Levansucrase (EC 2.4.1.10) was purified to homogeneity from cell-free extracts of Rahnella aquatilis JCM-1683 by strepotomycin treatment, ammonium sulfate fractionation, and HPLC with a DEAE-Toyopearl pak 650M, TSKgel G-3000SW, and TSKgel DEAE-5PW. The enzyme had optimum activity around pH 6.0 and 55-60°C. The molecular weight of the enzyme was 120, 000 on gel filtration and it was composed of two identical subunits (64, 000). The amount of levan synthesized by the purified enzyme was 2.95 g from 10.0 g sucrose. The enzyme had a broad acceptor specificity and gave transfer products. D-Xylose, D-arabibnose, L-arabinose, lactose, maltose, maltotriose, cellobiose, and melibiose were effective acceptors in the transfructosylation reaction of the enzyme.

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Oxalic acid was found to inhibit noncompetitively the Cα-Cβ bond cleavage of veratrylglycerol catalyzed by a lignin peroxidase (LiP) isozyme of the white-rot fungus P.chrysosporium. With greater amounts of oxalic acid in the LiP system, the substrate was not converted to veratraldehyde but was almost all recovered. Oxalic acid was shown to be decomposed to CO₂ during the enzymatic reaction. The results clearly indicate that oxalic acid reduced the cation radical intermediate formed in the reaction back to the substrate to block the production of veratraldehyde. A novel equation has been derived to explain the mechanism for this unique non-competitive inhibition that is different from the classical noncompetitive one. The inhibition constant K_i obtained here, which is different from the classical inhibition constant K_i, is defined as the ratio of the rate constant (K_p) for product formation to the rate constant (K_i) for the reduction of the cation radical to the substrate.

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Oxazolomycin diacetate, dipropionate, monobutyrate and dibutyrate were derived from oxazolomycin, a product of Streptomyces sp.KBFP-2025. These esters were potent inhibitors of crown gall formation on potato tuber disks upon infection with Agrobacterium tumefaciens. They showed neither antibacterial nor phytotoxic activity, whereas oxazolomycin showed both antibacterial and phytotoxic activities. Further, they had no inhibitory activity against A.tumefaciens on the potato tuber disk. The inhibitory activity of these esters against crown gall formation seems to be due to specific inhibition of the transformation of plants with A.tumefaciens.

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Bacillus stearothermophilus CGTase had a wider acceptor specificity than Bacillus macerans CGTase did and produced large amounts of transfer products of various acceptors such as D-galactose, D-mannose, D-fructose, D- and L-arabinose, D- and L-fucose, L-rhamnose, D-glucosamine, and lactose, which were inefficient acceptors for B.macerans CGTase. The main component of the smallest transfer products of lactose was assumed to be α-D-glucosyl O-β-D-galactosyl-(1→4)-β-D-glucoside.

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The D-aminoacylase produced by Alcaligenes denitrificans DA181 was a new type of aminoacylase which had both high stereospecificity and specific activity. The molecular weight and isoelectric point of this enzyme were 58, 000 and 4.4, respectively. The apparent K_m and κ_cat values of this enzyme for N-acetyl-D-methionine were estimated to be 0.48 mM and 6.24×10⁴ min^-1, respectively. The optimum temperature was 45°C. The enzyme was stable up to 55°C for 1 hr in the presence of 0.2 mg/ml bovine serum albumin. The enzyme was stable in the pH range of 6.0 to 11.0 with an optimum pH of 7.5. This enzyme contained about 2.1 g atom of zinc per mole of enzyme. Enzyme activity was inhibited by incubation with EDTA. The inhibition by EDTA was fully reversed by Co²⁺ and partially by Zn²⁺.

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The effects of dexamethasone (Dex) on insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-1 production were investigated in primary cultures of rat hepatocytes. Dex enhanced the secretion of IGFBP-1 as measured by ligand blot analysis but did not show any prominent effect on immunoreactive IGF-I secretion. EC₅₀ of Dex on IGFBP-1 secretion was calculated to be 3×10^-8M. The content of IGFBP-1 mRNA in the cells increased greatly in the presence of Dex but the IGF-I mRNA content did not change significantly under the same conditions. Insulin showed the opposite effect of Dex by decreasing the production of IGFBP-1 and the cellular content of IGFBP-1 mRNA. This effect of insulin was observed also with Dex in the medium. These results show that the gene expression of IGF-I and IGFBP-1 is differently regulated by glucocorticoids and insulin in primary cultures of rat hepatocytes. The results most possibly explain the in vivo effects of glucocorticoids and insulin in regulation of IGF-I and IGFBP-1 production by liver.

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An enzyme hydrolyzing nigeran (alternating α-1, 3-and α-1, 4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by precipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-100. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68, 000 by SDS-PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50°C. The enzyme activity was inhibited significantly by Hg⁺, Hg²⁺, and p-chloromercuribenzoic acid. The K_m (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the α-1, 4-glucosidic linkages in nigeran.

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A transglycosylation reaction with moranoline (1-deoxynojirimycin) was done with soluble starch as the glucosyl donor and Bacillus macerans amylase as a cyclodextrin glycosyltransferase [EC 2.4.1.19]. The resultant transglycosylation products with moranoline, obtained by treating the reaction mixture with a strong cation exchange resin, were hydrolyzed by β-amylase [EC 3.2.1.2] from sweet potatoes. The hydrolysate was treated with a strong cation exchange resin, and high purity maltose was obtained.

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Acetyl CoA : deacetylcephalosporin C O-acetyltransferase, which catalyzes the final step of the biosythetic pathway to cephalosporin C, was stabilized by a buffer solution containing 7-aminocephalosporanic acid and purified over 1300-fold from Acremonium chrysogenum. The purified enzyme has a molecular weight of 55, 000 as measured by gel filtrantion. SDS-polyacrylamide gel electrophoresis showed two subunit bands corresponding to molecular weights of 27, 000 and 14, 000. The enzyme has an isoelectoric point at pH 4.0 and optimum activity at pH 7.5.

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Methyl α-and β-D-galactopyranosides and 4-O-β-D-galactopyranosyl-3, 6-anhydro-L-galactose dimethylacetal were sulfated with sulfuric acid and dicyclohexylcarbodi-imide as a condensation reagent. The sulfated sugars were isolated by ion-exchange chromatography, characterized, and assigned by methylation analyses. On the basis of the yield of each sulfated product that was isolated, sulfation on O-6 appeared to be predominant.

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The naturally occurring (-)-enantiomer of a fatty acid isolated from the mushroom (Hericium erinaceum) was synthesized from (R)-(-)-benzyl glycidyl ether in 8 steps. A comparison of the specific rotation of the synthetic sample with that of the natural compound established the absolute configuration of the latter.

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Both α-tocopherol and a 1 : 1.7 mixture of α-tocopherol and tocotrienols at a 0.2% dietary level significantly depressed the age-related increase in the systolic blood pressure of spontaneously hypertensive rats (SHRs) after 3 weeks of feeding. The aortic production of prostacyclin was increased 1.5 times both by α-tocopherol and a tocotrienol mixture, suggesting a possible relevance to their hypotensive effect. These vitamins did not influence the Δ6- and Δ5-desaturase activities of liver microsomes, but fatty acid profiles of the liver phospholipids predicted a reduction of linoleic acid desaturation. These effects were in general more clear with tocotrienols than with α-tocopherol. Platelet aggregation by 5μM ADP remained uninfluenced. Thus, tocotrienols may have effects on various lipid parameters somewhat different from those of α-tocopherol.

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The structural and functional properties of lysozymes genetically deamidated at positions 103 (N103D) and 106 (N106D) were studied by a protein engineering technique. The wild-type and mutant lysozymes were expressed in Saccharomyces cerevisiae and purified from the cultivation medium in two steps by cation-exchange chromatography on CM-Toyopearl. The lytic activity of deamidated lysozymes was almost the same as that of wild lysozyme, although the optimal pH of activity was slightly shifted to lower pH by the deamidation. The Gibbs free energy changes of unfolding (ΔG) at 20°C for N103D and N106D were almost the same as that of wild-type. On the other hand, the structural flexibility of lysozymes, estimated by protease digestion, was significantly increased by the deamidation. The surface functional properties of deamidated lysozymes were considerably enhanced, compared to those of wild-type lysozyme. These results suggest that structural flexibility is an important governing factor in surface functional properties of proteins, regardless of their structural stability.

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A 20-kDa protein of Bacillus thuringiensis var. israelensis (Bti) has been shown to be necessary for the efficient expression of the 27-kDa mosquitocidal protein gene in Escherichia coli. We have investigated the effects of this 20-kDa protein on the expression of two 130-kDa mosquitocidal protein genes (cryIV A, cryIV B) in E.coli by supplying the 20-kDa protein gene in trans. When a recombinant plasmid, pLH4BX, which was constructed to express cryIV A under the E.coli lac promoter on pUC19, coexisted with the 20-kDa protein gene, a striking increase in production of the fused CryIV A was detected. This was not accompanied by an increase in the amount of intracellular mRNA, suggesting that 20-kDa protein exerts a posttranscriptional effect. We conclude that the 20-kDa protein also stimulates the production of 130-kDa protein in E.Coli.

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A zinc-binding protein was purified to homogeneity for the first time from the gonad parts of scallops, Patinopecten yessoensis, kept in filtered seawater to which no heavy metals were added. Based upon the elution profiles in two chromatographic systems, spectrophotometric analysis, and amino acid composition of the purified preparation, the protein met the criteria for classification as a metallothionein ; i.e., low molecular weight (about 9000), paucity of both aromatic amino acid residues and absorbance at 280 nm, and abundance of both cysteinyl residues (>25%) and absorbance at 215 and 254 nm. Furthermore, the results of chromatographies on a Sephacryl S-300 column and electrophoresis with or without SDS suggested that the protein molecules would be in several polymeric forms in vivo. The antiserum prepared with the purified protein as the antigen was shown to have immunocross-reactivity to neither an extract of the surf clam, Pseudocardium sybillae, nor the whelk, Neptunea arthritica, indicating the heterogeneity of the proteins in marine shellfishes. These results suggested that the Zn-binding protein purified in this study was characteristic of scallops and involved in zinc storage of this organism.

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New antibiotics, T1801 A, B, C, and D, were isolated from the culture broth of Pseudomonas sp.SC-1801. Their structures were found by spectroscopic analyses to be tri- and tetra (methylthio) derivatives of hydroquinone (T1801 A and C) or p-benzoquinone (T1801 B and D). They are new quinone and hydroquinone antibiotics and are active against Gram-positive bacteria, some fungi, and yeasts.

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An exo-inulinase was highly purified from the culture broth of Penicillium trzebinskii by anion exchange, hydrophobic, and gel filtration chromatographies. The enzyme was homogeneous by disc electrophoresis. The molecular weight was 8.7×10⁴, and the isoelectric point was pH 4.3. The enzyme hydrolyzed not only inulin and sucrose but also inulooligosaccharides [1^F(1-β-D-fructofuranosyl)_n-1fructose, F_n(n=2-5)] and fructooligosaccharides [1^F(1-β-D-fructofuranosyl)_n-1 sucrose, GF_n, (n=2-8)] liberating the nonreducing terminal fructose of the substrates. The substrate specificity was investigated. The K_m (mM) and κ₀ (sec^-1) were : inulin, 0.042 and 159 ; sucrose, 6.5 and 169 ; F₂, 2.1 and 62.8 ; F₃, 0.40 and 126 ; F₄, 0.47 and 171 ; and F₅, 0.47 and 131, respectively. Dependence of K_m and κ₀ values on the degree of polymerization of substrates was observed. The subsite affinities in the active site were 1.05, 4.57, 1.45, 0.09, and -0.16 kcal/mol for subsite 1, 2, 3, 4, and 5, respectively.

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New derivatives of pyridinyloxyphenoxypropionamide were synthesized, which have an unnatural α-amino acid or its decarboxylated structure as amide moieties. Some compounds such as N-(1-methoxycarbonyl-1-tert-butylamino) methyl, N-methoxymethyl and N-hydroxymethyl 2-[4-(3-chloro-5-trifluoromethyl-2-pyridinyloxy) phenoxy] propionamide were found to show stronger herbicidal activity against grass weeds than that of pyridinyloxyphenoxypropionamides which have already been synthesized.

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The productions of β-fructofuranosidase from Bifidobacterium longum A1, B.adolescentis G1, and four other strains of Bifidobacteria were investigated. All strains used in this study were grown in modified BL broth containing a mixture of fructooligosaccharides [1^F(1-β-D-fructofuranosyl)_n-1 sucrose, GF_n(n=2-5)] as the only carbon source. Hydrolyses of 1-kestose, sucrose, and inulin were detected in the extract of the cell. The highest activity on 1-kestose was detected in the extract of B.longum A1 followed by B.adolescentis G1. The other extracts weakly attacked 1-kestose. The relative activities of the extract of B.adolescentis G1 for 1-kestose, nystose, 1^F-fructosylnystose, sucrose, and inulin were 100, 82.5, 50.8, 28.3, and 15.0, respectively. The relative activities for various substrates differed from invertases (yeast β-fructofuranosidases) and exo-inulinase from Penicillium trzebinskii.

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The gene encoding an alkaline serine protease from alkaliphilic Bacillus sp.221 was cloned in Escherichia coli and expressed in Bacillus subtilis. An open reading frame of 1, 140 bases, identified as the protease gene was preceded by a putative Shine-Dalgarno sequence (AGGAGG) with a spacing of 7 bases. The deduced amino acid sequence had a pre-pro-peptide of 111 residues followed by the mature protease comprising 269 residues. The alkaline protease from alkaliphilic Bacillus sp.221 had higher homology to the protease from alkaliphilic bacilli (82.1% and 99.6%) than to those from neutrophilic bacilli (60.6-61.7%). Also Bacillus sp.221 protease and other protease from alkaliphilic bacilli shared common amino acid changes and 4 amino acid deletions that seemed to be related to characteristics of the enzyme of alkaliphilic bacilli when compared to the proteases from neutrophilic bacilli.

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Growth tests of type culture strains were done on interfaces between nutrient agar and hydrophobic organic solvents. All strains examined were able to grow against n-paraffin (C₁₁-C₁₅), and many could grow against isooctane, and pentyl- and heptylbenzene. The interface cultivation method was also successful even when the solvent layer contained compounds toxic for microorganisms.

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