Cyclomaltodextrin glucanotransferase was purified from B. circulans C31 through two successive steps of starch and Biogel column chromatography. The enzyme was purified up to 90-fold with a 30% yield. Its molecular weight was around 103, 000. The purified enzyme converted 28% of the soluble starch to β-cyclodextrin at pH 7.0 and a substrate concentration of 5%. The optimum pH for the enzyme was found to be 5.5. The optimum temperature was 60°C. The enzyme was stable from pH 5.5-9.0 and up to 50°C.

View full abstract

A simple and rapid method was developed for measurement and identification of the nucleotides in vegetable RNA. The reliability of the method was verified by analysis of yeast RNA of known nucleotide composition. Natural enzymes of the vegetables involved in degradation of RNA were inactivated by zinc chloride during vegetable homogenization followed by a heat treatment. The total endogenous RNA was then hydrolyzed in situ by nuclease Pl enzyme at 60°C for 1 hr. The hydrolysate was extracted with cold perchloric acid followed by a Freon-octylamine extraction step to remove the acid before HPLC analysis. The four major nucleotides in the RNA hydrolysate were then identified by their isocratic separation using a Partisil 10 SAX anionic column and 3% methanol-8 mM K-phosphate buffer of pH 4.15 as an eluent. An alternative alkaline rather than enzymatic hydrolysis of vegetable RNA in situ was found to be unsuitable for HPLC analysis.

View full abstract

The production of D-pipecolic acid from DL-pipecolic acid using a microbe was investigated. Some bacteria which assimilated L-pipecolic acid in preference to D-pipecolic acid and accumulated L-α-aminoadipic acid extracellularly when cultivated on DL-pipecolic acid were isolated. When one of these bacteria, which was designated as Alcaligenes sp. 309B1, was cultivated on a medium containing 5% of DL-pipecolic acid, it assimilated all of the L-pipecolic acid, with most of the D-pipecolic acid not being assimilated. Ninety-five % of the D-pipecolic acid remained after the complete disappearance of L-pipecolic acid from the culture broth. The amount of L-α-aminoadipic acid accumulated in the broth reached 56% of the total quantity of L-pipecolic acid.

View full abstract

L-Prolyl and other aminoacyl derivatives of ascamycin (1) were synthesized by a condensation reaction of N⁶-t-butyloxycarbonyl-2-chloro-9-(2', 3'-O-isopropylidene-5'-O-sulfamoyl-β-D-ribofuranosyl)adenine (9) with the corresponding t-butyloxycarbonylaminoacylimidazole in the presence of Cs₂CO₃ in DMF as the key step. The L-prolyl derivative (2) and L-phenylalanyl derivative (3) as well as 1 showed selective toxicity against Xanthomonas citri. The L-prolyl-L-prolyl derivative (5) as well as dealanylascamycin (6) showed broad toxicity against various Gram-negative and Gram-positive bacteria. The D-alanyl derivative (4) lost its antibacterial activity. 2 was the best substrate (15.3M/min per mg of protein) for an Xc-aminopeptidase (an ascamycin-dealanylating enzyme from X. citri cells) among these analogs. This enzyme scarcely hydrolyzed 4 (0.2 M/min per mg of protein). The substrate specificity of the enzyme accounts for antibacterial activity of the analogs. 2 showed greater selective toxicity against Kirsten sarcoma virus transformed Balb3T3 (KN-3T3) cells than against nontransformed cells (Balb 3T3).

View full abstract

A recombinant plasmid containing a DNA segment complementary to the mRNA of the North American firefly, Photinus pyralis, luciferase was constructed and cloned into Escherichia coli. The cDNA was inserted into a yeast expression vector, AAH5, designed to express active luciferase under the control of the yeast alcohol dehydrogenase promoter. When introduced into the yeast, Saccharomyces cerevisiae, the resultant plasmid directed the synthesis of enzymatically active firefly luciferase, the activity of which was inhibited by antiserum raised against the purified firefly luciferase.

View full abstract

The L-leucine production by an L-leucine producing mutant, H-1204, of Corynebacterium glutamicum was unstable because the strains (M- and V-types) exhibiting reduced L-leucine productivity appeared during cultivation. Stabilization of the L-leucine production was attempted on the basis of the properties of various strains: L-type, an original L-leucine producer (valine leaky strain); M-type, a prototrophic strain; and V-type, a leucine auxotrophic strain. The addition of L-valihe to the medium was found to be most effective. L-Valine had a double effect in that it not only stimulated the growth of the L-type but also inhibited the growth of the V-type as an antagonist as to its leucine uptake. It was also revealed that C. glutamicum has two different transport systems for branched chain amino acids and dipeptides, respectively.

View full abstract

A comparative investigation was carried out on functional properties of three kinds of soymilk curds, which were prepared with an enzyme, calcium chloride and an acid. The enzyme-curd showed high emulsion stability over wide ranges of pH and temperature, whereas the Ca- and acidcurds produced lower values at pH 4-6 and temperatures higher than 40°C. The foam stability of each soymilk curd was low, but the enzyme-curds prepared from defatted soymilk had higher foam stability than the others over wide ranges of pH and temperature. The lyophilized enzyme soymilk curd solubility in water was higher than the others, although its water-holding capacity was lower than the others. None of the soymilk curds showed gelation. The experimental results suggest that enzyme-curd could be widely applicable to various food items with its properties of a remarkably high emulsion stability and a very smooth texture.

View full abstract

Oil-in-water (4:6) emulsions stabilized with bovine serum albumin (BSA) (0.225 HIM), at pH 7, were used to study the effects of molecular association on the viscosity of emulsions and emulsifying activity (EA). Anions -TCA^- and -SO₄^- had different effects on the intermolecular association of BSA. Aggregates (A) of BSA comprising A₁, A₂, A₃, A₄, A₅, A₆ and A₇ were detected in the control and the anion (100 mM) containing emulsions. The extent of association of BSA was in the order; control > -SO₄^- > -TCA^-. The EA also showed the same order whereas the viscosity of emulsions showed the reverse order. High energy input per unit volume (>684 10⁶ J•m^-3) caused molecular dissociation and a concomitant decrease in EA. Apparently, a certain degree of association is required for EA of BSA.

View full abstract

A gene encoding human cardiodilatin (hCDD; a vasodilating polypeptide located on the N-terminal portion of γ human atrial natriuretic polypeptide) was fused to the secretion signal coding sequence of the Escherichia coli outer membrane protein F (OmpF). This hybrid gene was preceded by a chemically synthesized consensus ribosome binding sequence and was expressed in E. coli under the transcriptional control of the tac (trp:lac fusion) promoter. On the addition of isopropy-β-D-thiogalactopyranoside (IPTG), cells secreted about 17-30mg of hCDD per liter broth. On induction at OD₆₅₀ = 2.90, the majority of the hCDD produced (75%) was processed precisely and secreted into the periplasmic space. These results demonstrate that E. coli cells are able to synthesize and secrete high levels of this human protein with a prokaryotic signal sequence.

View full abstract

The transxylosylation reaction products of β-xylosidase-1, excreted by Penicillium wortmanni IFO 7237 using β-(1→4)-xylobiose as substrate, have been separated by chromatography on activated charcoal into four fractions, designated as P-1, P-2, P-3, and P-4, respectively. They were further purified by preparative paper chromatography. The characterization and structural analysis were done by measurement of the degree of polymerization (DP) and specific rotation followed by methylation analysis. Moreover, the enzymatic structural analysis of transxylosylation products, with high performance liquid chromatography (HPLC), allowed the confirmation of each structure. The first product, P-1, was β-(1→3)-xylobiose and the second, P-2, was β-(1→4)-xylotriose, but, P-3 was O-β-D-xylopyranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose or isomeric xylotriose and P-4 was assumed to be O-β-D-xylopyranosyl-(1→4)-[O-β-D-xylopyranosyl-(1→3)]-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose.

View full abstract

The extracellular glutaminase from Aspergillus oryzae MA-27-IM was found to have γ-glutamyltranspeptidase (GGT) activity, and the properties of the enzyme as to this reaction were investigated.
Temperature, *pH and various reagents affected the GGT reaction in the same way as the glutaminase reaction. As the concentration of glycylglycine increased, the formation of γ-glutamylglycylglycine was enhanced, while the hydrolysis of glutamine was depressed. When L-γ-glutamyl-p-nitroanilide was used as a γ-glutamyl donor, the enzyme could utilize some peptides containing glycine residues at their C-termini as γ-glutamyl acceptors, but the reaction rates were lower than with glycylglycine, while free amino acids could not serve as γ-glutamyl acceptors. Further investigation suggested that the GGT reaction proceeded through a ping-pong Bi Bi mechanism.

View full abstract

The effects on lipolysis in fat cells of synthetic peptides with different amino acid residues added to the N-terminal region of the sequence Phe-Phe-Phe were examined. Several synthetic peptides elicited free fatty acid (FFA) release from fat cells, the order of efficacy of their lipolytic actions being, Arg-Glu--Arg-Glu-Gly- > Glu-Arg-Gly- > Lys-Asp- - Lys-Asp-Gly- - Glu-Arg- > Asp-Lys-Gly-. Arg(NO₂)-Glu-Gly-Gly-Phe-Phe-Phe did not cause any release of FFA from fat cells.
Arg-Glu-Phe-Phe-Phe stimulated lipolysis of fat cells even when calcium ions were not present in the reaction mixture, but other peptides required calcium ions for their lipolytic actions. The β-adrenergic blocker propranolol completely inhibited the lipolysis stimulated by Arg-Glu-Gly-Phe-Phe-Phe or Arg-Glu-Phe-Phe-Phe. The α-adrenergic blocker phenoxybenzamine had little effect on the lipolytic action stimulated by these two peptides.
Lys-Asp-Gly-Phe-Phe-Tyr and Asp-Lys-Gly-Phe-Phe-Tyr also stimulated lipolysis, the stimulatory effect of the latter being about 58% of that of the former. Glu-Arg-Gly-Phe-Phe-Tyr and Arg-Glu-Gly-Phe-Phe-Tyr did not cause any release of FFA from fat cells.

View full abstract

We isolated a bacterial strain, NK-330, from Japanese soil which inhibited the growth of Aspergillus parasiticus NRRL 2999 on a PDA plate. Later, we isolated an inhibitory strain, NK-C3, as a contaminant. Both strains inhibited not only the growth of A. parasiticus NRRL 2999 but also that of A. flavus NRRL 3357. These strains were gram-positive rods, and were identified as Bacillus subtilis. They inhibited the growth and aflatoxin-production of NRRL 2999 or NRRL 3357 in peanuts or corn. Moreover, cultures of these strains markedly inhibited the growth of NRRL 2999 or NRRL 3357, and also inhibited the aflatoxin-production completely.

View full abstract

The fructosyl transfer to sucrose was investigated, and Aspergillus niger ATCC 20611 was selected as the most suitable strain for fructooligosaccharide production. This strain showed very high enzyme productivity, and its transfructosylating activity was very strong compared to its hydrolyzing activity. Fructooligosaccharides could be more effectively prepared with a higher concentration of sucrose if use could be made of an enzyme having higher transfructosylating ability. Treatment of 50% (w/v) sucrose with the A. niger enzyme afforded a mixture of fructooligosaccharides with inulin-type structures of 1^F(1-β-fructofuranosyl)_n-sucrose (n=1 to 3). The individual saccharides could be separated by the combination of a carbon column chromatography and preparative HPLC.

View full abstract

Changes in the kind and level of endogenous gibberellins (GAs) in the shoot and ear of rice (Oryza saliva L. japonica variety, cultivar Nihonbare) through its life cycle were investigated. Gibberellin A₁₉ (GA₁₉) was the major GA, and GA₁, GA₂₀, GA₂₉ and GA₅₃ were minor GAs in the shoot of rice. Besides these 13-hydroxylated (13-OH) GAs, two non-13-hydroxylated (13-H) GAs, namely GA₄ and GA₃₄, were identified in the shoot (containing the ear) collected at the heading stage.
Further studies on the distribution and changes in the level of GAs in reproductive organs revealed that GA₄ was highly concentrated in anthers collected just before anthesis, but was not detected in immature seeds collected about a week after anthesis. These results suggest that 13-H GAs may be biosynthesized in specific organs at a specific stage of the life cycle, and that they may be concerned with the regulation of reproductive growth, while vegetative growth is regulated by 13-OH GAs, probably GA₁.

View full abstract

A new ubiquinone homologue was isolated from Chaetomium funicola JS 525. Its chemical structure was found to be 2, 3-dimethoxy-5-methyl-6-IX, X-tetrahydrofarnesylfarnesylgeranylgeranyl-1, 4-benzoquinone based on the results of mass and ¹H-NMR spectra containing a spin decoupling experiment. This fungus also contains dihydrogenated ubiquinone with ten isoprene units as a minor component, and this ubiquinone was presumed to be composed of two different homologues saturated at the 9th and 10th (terminal) units from a benzoquinone ring.

View full abstract

Ester synthesis by the purified lipase from Pseudomonas fragi 22.39 B was investigated. The lipase could synthesize esters from oleic acid and primary or secondary alcohols, but it did not react with tertiary alcohols. Also, the enzyme could use the fatty acids with straight carbon chains as substrates. The activity was enhanced by increasing the carbon number of the fatty acid, but this is not the case for alcohol. The lipase synthesized glycerides from glycerol and oleic acid. 1(3)-Monoolein and 1, 3-diolein were the main products and triolein was minor. Synthesis of monoester such as butyl oleate was scarcely affected by the water content in the reaction mixture, while that of glyceride of oleic acid was much affected.

View full abstract

The addition of F₂ to methyl 5-acetamideo-2, 6-anhydro-4, 7, 8, 9-tetra-O-acetyl-2, 3, 5-trideoxy-D-glycero-D-galacto-non-2-enopyranosonate (6) in acetic acid gave a mixture of methyl 5-acetamido-4, 7, 8, 9-tetra-O-acetyl-2, 3, 5-trideoxy-2, 3-difluoro-β-D-erythro-L-gluco-2-nonulopyronosenate (7), methyl 5-acetamido-2, 4, 7, 8, 9-penta-O-acetyl-3, 5-dideoxy-3-fluoro-β-D-erythro-L-gluco-2-nonulosopyranosonate (8) and methyl 5-acetamido-2, 4, 7, 8, 9-penta-O-acetyl-3, 5-dideoxy-3-fluoro-β-D-erythro-L-manno-2-nonulopyranosonate (9). On the other hand, the addition of acetylhypofluorite to 6 in acetic acid gave 8 concomitant with small amounts of 7 and 9. The structural elucidation of 7, 8 and 9 on the basis of their ¹H-, ¹³C- and ¹⁹F-NMR spectra are described. Saponification of the ester groups of 7, 8 and 9 gave the corresponding N-acetyl 3-fluoro-neuraminic acids (14, 15 and 16). Compound 14 was a potent inhibitor against neuraminidase.

View full abstract

Abrus precatorius agglutinin (APA) bound to an acid-treated Sepharose 4B column was reduced with 2-mercaptoethanol and the A-chain released from APA was eluted. After repeating this procedure three times, the remained B-chain was eluted from the column with lactose. Both the chains were put through fast protein liquid chromatography using a Mono Q column and pure A-, B₁-, and B₂-chains were isolated.
The A-, B₁-, and B₂-chains were acidic glycoproteins having molecular weights of 30, 000, 32, 000, and 33, 000, and isoelectric points of 3.8, 5.4, and 5.6, respectively. The B₁ and B₂-chains have identical N-terminal sequences and similar amino acid compositions, and contain 6.80 and 9.12% sugar, respectively. These results indicated that the difference in their molecular weight might be due to the variation in sugar contents. Furthermore, the very low cytoagglutinating activity of the isolated B-chains suggested their monovalent nature.

View full abstract

Two protein-biosynthesis inhibitory proteins, luffin-a and -b, were isolated from the seeds of the sponge gourd (Luffa cylindricd). Luffins were extracted from defatted meal of seeds of the sponge gourd with water at pH 4.0 and purified by gel filtration through a Sephadex G-75 column followed by CM-cellulose column chromatography and FPLC with Mono S column. Luffin-a and -b were basic proteins with isoelectric points of about 10, with molecular masses of 28, 000 Da and 29, 000 Da, respectively, as judged by SDS-PAGE. Luffin-a and -b were found to be homologous proteins having similar amino acid compositions (except threonine and proline), and having N-terminal sequences of Asp-Val-Arg-Phe-Ser-Leu-Ser-Gly- and Ala-Asn-Val-Ser-Phe-Ser-Leu-Ser-Gly-, respectively. Inhibitory activities of luffin-a and -b on cell-free protein synthesis were approximately 5.5 and 1.5 fold, respectively, stronger than that of ricin A-chain.

View full abstract

6-Arylamino-1, 2, 4-triazolo[3, 4-b][1, 3, 4]thiadiazoles (IV) were synthesized by cyclo-dehydrosulphurization of corresponding thioureas (III), which in turn were prepared by the condensation of N-amino-3-mercapto-1, 2, 4-triazoles (II) and aryl isothiocyanates (I) in dry dimethylformamide. These triazolo thiadiazoles (IV) were screened for their fungicidal activities of A. niger and H. oryzae.

View full abstract

Microorganisms that produce ribavirin(1-β-D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide; virazole®) directly from pyrimidine nucleosides and TCA (1, 2, 4-triazole-3-carboxamide) were screened from our stock cultures. Of the 400 strains tested, 16 were isolated as ribavirin-producers from uridine or cytidine. In particular, Enterobacter aerogenes AJ 11125, Bacillus brevis AJ 1282 and Sarcina lutea AJ 1212 were found to possess potent activities of ribavirin production from them. In the presence of intact cells of Enterobacter aerogenes AJ 11125, which was selected as the best strain, 110.2mM and 67.6mM ribavirin were produced from uridine and cytidine, respectively, on 96 hr reaction at 60°C. In addition, this strain could also produce ribavirin from guanosine, but could not produce it from orotidine, which is also a pyrimidine nucleoside.

View full abstract

Mixed oils and the corresponding interesterified oils of soybean, palm and coconut were investigated as to their thermal deterioration during continuous heating at 180°C. The extent of thermal deterioration of each oil increased in proportion to the degree of unsaturation, there being nearly no difference at all between the mixed oils and the corresponding interesterified oils. However, the refractive index of the interesterified oils increased more rapidly than that of the others. We supposed that the rapid increase in this value in the case of interesterified oils may be caused by an increase in the total number of triacylglycerol molecules, which were composed of easily oxidizable unsaturated and fairly stable saturated fatty acids, resulting in polymerization.

View full abstract

The trypsin and chymotrypsin inhibitory activities of two families of inhibitors, the Kunitz family and the Bowman-Birk family, were screened for in seeds of 34 leguminous species by gel nitration. Results were compared with the morphological classification of leguminous plants. There seemed to be a relationship between the inhibitors found and the evolution of leguminous plants. That is, the seeds of the more primitive leguminous species contained mainly the Kunitz family inhibitors and those of the more advanced ones had the Bowman-Birk family inhibitors. Results suggested that the Kunitz family inhibitors in leguminous seeds have gradually been replaced by the Bowman-Birk family inhibitors in the process of evolution.

View full abstract

From an unidentified Fusarium sp., eight substances possessing a 1, 4-naphthoquinone skeleton were isolated (F2-F9) and their structures were elucidated from the physical and chemical evidence. The effect of these compounds together with 1, 4-naphthoquinone, 5-hydroxy-1, 4-naphthoquinone and 5, 8-dihydroxy-1, 4-naphthoquinone on the pollen germination of Pinus thunbergii Parl was examined. 5, 8-Dihydroxy-1, 4-naphthoquinone showed marked inhibitory activity.

View full abstract

Benzyl 6-O-β-D-apiofuranosyl-β-D-glucopyranoside and benzyl 6-O-α-D-apiofuranosyl-β-D-glucopyranoside were synthesized to determine the stereoschemistry of the glycosidic bond of a metabolite of benzoic acid isolated from Lemna paucicostata 151.

View full abstract

The gag gene of human T cell leukemia virus type I (HTLV-I) was fused to a lac'Z segment and fused proteins with gag-β-galactosidase were efficiently expressed under the control of the tryptophan promoter in Escherichia coli. It was found that not only the sequence around the initiation codon, but also the 3'-part of the gag gene segment greatly affected the efficiency of expression for these hybrid proteins. Modifying both the 5' and 3' regions, high level expressions of two forms of gag antigens have been achieved.

View full abstract