Kinetic analysis was done on chitinase E3 (EC 3.2.1.14) from yam, Dioscorea opposita THUNB, using both series of N-acetylchitooligosaccharides (GlcNAc_n, n=2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-GlcNAc_n, n=1 to 5) as substrates. The enzyme cleaved GlcNAc₃ to GlcNAc plus GlcNAc₂, GlcNAc₄ to two molecules of GlcNAc₂, GlcNAc₅ to GlcNAc₂ plus GlcNAc₃, and GlcNAc₆ by three ways to GlcNAc plus GlcNAc₅ (32%), GlcNAc₂ plus GlcNAc₄ (42%) and two molecules of GlcNAc₃ (26%). The speed of the reaction was observed in the following order, GlcNAc₄>GlcNAc₅>GlcNAc₆>GlcNAc₃. Stronger substrate inhibition was observed in the longer chain substrates. The reactions of pNp-GlcNAc_n (n=1 to 5) were similar to those of GlcNAc_n (n=2 to 6), respectively. The cleavage sites of pNp-GlcNAc₂ and pNp-GlcNAc₃ were the second β-1, 4-linkages from the reducing end side, and the main cleavage sites of pNp-GlcNAc₄ and pNp-GlcNAc₅ were the third linkages. p-Nitrophenol was not released from all p-nitrophenyl derivatives. The speed of the reaction was observed in the following order, pNp-GlcNAc₄ > pNp-GlcNAc₅ > pNp-GlcNAc₃ > pNp-GlcNAc₂. Neither GlcNAc₂ nor pNp-GlcN Ac was cleaved. These results suggest that yam chitinase E3 acts in a random fashion.

View full abstract

Gum arabic, a widely used emulsifier, stabilized the substrate of lipase in an emulsion of oil and enhanced the lipase reaction. Pectin, a well-known gelling agent, destabilized the emulsion, and decreased the plasma levels of cholesterol in rats. Little effect of gum arabic on the cholesterol concentration of plasma and liver was observed. These findings might indicate the differences between the effects of gum arabic and pectin on the stability of emulsification and the activity of lipase in vivo.

View full abstract

For the cross-linking of Taka-amylase A (TAA) to carboxymethyl dextran (CM-Dex), the use of water-soluble carbodiimide (EDC, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide) was examined. CM-Dex was treated with EDC and the carboxyl groups were converted into the activated form, and then, the activated CM-Dex was attached to TAA. Purification of the modified TAA was done by a column of DEAE-Sephacel, where 80% of the enzyme activity was eluted at the breakthrough fraction (M-TAA I). About 14% of the activity was eluted with 0.25 MNaCl (M-TAA II). Polyacrylamide gel electrophoresis showed an increase in the carbohydrate at the position of native TAA. The Km values of M-TAA I and II for the soluble starch were 2 - 6-fold higher than that of native TAA, while Km values for γ-cyclodextrin were at the same level as the native TAA. Although the pH stability of M-TAA I at the acidic limb of pH 3.0 - 4.5 decreased slightly, the stability at the alkaline limb of pH 10.0 - 11.0 was improved. There were some increase in the stability against the denaturation by methanol.

View full abstract

A dual immobilized enzyme system composed of immobilized β-amylase [EC 3.2.1.2] and pullulanase [EC 3.2.1.41] was studied for the continuous production of maltose. Porous chitosan beads were selected from among many commercial carriers as the best carrier to immobilize β-amylase.
The properties of the immobilized enzyme were examined and compared with those of the native enzyme. The optimum pH of the immobilized β-amylase shifted slightly to the alkaline side and the pII stability was improved. The optimum temperature of the immobilized enzyme increased by about 5°C and the thermostability of the enzyme did not change compared to the native enzyme.
Using a dual immobilized enzyme system composed of immobilized β-amylase and pullulanase, the effects of operating conditions on the maltose production reaction were examined. To obtain a maltose content of higher than 75% (w/w), it was necessary to operate at a space velocity of below 0.6hr^-1 with a substrate concentration of below 25% (w/w) under the optimum reaction conditions. Also, the dual enzyme system proved to be effective in increasing the maltose content in the product.

View full abstract

An isolated bacterial strain, Corynebacterium sp. C5, could produce trans-4-cyanocyc-1-ohexane-lcarboxylic acid (t-MCC), an intermediate in the chemical synthesis of tranexamic acid, from trans-1, 4-dicyanocyclohexane (t-DCC), through catalysis by two enzymes, nitrile hydratase and amidase. The two enzymes were constitutively formed in cells. The activity of nitrile hydratase increased with the addition of FeSO₄ and isobutyronitrile to the culture medium. The activity of amidase increased with the addition of isobutyronitrile. Peptone, as the nitrogen source, was effective for production of the two enzymes without a decrease in their activities in the stationary growth phase. The cells could produce 92.1 mg/ml t-MCC from 100mg/ml t-DCC on 20-hr incubation in a resting cell system.

View full abstract

Two enzymes, nitrile hydratase and amidase, which participate in the conversion of trans-1, 4-dicyanocyclohexane (tDCC) to trans-4-cyanocyclohexane-1-carboxylic acid (tMCC), a tranexamic acid intermediate, were purified and characterized. Nitrile hydratase was obtained in a homogeneous state. The molecular weight of the native enzyme was 61, 400 and that of the subunit 26.900, indicating a dimer structure. Valeronitrile and butyronitrile were good substrates for the enzyme. The enzyme could also hydrate benzonitrile, p-hydroxybenzonitrile and 4-cyanobenzoic acid. tDCC was exclusively hydrated to trans-4-cyanocyclohexane-1-carboxyamide (tMCMA), further hydration of the nitrile group of tMCMA and t-MCC not being observed. The presence of py rroloquinoline quinone in the enzyme was confirmed. The presence of iron was also confirmed. The amidase of the strain was also purified. The latter enzyme could hydrate tMCMA, yielding tMCC. The enzyme was highly resistant to SH reagents.

View full abstract

To find ammo acid residues which are required for glucoamylase activity, mutant glucoamylase genes were constructed by in vitro mutations of GLU1 DNA encoding Saccharomycopsis fibuligera glucoamylase and introduced into Saccharomyces cerevisiae, and the resulting mutant proteins were assayed for enzymatic activities. Eighteen mutant proteins were obtained by random insertions of a BamHI linker DNA. Six out of 7 proteins with mutations in conserved regions among divergent glucoamylases showed no activities, while 8 out of 11 proteins with mutations in unconserved regions had similar specific activities to a wild-type value, suggesting that the conserved regions are important to the activity. A series of amino-terminal deletion mutants were also constructed. A mutant protein with a deletion of only two amino acid residues from the amino terminus had a significant reduction in the activity, suggesting an essential role for the amino-terminal peptide. Ten mutant proteins with single amino acid replacements were produced by site-directed mutagenesis. Analyses for thermal stability and temperature dependency of these mutant proteins revealed that Ala81, Asp89, Trp94, Arg96, Asp97, and Trp166 are required for wild-type levels of activities, and that at least Ala81 and Asp89 are not essential to catalytic activities, but act in thermal stability.

View full abstract

The Saccharomyces diastaticus glucoamylase encoded by STA1 contains two signal sequences for potent secretion of the enzyme, a hydrophobic leader peptide (HL), and a tract consisting of threonineand serine-rich sequences (TS); hybrid proteins of Escherichia coli β-galactosidase carrying both HL and TS are secreted through the cytoplasmic membrane to the cell-surface fraction of yeast cells, but those carrying either HL or TS are not. To investigate the molecular mechanisms for these signal sequences, we have isolated a dominant mutation, SSD1, which suppresses a secretory defect caused by deletion of these sequences. Yeast cells harboring the mutation secreted hybrid β-galactosidase proteins carrying either HL or TS into the cell-surface fraction. Even β-galactosidase itself was secreted to the cell surface in the mutant. These results suggest that HL and TS interact with a wild-type ssd1⁺ gene product to promote protein secretion.

View full abstract

Purified N-acetylmuramidase (EC 3.2.1.17) produced by Streptomyces rutgersensis H-46 was used as a preservative for bean paste made of adzuki beans (Vigna angularis) without addition of sugar, called raw an. The enzyme was stable during the storage of the sample. The viable cell count in the enzyme-treated sample first decreased as susceptible microorganisms were killed as a result of cell wall degradation by the enzyme and then increased as resistant microorganisms grew. The shelf life of raw an stored at 10°C can be retarded from 48 hr to 109 hr by the addition of enzyme at a concentration of 0.109%. Differences in microflora between control and the enzyme-treated sample was described. Synergistic effects of glycine, EDTA, acetic acid, and lactic acid with the enzyme were also investigated.

View full abstract

Two strains of thermophilic cellulolytic anaerobes, Nos. 138 and 183, were isolated from soil and compost, respectively, and identified as Clostridium thermocellum, based on their morphological, physiological and genetic characteristics. These two isolates decomposed cellulose more efficiently than the type strain, C. thermocellum ATCC 27405. The celA gene of strain No. 138 was cloned onto vector plasmid pBR322 and the hybrid plasmids obtained were introduced into Escherichia coli cells. The cleavage map of the cloned celA gene and the extent of CM-cellulase expression of the cloned gene in E. coli were the same as those of the celA gene from ATCC 27405.

View full abstract

The antioxidative ability of inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) at various water activities (A_w, 0-0.75) and at 40°C was investigated. IMP and GMP exhibited antioxidative ability above A_w 0.25, and this ability was strongest at A_w 0.5. There was no obvious difference between the ability of IMP and GMP. IMP-related substances (hypoxanthine, inosine and ribose 5-phosphate) did not have antioxidative ability, suggesting that both the base and phosphate moieties of 5'-ribonucleotide are necessary to exhibit this ability. The ability of IMP and GMP was weaker than of 2, 6-di-tert-butyl-4-methylphenol (tert-butyl-hydroxytoluene; BHT) and ethylenediamine tetraacetic acid (EDTA). The possible mechanism for the antioxidative ability of IMP and GMP was assumed to be their chelating action.

View full abstract

The distribution coefficients of maltooligosaccharides with degrees of polymerization (d.p.) of 1 to 7 on cation-exchange resins (Amberlites HFS-471X and CG-60) with various divinylbenzene (DVB) contents and alkali-ion forms were determined by the moment analysis of elution curves of the saccharides. The distribution coefficients were largely dependent on both the DVB content and the ionic form, as well as on d.p. The resin with the higher DVB content gave the smaller coefficient. As the radius of the hydrated ion became larger, the distribution coefficient was reduced. The resin-phase diffusion and axial dispersion coefficients were also determined for maltooligosaccharides with d.p. of 1 to 3. Regardless of the DVB content and the ionic form, the resin-phase diffusion coefficient was correlated with the molecular diffusivity of each solute by a parallel pore model with a tortuosity factor of 4.0. The mixing lengths of flow for Amberlites HFS-471X and CG-60 were 1.9 and 1.5 times as long as their mean diameters, respectively. By using the parameters evaluated, the effects of some operating variables on the resolution of maltooligosaccharides with d.p. of i and i+1 (i=1 to 6) are discussed.

View full abstract

The distribution coefficients of maltooligosaccharides with a degree of polymerization of 1 to 7 in cation-exchange resins (Amberlites HFS-471X and CG-60) with various crosslinkages and of counterion forms were used to estimate the swelling pressure of the resins. The distribution coefficient of glucose in the resin with 6% divinylbenzene of various ionic forms was measured by varying the ionic strength of the eluent. Although the resins shrank with increasing ionic strength, the distribution coefficient became larger. These results suggest that the swelling pressure of an ion-exchange resin plays an important role in the distribution of a non-electrolyte such as maltooligosaccharide to the resin. A simple method to estimate the swelling pressure is also described.

View full abstract

Microorganisms that produce 5-methyluridine (ribothymidine) directly from purine nucleosides and thy mine were screened from our stock cultures. Of the 400 strains tested, Erwinia carotovora AJ-2992 was found to possess the most potent ability as to production of 5-methyluridine from guanosine and thymine. In the presence of intact cells of Er. carotovora AJ-2992 as the enzyme source, 222 mM 5-methyluridine was produced from 300 mM guanosine and 300 mM thymine at 60°C on 48 hr incubation. The enzymatic production of 5-methyluridine by Er. carotovora AJ-2992 was found to involve the following two successive reactions via ribose-1-phosphate as an intermediate: phosphorolysis of purine nucleosides to ribose-1-phosphate and purine bases by purine nucleoside phosphorylase, followed by condensation of ribose-1-phosphate and thymine into 5-methyluridine by pyrimidine nucleoside phosphorylase.

View full abstract

Thermostable purine nucleoside phosphorylases, PUNPI and PUNPII, have been purified from Bacillus stearothermophilus JTS 859. The characterization of PUNPI was reported previously. [Hori et al., Agric. Biol. Chem. 53, 2205 (1989)] PUNPII had a molecular weight of 113, 000, consisting of 4 identical subunits (M_w 28, 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, PUNPII was not a SH-enzyme because the enzyme reaction was not inhibited by PCMB and iodoacetic acid, the optimal pH of the enzyme reaction was from 7.0 to 11.0, 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 50.6μ/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.

View full abstract

The effects of eicosapentaenoic acid (EPA, 20:5n-3) on essential fatty acid (EFA)-deficient rats were studied. After low growth and scaly dermatitis in the hind legs due to dietary EFA deficiency were induced by feeding rats an EFA-free 25% casein diet (25C) containing 30% hydrogenated coconut oil with 1% cholesterol (HCO•CHOL) for 8 weeks, they received the 25C diet with 0.19 or 0.57% EPA ethyl ester concentrate added, or 0.02% or 0.38% linoleic acid (LA, 18:2n-6) concentrate (Exp. I), and the HCO CHOL meal including any one of 0.25, 0.50, or 1.00% EPA concentrate, and 0.12 and 0.48% LA concentrate (Exp. II) for an additional 6 weeks. When EFA-deficient rats were fed the EPA in both experiments, body weight was gained to almost reach those of the 0.38 or 0.48% LA-fed group (control), and the dermal symptoms of the hind legs were relieved, though the degree of healing was less than those of the controls. The ratios of eicosatrienoic acid (20:3n-9) to arachidonic acid (20:4n-6) characteristically increased due to EFA deficiency were reduced to the level of the control in the liver and heart by addition of the EPA concentrate.

View full abstract

The effects of Freund's adjuvants on antibody production in chickens against E. coli whole cells were examined. The levels of anti-E. coli IgG antibodies in serum were higher when Freund's complete (FCA) or incomplete adjuvant (FIA) was administered than that without adjuvant. Production of antibodies recognizing E. coli cells and their lipopolysaccharide was enhanced by FIA, while both FIA and FCA enhanced production of antibodies recognizing outer membrane components. In contrast, serum IgM antibody levels were higher when no adjuvant was used. Anti-E. coli IgG antibodies in serum were efficiently transferred to egg yolk, giving antibody activity in egg yolk similar to that in serum. However, anti-E. coli IgM antibodies were not detected in the egg, suggesting that egg (white) IgM was not influenced by antigenic stimulation of the humoral immune system. Antimicrobial activity of the egg yolk IgG was highest when the bacteria antigen was injected with FIA.

View full abstract

The formation of γ-glutamyltaurine (γ-GluTau) from L-glutamine and taurine was carried out with γ-glutamyltranspeptidase (GGT) of Penicillium roqueforti (IFO 4622). For GGT preparation, a wheat bran koji extract prepared with P. roqueforti (IFO 4622) was used. 180mM γ-GluTau was formed, with a yield of 36%, in a reaction mixture comprising 500 mM L-glutamine, 500 mM taurine, 100 mM Tris-HCl buffer (pH 8.5) and 0.6 units/ml of GGT. The γ-GluTau formed was isolated by ion exchange column chromatography, and identified by infrared, ¹H-NMR and mass spectroscopic analyses.

View full abstract

Polymyxin acylase from Pseudomonas sp. M-6-3 can deacylate not only polymyxin antibiotics, but also N-fatty acyl-peptides and N-fatty acyl-amino acids. We found that this enzyme causes intramolecular N^2→_←N⁶ acyl transfer in monooctanoyl-L-lysine; when N²-octanoyl-L-lysine is the substrate, N⁶-octanoyl-L-lysine is produced at pH 10.5, but when N⁶-octanoyl-L-lysine is the substrate, N²-octanoyl-L-lysine is produced at pH 8.0. In these reactions, the deacylation proceeded gradually at the final stage and eventually, both N²-octanoyl-L-lysine and N⁶-octanoyl-L-lysine were hydrolyzed to L-lysine and octanoic acid. Furthermore, this enzyme showed intermolecular acyltransferase activity, transferring several N-octanoyl-DL-amino acids to N-octanoyl-hydroxylamine. This acyltransfer ability of polymyxin acylase offers a new method of enzymic N-acylation of compounds containing amino components.

View full abstract

Bacillus thuringiensis var. kurstaki HD-255 was found to produce an extracellular, thermostable protease after the end of the vegetative growth phase. The purified enzyme had a molecular weight of 34, 000 according to sodium dodecyl sultate-polv acrylamide gel electrophoresis and an isoelectric point of 9.0.
Its proteolytic activity was inhibited by an active-site inhibitor of serine protease, phenylmethyl-sulfonyl fluoride, and also by an SH-modifying reagent, p-chloromercuribenzoic acid, suggesting that the enzyme is one of a subfamily of SH-containing serine proteases.
The enzyme showed maximal proteolytic activity at 70°C and pH 8.5-9. The most interesting characteristic was its thermostability; it retained 88.4% of its initial activity at pH 8.7 and 60°C after more than 7hr incubation in the presence of 2mM CaCl₂.

View full abstract

α-Tocopherol was reacted with an alkylperoxyl radical at 37°C in benzene. 2, 2'-Azobis(2, 4-dimethylvaleronitrile) was used to generate the alkylperoxyl radicals. The reaction products of α-tocopherol were isolated by reverse-phase and normal-phase high performance liquid chromatography, and their structures were characterized. They were four stereoisomers of 8a-(l-cyano-1, 3-dimethyl)butylperoxy-α-tocopherone, spirodiene dimer and two geometrical isomers of the trimer. When α-tocopherol at a low concentration was reacted with AMVN, the major products were 8a-alkylperoxy-α-tocopherones. On the other hand, the products of the alkylperoxyl radical with α-tocopherol at a high concentration were spirodiene dimer and trimer in addition to the 8a-alkylperoxy-α-tocopherones.

View full abstract

Soybean proteins were subjected to phosphorylation with cyclic adenosine monophosphate-dependent protein kinase (A-kinase). As a result, acidic subunits of the 11S fraction were found to be phosphorylated by A-kinase. To estimate the effect of the phosphorylation, 11S acidic subunits were isolated and subjected to A-kinase phosphorylation. The optimal enzyme amount and Mg²⁺ concentration for the phosphorylation of 11S acidic subunits were determined to be 1.51/ml and 1.6mM, respectively. The rate of phosphorylation was 2mol/mol acidic subunits (MW 38, 000) under the above conditions. The protein structures of 11S acidic subunits, as determined from UV and CD spectra, were slightly affected by the enzymatic phosphorylation.

View full abstract

In studies on the production of S-adenosyl-L-methionine (AdoMet) by Saccharomyces sake Kyokai No. 6 in a bench-scale fermentor, the rate of growth of the organism in a medium containing sucrose and urea as carbon and nitrogen sources, respectively, increased with a higher agitation speed (500 rpm) or sucrose feeding, but the cellular content of AdoMet was lower than that with a lower agitation speed (300 rpm) or ethanol feeding. The additin of L-methionine was necessary for enhancement of both growth and AdoMet production. The L-methionine added (15g/1) was efficiently incorporated into the cells during the course of the cultivation, about 20% of the L-methionine being found as AdoMet in the cells. The ultraviolet absorbance (258 nm) based on the extracted AdoMet comprised 73% of the total ultraviolet absorbance of material extracted from the cells, whereas that based on S-adenosylhomocysteine was very low (1.4%).

View full abstract

DNA-synthesis stimulatory activity was found in a tryptic hydrolysate of bovine β-caseins using BALB/c3T3 cells, and an active peptide was identified to be the fragment Ala[177] to Arg[183]β-CN(f177-183). The activity of this casein fragment was confirmed using a synthetic peptide of β-CN(f177-183).

View full abstract

Rosefuran and per ill en e were isolated from the opisthonotal glands secretion of the Tyrophagus neiswanderi mite. A facile synthesis of the former was accomplished by the lithiation of 3-methylfuran with butyllithium-tetramethylethylenediamine, and a subsequent reaction with 3-methyl-2-butenylbromide. A mixture of rosefuran, 4-methyl-2-(3-methyl-2-butenyl)-furan and 3-methyl-2, 5-bis(3-methyl-2-butenyl)-furan was obtained in a high yield. The occurrence of these and other recently identified monoterpenoids, namely, α- and β-acaridial and 2, 3-epoxyneral, is discussed relative to the CP-Sil 19 CB column profiles as worthy for the identification of economically important mites of the genus Tyrophagus, which is a difficult but essential task for the successful application of the speciesspecific alarm pheromones in the management of these pests.

View full abstract

Iron(II)/EDTA/ascorbate-mediated oxidative damage to specific amino acid residues (tryptophan) of serum albumin was studied. The active species generated by Fe(II)/EDTA/ascorbate preferred to react with tryptophan residues rather than histidine or other amino acids. The observation of preferential damage to tryptophan residues of the protein was fully suported by a model experiment using a tryptophan analogue. The reaction of Fe(II)/EDTA/ascorbate to the protein was significantly suppressed by mannitol and dimethysulfoxide, suggesting the participation of the hydroxyl radical generated via Fenton's reaction. The result was supported by the hydroxyl radical assay using 2-deoxyribose.

View full abstract

We have investigated the utilization of a variety of alkylbenzenes by P. putida strains and found that a strain harboring the OCT plasmid assimilated ethylbenzene. The linkage between the determinant for the degradation of ethylbenzene (Etb⁺ phenotype) and the OCT plasmid was inferred from conjugation experiments. The growth characteristics of the strains carrying mutations in the alk genes of the OCT plasmid which determine the assimilation of n-alkanes indicated that alk B, alk A, and alk R should be responsible for the degradation of ethylbenzene. The exposure of ethylbenzene to the P. putida strain harboring the CAM-OCT plasmid resulted in the accumulation of β-phenylethyl alcohol. A possible degradation pathway for ethylbenzene including the terminal oxidation of the alkyl side chain was proposed.

View full abstract