliposomes together with natural ubiquinone and a terminal ubiquinol oxidase. The reconstitution was done by binding the detergent-free dehydrogenase at room temperature to proteoliposomes that had been prepared in advance from a ubiquinol oxidase and phospholipids containing ubiquinone by detergent dialysis using octyl-β-D-glucopyranoside; the enzyme of A. aceti was reconstituted together with ubiquinone-9 and A. aceti cyochrome a₁ while G. suboxydans alcohol dehydrogenase was done into proteoliposomes containing ubiquinone-10 and G. suboxydans cytochrome o. The proteoliposomes thus reconstituted had a reasonable level of ethanol oxidase activity, the electron transfer reaction of which was also able to generate a membrane potential. Thus, it has been shown that alcohol dehydrogenase of acetic acid bacteria donates electrons directly to ubiquinone in the cytoplasmic membranes and thus the ethanol oxidase respiratory chain of acetic acid bacteria is constituted of only three membranous respiratory components, alcohol dehydrogenase, ubiquinone, and terminal ubiquinol oxidase.

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Spermidine dehydrogenase was crystallized for the first time from the cell-free extract of Citrobacter freundii IFO 12681. The enzyme contained one mole of heme b as the prosthetic group and the heme was autooxidizable. Absorption maxima in the visible region were at 561, 530, 428, and 335 nm with the reduced enzyme, while only one peak at 416nm predominated with the oxidized enzyme. No flavin prosthetic group was detected with the enzyme and PQQ was detected in acid hydrolysate of the enzyme. PQQ appeared to be bound covalently to the enzyme. Thus, the spermidine dehydrogenase of C. freundii has been characterized to be a quinohemoprotein. This is the first instance of amine dehydrogenase in which both heme b and PQQ are involved in enzyme activity.

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Yeast cells carrying intronless mono- and diacylglycerol lipase (MDGL) genes, constructed by recombination of the genomic gene and cDNA, secreted MDGL into the culture supernatant. Most of the yeast MDGL were extensively glycoslated while they had a similar glyceride specificity to that of native MDGL. Site-directed mutagenesis was used to directly confirm the involvements in enzyme activity of the presumptive amino acid residues to form the catalytic center of MDGL. These residues were conserved in the primary structure alignment of a lipase family from filamentous fungi. Mutant lipase proteins in which Ser⁸³, Ser¹⁴⁵, or His<259> was replaced with glycine were secreted by yeast transformants as inactive proteins. Mutant proteins replacing Asp¹⁹⁹ with glycine or asparagine were not detected in the culture supernatant. Replacing other two highly conserved aspartic acids (at positions 232 and 243) with glycine did not render the enzyme inactive. These results indicate that Ser⁸³, Ser¹⁴⁵, and His²⁵⁹ in MDGL, are essential to enzyme activity. Asp¹⁹⁹ is also likely to be involved.

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Various yeast strains were screened for production of 3-hydroxybutyric acid (3-HBA) from 1, 3-butanediol (1, 3-BD) by a resting cell system. Many yeasts were found to oxidize 1, 3-BD to 3-HBA. Among them, Hansenula anomala IFO 0195 produced (S)-(+)-3-HBA of the highest optical purity. Reaction temperature and addition of glucose were significantly effective on the optical purity and production of the acid. When resting cells of the strain were incubated at 27°C in an optimal reaction mixture containing 60.0mg/ml 1, 3-BD, 2.0% CaCO₃, and 1.0% glucose, 26.7 mg/ml of 3-HBA were produced with 88% enantiomer excess for 2 days. Dominant accumulation of (S)-(+)-3-HBA might be due to enantioselective degradation of (R)-(-)-3-HBA, though both (S)-(+)-and (R)-(-)-1, 3-BD are oxidized by the strain.

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