The intense CD bands of marmelo lactones and the related γ-lactones with unsaturation at the C-5 position were studied in terms of the possible interactions between the n→π^* transition of the carbonyl and the π→π^* transition of the double bond at C-5, together with the preferred conformations about the C4-C5 axis. The C4-C5 axis of marmelo lactones takes a preferred conformation which is characteristic for the acyclic allylic alcohols and seems to be an important factor for the (4S)-γ-lactones with a double bond at the C-5 position to give a negative coupling between the two chromophores.

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Protein synthesis in cells transformed by the E1A gene of adenovirus type 12 (Ad 12) was investigated using rat fibroblast cells (3Y1), these cells transformed by the E1A gene of Ad 12 (HY1), human epithelial carcinoma cells (KB) and their transformant (KB-E1A), and mouse mammary tumor cells (FM3A) and their transformant (FM3A-E1A). The proteins labeled with (³²S)-methionine in the intact cells or synthesized using an in vitro translation system were analyzed by two dimensional gel electrophoresis.
The E1A gene of Adl2 increased the number of protein spots in the gel. On the other hand, TPA (12-O-tetradecanoyl-phorbol-13-acetate) increased the number of protein spots in 3Y1 cells while TPA did not seem to have the same effect in HY1 cells. In vitro translation of the mRNAs from 3Y1, HY1, KB, and KB-E1A cells have shown that the number of protein spots in the gel were greater in the E1A-transformed cells than non-transformed cells.
FM3A cells transformed by the E1A gene of Ad 12 drastically suppressed their induction of interferon as compared with that in FM3A cells.
Since E1A gene encodes for proteins, it is suggested that the proteins are trans-acting components (enhancers or suppressors) at the level of transcription of cellular genes as well as viral genes and consequently protein synthesis is affected in the transformed cells.

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A thermotolerant yeast, Hansenula polymorpha CK-1, which had an optimum growth temperature of 40°C, was isolated from bagasse. The fatty acid composition of the yeast grown at 40°C was characterized by the presence of oleic (18:1, 25 to 27%), linoleic (18:2, 42 to 43%) and linolenic (18:3, 3 to 6%) acids. An increase in the growth temperature from 20 to 50°C resulted in a decrease in the phospholipid content of cells from 2.84 to 1.24% on a dry weight basis, and reduced the proportion of 18:3 acid from 17.5 to 1.3% in phospholipids. The sterol content ranged between 0.34 and 0.53% of the dry weight of the cells. An increase in the molar ratio of sterol/phospholipid from 0.26 to 0.54 was observed with increasing temperature from 20 to 40°C, although no further increase in this molar ratio was found at 50°C. Since the decrease in fatty acid unsaturation in the phospholipids was not significant, the increase in the molar ratio of sterol/phospholipid may play an important role in the adaptation of H. polymorpha CK-1 to high temperatures.

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The effects of membrane permeability with several types with molecular weight cut-off levels of from 5, 000 to 30, 000, on the flux and quality of raw and limed cane juice on ultrafiltration were studied by long-term testing for 2-3 hr. The flux of raw juice showed a remarkably high value when ultrafiltration was performed with a YM-30 membrane, for which the sucrose rejection was 5-7%. The flux through a YM-5 membrane was the greatest, and the value did not drop during ultrafiltration of juice limed to pH 7.0 at 85°C. The sucrose rejection for the permeate with the YM5 membrane was very low, i.e. 0-1.2%, for juice limed to pH 7.0 at 85°C.
The YM-30 membrane was suitable for the ultrafiltration of raw juice, while for juice limed under the optimum conditions of pH 7.0 at 85°C, the YM-5 membrane was more suitable.

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The synthesis of 4-substituted monocyclic β-lactam is described. The key step in this method was accomplished by nucleophilic replacement in the presence of Lewis acid, that is, (3S, 4S)-4-acetoxy-3-phenylacetamido-2-azetidinone derivatives or oxazolinoazetidinone derivatives were reacted with nucleophiles (5-mercapto-l-methyltetrazole, 5-methyltetrazole, l-ethyl-2, 3-dioxopiperazine, l-ethyl-4-trimethylsilyl-2, 3-dioxopiperazine) in the presence of Lewis acids (BF₃•Et₂O, SnCl₄, TiCl₄, ZnCl₂) to afford the title compounds. The antibacterial activities of the compounds thus obtained were compared with those of the reference compound (pyridium (3S)-3-phenylacetamido-2-azetidinone-1-sulfonate).

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Colloidal rhodium catalysts in water were prepared by the reaction of rhodium chloride and sodium borohydride in the presence of Celite, White Carbon and Avisel. Hydrogenation of paraquat by the catalysts afforded 1, 1'-dimethyl-4, 4'-bipiperidine in good yields. The reactions using the catalysts supported with Celite or White Carbon were superior to the reaction of nonsupported catalysts.

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Transglutaminase catalyzes the formation of ε-(γ-glutamyl)lysyl cross-links within and between protein molecules. Therefore, transglutaminase polymerizes proteins through the formation of isopeptide bonds. We have reported that several high-concentration protein solutions formed firm gels when they were incubated with transglutaminase. This paper presents unambiguous evidence that α_s1-casein solution is gelled by the formation of ε-(γ-glutamyl)lysyl cross-links. Gelation of α_s1-casein was effected by the amount of transglutaminase and pH value, and α_s1-casein gel was not dissolved by various denaturants. Succinylated α_s1-casein, in which E-amino groups of lysine residues are blocked, was not gelled by transglutaminase. Therefore, it is confirmed that α_s1-casein is formed by the action of transglutaminase.

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Antioxidative constituents in roasted sesame seed oil were studied mainly by an antioxidative assay and HPLC analysis, in comparison with unroasted sesame seed oil. The main active constituent in fresh roasted seed oil was γ-tocopherol, but after heating at frying temperature for 1-2 hr, this was identified as sesamol, which was produced by hydrolysis of sesamolin that is present to a large degree in roasted sesame seed oil. This conversion of sesamolin to sesamol is catalyzed by acids.

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When incubated for 3 days with a culture of Aspergillus flavus, the diprenylated isoflavonc 2'-hydroxylupalbigenin [5, 7, 2', 4'-tetrahydroxy-6, 3'-di-(3, 3-dimethylallyl)isoflavone] was converted to ten phenolic compounds which were isolated and purified by a combination of column and thinlayer chromatography. The aromatic (A/B) rings of these isoflavone-like metabolites were found to be variously substituted with acyclic dihydrohydroxy (ring A) and dihydrodihydroxy (rings A and B) sidechains, as well as with cyclic dihydrofuran (both rings) and dihydropyran (ring B) attachments resulting from fungal modification of the prenyl groups located at C-6 and C-3' on the substrate molecule. One of the metabolites (denoted AF-d-1) is spectroscopically (UV, MS and 1H-NMR) and chromatographically indistinguishable from lupinisoflavone F, a compound already known to occur constitutively in roots of the white lupin (Lupinus albus).

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Starch-utilizing mutants of Escherichia coli which can grow well on starch or amylose as the sole carbon source were isolated. The maximal viable cell number of the starch-utilizing mutants on the polysaccharide media reached the same level (4×10⁹ cells/ml) as that with glucose medium after incubation for 24 hours at 37°C. The isolated mutants could produce more intracellular α-amylase than the wild-type strain, and the enzyme activity was detected in the extracellular fluid. Polyacrylamide gel electrophoresis showed that the intracellular and extracellular enzymes had similar electrophoretic mobilities. These observations suggested that the ability of growth on the polysaccharide media' was due to the excreted α-amylase, which appeared to be identical with the intracellular enzyme.

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Glucose oxidase (GOD) was immobilized on the surface of a benzoquinone (BQ)-mixed carbon paste electrode (CPE) by coating the enzyme-loaded electrode surface with a nitrocellulose film. This film-coated glucose oxidase-immobilized benzoquinone-mixed carbon paste electrode (GOD-BQ-CPE) served as a biocatalyst electrode for electro-oxidation of D-glucose. The catalytic oxidation current at 0.5V vs. SCE was analyzed as a function of the concentration of D-glucose, the amount of the mixed benzoquinone in carbon paste electrode, the amount of the immobilized enzyme on the carbon paste electrode surface and the thickness of the coating film. The kinetics of the electrocatalysis at the film-coated GOD-BQ-CPE were interpreted by the concept of the enzyme reaction layer on the carbon paste electrode surface.

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A photosynthetic bacterium, Rhodospirillum rubrum G-9 BM, was immobilized in agar or carrageenan gel. The immobilized cells produced hydrogen from a wide range of organic compounds in the light. The highest activity was seen for mid-log phase cells. The immobilized cells also consumed organic compounds in the dark. Inhibition experiments suggested that the photoproduction of hydrogen was mostly catalyzed by nitrogenase. By using the immobilized cells, continuous photoproduction of hydrogen was carried out successfully for 60 days. After the operation, the hydrogen-producing activity of the cells was still about 40% of the original level.

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An ATP production system involving the oxidation of methanol, oxidative phosphorylation of ADP and phosphorylation of adenosine with sorbitol-treated cells of Candida boidinii (Kloeckera sp.) No. 2201 was studied with regard to the metabolism of adenosine and the role of sorbitol.
Analysis by high performance liquid chromatography showed the accumulation of IMP in the reaction mixture, possibly due to catalysis by AMP deaminase. The efficiency of energy conversion from methanol to nucleotides was improved 30% by preventing the evaporation of methanol. Among osmoregulatory compounds that plasmolyzed the yeast cells, only sorbitol caused high ATP production activity on addition to the reaction mixture. The sorbitol effect was attributed to the supplying of NADH by the oxidation in parallel with the methanol oxidation.
The ability of ATP production from adenine and adenosine was found in many methanol yeasts belonging to the genera, Hansenula, Pichia, Candida and Torulopsis. C. boidinii had especially high ability to accumulate ATP from adenosine.

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Carotenoid-carrying lipoproteins (CCLs) were found in the high density lipoprotein (HDL, d=1.063-1.210g/ml) and the very high density lipoprotein (VHDL₂, d>1.210g/ml) fractions of the serum of chum salmon, Oncorhynchus keta. A CCL was isolated from the HDL fraction by a sequential ultracentrifugal technique, and DEAE-cellulose and gel nitration column chromatography. Gel filtration chromatography showed that the molecular weight of the CCL ranged from 30, 000 to 500, 000, with a peak at 70, 000 dalton. The CCL gave rise to two subunits whose molecular weights were 24, 000 and 12, 000 by SDS-PAGE. No disulfide bond was detected between the two subunits. The CCL was considered to be a cluster of molecular species of various particle sizes any of which was composed of the two subunits. The CCL had a pi at 5.1, and was rich in glutamic acid, alanine, leucine, and lysine. The CCL showed a high content of lipid (54%), which consisted mainly of cholesterol ester, cholesterol, triglyceride, phosphatidylcholine, and sphingomyelin. The CCL could maintain a consistent function irrespective of the physiological state of chum salmon.

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The perfect stage of Rhizoctonia solani Kühn R-44 (Thanatephorus cucumeris) was confirmed by microscopic observation of basidiospores after three successive single spore isolations. A morphological mutant, ML-19, was induced by UV-light irradiation of a mycelial-fragment suspension from a single basidiospore culture of the fourth generation, and characterized.
Different from in the cases of some kinds of morphological mutants of Neurospora, the morphology of this mutant was not responsible for conditional morphologicals, such as choline, inositol, linolenic acid and cAMP or hyphal extension factor.
The characterization of the cell wall by fractionation, acid hydrolysis, Smith degradation and methylation analysis revealed that fraction III (glycan fraction) was composed of 1, 3-glucomannan containing small amounts of pentoses. The major difference in the fractions III from the original strain and the mutant was the change in the glucose/mannose ratio from 5/3 to 10/3.

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To clarify the substrate specificity of the milk-clotting enzyme excreted by Irpex lacteus (IR), various peptide hormones were used as substrates to find the cleavage sites and to compare the specificity with those of other milk-clotting enzymes such as calf chymosin (CR), Mucor miehei enzyme (MR), and Endothia parasitica enzyme (ER).
IR hydrolyzed substance P, physalaemin, β-neo-endorphin, neurotensin, angiotensin I, LHRH, and DSIP, but not bradykinin. Although IR generally require hydrophobic amino acids in the P₁ and P₁' sites, it also hydrolyzed peptide bonds which have only one hydrophobic amino acid either in the P₁ or P₁' site.
CR did not hydrolyze either angiotensin I or DSIP, and MR did not hydrolyze DSIP, but both enzymes hydrolyzed the Tyr(5)-Glu(6) bond of LHRH slightly. On the other hand, ER hydrolyzed these substrates at several bonds. Besides the common cleavage sites with IR, ER hydrolyzed the His(6)-Pro(7) bond of angiotensin I, the Leu(7)-Arg(8) bond of LHRH, and the Ala(2)-Gly(3) and Gly(3)-Gly(4) bonds of DSIP. Therefore, these milk-clotting enzymes can be classified into two groups according to their substrate specificities on peptide hormones. CR and MR belong to the first group which has restricted specificity, while IR and ER belong to the second group which shows relatively broad specificity.

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A 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA) hydrolyzing enzyme was purified to a homogeneous state from P. cruciviae S93 Bl grown on biphenyl as the carbon source. The enzyme hydrolyzed HOPDA between the C₅-C₆ bond to produce benzoic acid and 2-oxopent-4-enoic acid. The HOPDA hydrolyzing enzyme had a molecular weight of about 160, 000, and was dissociated in SDS, with prior treatment with mercaptoethanol, into a single subunit with an approximate molecular weight of 29, 000. The optimum pH of this hydrolase was 4.7, and it was relatively stable in an alkaline solution. It attacked the meta ring-fission products of biphenyl-2, 3-diol, 3-isopropylcatechol, 3-methylcatechol and catechol. It was proposed that this enzyme is called 2, 6-dioxo-6-phenylhexa-3-enoic acid (DPEA) by hydrolase and is classified under the number EC 3.7.1.

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A new triterpenoid glycoside named SI was isolated from the aerial part of Bupleurum chinense. Based on chemical degradation and NMR studies, its structure has been determined as 3-0-[α-L-arabinopyranosyl (1→3)-β-D-glucuronopyranosyl]-oleanolic acid β-D-glucopyranosyl (1→28) ester.

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Five new triterpenoid glycosides named S3, S4, S8, S9 and S10 were isolated from the root of Bupleurum kunmingense, and their structures were elucidated by NMR spectral analysis as 16-epi-chikusaikoside 1, 3''-O-acetylsaikosaponin-a, 4''-O-acetylsaikosaponin-a, 2''-O-acetylsaikosaponin-d, and 2''-O-acetylsaikosaponin-a, respectively.

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Ricin D was cross-linked with glutaraldehyde. Cross-linked (CL-)ricin D was divided into three fractions, which contain the oligomer (trimer and tetramer), dimer, or monomer as the main components, by gel filtration on a Sephacryl S-200 column followed by DE-32 cellulose column chromatography. From the analyses of the unmodified lysyl residue in each fraction and the reduction product of the dimer-fraction on SDS-PAGE, it was suggested that ricin D was crosslinked by a single glutaraldehyde-link between any two chains, but more easily between two A-chains.
Cytoagglutinating activity of the monomer, dimer, and oligomer fractions of CL-ricin D toward human erythrocytes were 2.2, 9, and 18 fold that of native ricin D, but the Cytoagglutinating activity toward SAT cells was identical to that of native ricin D.
The inhibitory activities of these fractions on protein synthesis in HeLa cells were 38.6%, 4.9%, and 2.4% and those in SAT cells were 85%, 90%, and 42%, respectively. Toxicity toward mice was approximately 80%, 20%, and 7%, respectively.

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A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65, 000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.

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Amino acid sequences of three glucoamylases from Rhizopus, Aspergillus, and Saccharomyces were compared and the overall homology was from 25 to 36%. A greater homology was observed in four distinct regions suggesting that they may be functionally constrained amino acid sequences. Rhizopus and Aspergillus glucoamylases were more closely related among the three. The structure-function relationships of glucoamylase are discussed based on sequence comparison, probable secondary structures, and the hydropathy profiles.

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Monodansylcadaverine (250μM), an inhibitor of transglutaminase, completely inhibited aggregation of human platelets induced by 2 μg/ml of collagen. Platelet responses necessary for aggregation (shape change, serotonin release, and phosphorylation of the 47, 000-Da protein) were also inhibited by this reagent. However, incorporation of [³H]putrescine into platelet proteins was very low and independent of the stimulation by collagen. It seems unlikely, therefore, that the transglutaminase reaction participates in platelet aggregation. Monodansylcadaverine inhibited the formation of thromboxane A₂ with a similar median inhibitory concentration (about 120μM) for aggregation. When platelets prelabeled with [¹⁴C]arachidonic acid were stimulated by collagen, the radioactivities in thromboxane B₂, hydroxyheptadecatrienoic acid, and hydroxyeicosatetraenoic acid increased. The increase was completely suppressed by the addition of monodansylcadaverine. Radioactivity in free arachidonic acid was very low throughout the experiments. From these results, it was concluded that monodansylcadaverine inhibited platelet aggregation by suppressing the release of arachidonic acid from phospholipids during platelet activation by collagen.

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An ethylene-forming enzyme was partially purified from Penicillium digitatum IFO 9372 by means of DEAE-Sepharose CL-6B ion exchange chromatography, and a cell-free ethylene-forming system was constructed. Using this system as a tool for elucidation of the biochemical basis of the ethylene formation, the immediate precursor of ethylene was found to be α-ketoglutarate and essential factors were L-arginine and the ferrous ion under reduced conditions. It was strongly suggested that this cell-free ethylene-forming system operates in living cells of P. digitatum on comparison of the time course of ethylene-forming activity of the cell-free system with that of living cells.

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On SDS-polyacrylamide gel electrophoresis, sesame seed 13S globulin was separated into three intermediary subunits termed IS₁, IS₂ and IS₃. Following a treatment with 0.2 M 2-mercaptoethanol, the globulin was separated into three acidic subunits termed AS₁, AS₂ and AS₃, and four basic subunits termed BS₁, BS₂, BS₃ and BS₄. Two dimensional SDS-gel electrophoresis before and after treatment with 0.2 M 2-mercaptoethanol revealed that IS₁ was composed of two combinations of acidic and basic subunits, these being AS₁, and BS₂, and AS₂ and BS₂. IS₂ was found to be composed of AS₃ and BS₁; and IS₃ was composed of AS₂ and BS₃, and AS₂ and BS₄. These combinations were consistent with the reactivity of each subunit to a fluorescent thiol reagent. The amino acid compositions were similar among the three acidic subunits and also among the four basic subunits. However, between the acidic and basic subunits, there were great differences in the amino acid composition, especially in the amount of glutamic acid.

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The secondary alcohol oxidase from Pseudomonas sp. catalyzed the oxidation of various vinyl alcohol oligomers with the molecular weight of 220 to 1500 and of β-ketols such as 5-hydroxy-3-heptanone, 4-hydroxy-2-nonanone, 3-hydroxy-5-nonanone, 6-hydroxy-4-nonanone, 7-hydroxy-5-dodecanone, and 8-hydroxy-6-tridecanone. β-Diketone hydrolase from the same strain catalyzed the hydrolysis of various aliphatic β-diketones and some aromatic β-diketones such as l-phenyl-1, 3-butanedione and 1-phenyl-2, 4-pentanedione. 4, 6-Nonanediol, used as a low molecular weight model of poly(vinyl alcohol) (PVA), was oxidized to 4, 6-nonanedione by way of 6-hydroxy-4-nonanone by secondary alcohol oxidase. 4, 6-Nonanedione was hydrolyzed to 2-pentanone and nbutyric acid by β-diketone hydrolase. These reactions were stoichiometric.
The presence of the β-diketone structure in PVA oxidized by secondary alcohol oxidase was confirmed by spectral experiments. The absorption due to β-diketone structure in the oxidized PVA decreased as it was hydrolyzed by β-diketone hydrolase. The ratio of the amount of carboxyl groups in the degraded PVA to that of carbonyl groups in the oxidized PVA became more than 0.5. A pathway for the enzymatic degradation of PVA was proposed.

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Valsa ceratosperma, which is the pathogenic fungus of apple canker, was grown in a synthetic medium. The neutral extract from the culture filtrate was chromatographed on a silica gel column to give five isocoumarins. Their structures were determined by MS, UV, IR, ¹H and ¹³C NMR, and CD spectra. Three of them were known compounds; (-)-5-methylmellein (1), (-)-5-carboxylmellein (2) and (-)-5-hydroxylmethylmellein (3). Since the absolute configurations at C-3 in 2 and 3 were not known until now, both were determined to be R by chemical correlations. The two were new compounds; (+)-(3R, 4S)-trans-4-hydroxy-5-methylmellein (4) and (-)-(3R, 4R)-cis-4-hydroxy-5-methylmellein (5). All the five compounds showed phytotoxicity in a bioassay using detached apple shoots and lettuce seedlings.

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Sinefungin inhibited the S-adenosylmethionine-dependent farnesoic acid methyltransferase in a cell-free system containing a homogenate of corpora allata from female locusts, Locusta migratoria. The enzyme catalyzed the penultimate step of juvenile hormone biosynthesis in the insects. Culturing corpora allata in the presence of sinefungin greatly suppressed juvenile hormone production. The following in vivo effects were visible after injection of the inhibitor: increase in mortality and reduction of total haemolymph protein liter and ovary fresh weight, as well as length of terminal oocytes. Attempts to reverse these effects by topical application of the juvenile hormone analog ZR-515 (methoprene) were only partly successful. Therefore, the in vivo effects may be due to a general inhibition of methyltransferase enzymes in the insect. Sinefungin appeared to be of potential interest as the first representative of a new class of insect growth regulators.

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The effects of various concentrations of succinate in the incubation system on ARA and oxidative succinate degradation by nodule bacteroids were analyzed. When the bacteroids' ARA was measured in various concentrations of succinate the highest ARA was observed at 2mM succinate, and the activity was lower at higher succinate concentrations.
To monitor the oxidative degradation of exogenous 2, 3-¹⁴C-succinate by nodule bacteroids the formation of ¹⁴CO₂ was measured continuously with or without Lb.
In 1% oxygen ¹⁴CO₂ was formed actively by bacteroids at 0.5 to 3mM succinate, but at 4 or 8 HIM succinate the bacteroid's ¹⁴CO₂ formation was almost negligible. The bacteroids did not release ¹⁴CO₂ with no oxygen in the incubation system.
Addition of Lb (100mM) to the incubation system stimulated the ¹⁴CO₂ formation at all concentrations of succinate in 1% O₂, especially at 4 or 8 mM succinate the succinate oxidation was greatly improved.
The pathway of succinate degradation in the bacteroids was analyzed when the incubation system contained 2, 3-¹⁴C-succinate and 100μ Lb in 1% O₂. The succinate fed was metabolized actively, and the labelling was incorporated mainly into TCA cycle member organic acids, though one major peak was not identified.
The data suggest that the exogenous 2, 3-¹⁴C-succinate was mainly oxidized through the TCA cycle in bacteroids, and Lb is important in stimulating succinate oxidation and ARA when the succinate concentration is higher than 3 mM.

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We previously constructed an L-threonine-producing strain of E. coli W, KY8280, which is an Ile⁺ revertant of KY8279 which requires L-methionine, α, ε-diaminopimelic acid and L-isoleucine [H. Kase et al., Agric. EM. Chem., 35, 2089 (1971)]. From KY8280, another L-threoninehyperproducing strain, KY8366, was obtained as an α-amino-β-hydroxyvaleric acid (AHV, a threonine analog)-resistant mutant. Enzymatic analysis revealed that KY8280 constitutively expressed 8-fold higher L-threonine-sensitive aspartokinase I activity than KY8279. In addition, KY8366 constitutively expressed 13-fold higher L-lysine-sensitive aspartokinase III activity than KY8280. Such elevated levels of aspartokinases may contribute to the hyperproduction of L-threonine by these mutant strains. KY8366 produced 28 mg/ml of L-threonine in a culture medium fed with 12% glucose.

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An L-threonine-hyperproducing strain of E. coli W, KY8366, which constitutively expresses L-threonine-sensitive aspartokinase (AKI) and L-lysine-sensitive aspartokinase (AKIII) was constructed [T. Mizukami et al., Agric. Biol. Chem., 50, 0000 (1986)]. In order to further improve the L-threonine productivity of KY8366, we cloned the E. coli thr operon from an α-amino-β-hydroxyvaleric-acid resistant (AHV^r) mutant of E. coli K-12 and introduced various types of recombinant plasmids carrying the AHV^r-thr operon into KY8366. Most ol them were unstable in KY8366; the loss of the whole plasmid molecules from the host cells or the deletion of the thr operon from the plasmids was observed. However, pGH2B1, a recombinant plasmid which is composed of the whole thr operon, the kanamycin-resistance (Km^r) gene and the replication origin of p15A, was stably maintained in KY8366 in the presence of kanamycin. KY8366 harboring pGH2B1 expressed 4.5-fold higher AKI activity than parental strain KY8366, and its L-threonine productivity was elevated to 20% higher than that of KY8366.

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A carboxyl proteinase was purified from rice koji of Aspergillus kawachii to a homogeneous state on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were 35, 000 and 3.9, respectively. The enzyme was most active between pH 2.8 and 3.4 with hemoglobin as substrate, and stable in the pH range of 2.2-6.4. The optimum temperature of the enzyme reaction was at 50°C. The serine content was highest and no methionine was found in the enzyme. The enzyme was inhibited by typical inhibitors for carboxyl proteinases such as DAN, EPNP, and SPI.

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Mass spectrometric determination of endogenous cytokinins of an agriculturally-important green manure, Azolla, was carried out on the bases of stable isotope dilution and selected ion monitoring. Several cytokinin species were detected and their levels were estimated.

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A mutant strain of Euglena gracilis Z isolated in our laboratory is highly tolerant to excess zinc and was proved by X-ray microanalysis to accumulate the element in micro-spherical bodies in the cytoplasm. On the other hand, the wild strain which is less tolerant to zinc had a uniform distribution of zinc within the cell when cultured in the zinc enriched medium. The production of the zincaccumulating globular substances in the mutant cells is considered to be the main protective mechanism against the metal toxicity.

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