The 130-kDa insecticidal protein (IP) of Bacillus thuringiensis subsp. aizawai is proteolytically processed in the gut juice of susceptible insect larvae to yield an insecticidally active 60-kDa fragment. Twenty-seven mutant IP genes with the replacement of codons for Arg and Lys with codons for Gln in the active fragment and its adjacent regions of the 130-kDa IP were constructed by site-directed mutagenesis and expressed in Escherichia coli cells. The produced mutant IPs at Arg87, Arg131, Arg198, Arg311, Arg368, Arg402, Arg458, Arg502, Arg512, Arg524, Arg526, Arg528, and Arg601 had reduced insecticidal activity against Spodoptera litura larvae. The mutant at Arg601 was sensitive to proteolytic digestion in the gut juice of S.litura larvae. Although the mutants at Arg619, Lys622, and Lys637 had nearly the same activity as that of the wild type, the mutant with the triple replacement at Arg619, Lys622, and Lys637 was 2.5 times more active against S.litura larvae than the wild type. This triple mutant showed a slightly different processing profile in the gut juice than that of the wild type.

View full abstract

A protease- and glycosidase-negative mutant F-2035, of Aspergillus awamori var. kawachi has been isolated that produces a glucoamylase that contains a large carbohydrate moiety and has enhanced ability to digest raw starch (GA MU-H ; MW, 110, 000). This enzyme digested raw corn starch 2.5 times faster than did the parental glucoamylase I (GA I ; MW, 90, 000). When grown from an enriched seed culture, this mutant also produced a glucoamylase with less ability to digests raw starch (GA MU-L ; MW, 110, 000). Its activity was 25% of that of GA MU-H with raw starch. Both GA MU-H and GA MU-L proved to be identical to GA I in terms of adsorption to raw starch, molar activity against gelatinized starch, amino acid composition, and terminal amino acid sequence. The carbohydrate contents of GA I, GA MU-H, and GA MU-L were 17%, 33%, and 33% by weight, respectively. The carbohydrates of GA I and GA MU-H were mostly mannose, but that of GA MU-L was composed of mannose (71%) and glucose (26%). Partial removal of the carbohydrate from GA I and GA Mu-H by Turbo glycosidase caused a parallel decrease in the ability to digest raw starch. Thus, the carbohydrate moiety of the glucoamylase molecule, in particular the mannose residues, appears to be important in the digestion of raw starch, and promote the hydration of micelles of raw starch but not the actual adsorption of the enzyme to raw starch.

View full abstract

We have reported that insulin-like growth factor II (IGF II) was produced as a fusion protein in Bombyx mori (silkworm) larval bodies infected with recombinant B.mori nuclear polyhedrosis virus [J.Gen.Virol., 68, 2599-2606 (1987)]. In this study, the purification of IGF II from the infected silkworms is reported. The fusion protein was extracted with 6.0M guanidine-HCl from the infected larval bodies homogenized in water. The use of organic solvents to remove the impurities, such as lipid derived from the larval bodies, was a very effective method of purification. IGF II was released from the partially purified fusion protein by treatment with CNBr, purified by HPLC, and refolded by air-oxidization. Refolded IGF II had an identical primary structure including disulfide bonds and showed identical thymidine uptake stimulation activity with human IGF II. Furthermore, protein disulfide-isomerase was shown to be able to refold scrambled IGF II rapidly.

View full abstract

Immature seeds of loquat (Eriobotrya japonica) were collected from developing fruits 90 days after full bloom. After purifying the acidic ethyl acetate-soluble fraction by several chromatographic procedures, gibberellin fractions were surveyed by combined gas-liquid chromatography/mass spectrometry. Consequently, GA₉, GA₁5, GA₂4, GA₃5, and GA₅0 were identified. Additionally, six GA-like substances were detected, and two were determined to be new GAs, GA₈0 (11β-hydroxy-GA₇), and GA₈4 (11β-hydroxy-GA₉). Based on these results, an early-11-hydroxylation biosynthetic pathway in the seed of loquat is suggested.

View full abstract

Two kinds of melanoidins formed between glucose and glycine with average molecular weights of 800 (low-MW MEL) and 3000 (high-MW MEL) were investigated for their absorption and distribution in rats. Seven hours after its oral administration, [¹4C] low-MW MEL underwent absorption by 12.8%, which was distributed in liver, kidney, urine, blood, and breath (CO₂) with percentages of 2.2, 0.5, 5.7, 1.2, and 3.1, respectively. On the other hand, [¹4C] high-MW MEL was absorbed to a lesser extent (less than 1% of the initial administration). The results of gel permeation chromatography analyses for melanoidins showed that [¹4C] high-MW MEL and [¹4C] low-MW MEL with average MWs of 2000 and 500, respectively, were excreted into the urine, and that 80% of [¹4C] low-MW MEL was present in a from bound to protein in the plasma. The protein-bound MEL had a desmutagenic activity against Trp-P-1. It thus is reasonable to consider that absorbed MEL (MW above 500) and protein-bound MEL may actually act as desmutagens in vivo.

View full abstract

The influences of radical generators, radical scavengers and chelating agent on crude actomyosin, which was prepared from frozen surimi (raw fish meat paste) of Alaska pollack, were investigated by incubating crude actomyosin solutions at pH 7.0 containing 0.45M KCl at 40°C for 30min, and subsequently analyzing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polymerization of crude actomyosin was significantly increased by radical generators (1mM 2, 2'-azobis (2-amidinopropane) dihydrochloride and 0.1mM H₂O₂ plus 9μM FeSO₄). Although polymer formation by AsA was significantly inhibited by removing metal with a chelating agent (4mM diethylenetrinepentaacetic acid), it wasn't inhibited by radical scavengers. Further, it was clarified that an increase in the ionic strength of the solution decreased the ability of the radical scavenger, and that the formation of polymers by radical generators was not suppressed by radical scavengers in the presence of 0.45M KCl. These results suggest the possibility that radicals generated from AsA participated in the polymerization of crude actomyosin. Moreover, the occurrence of radicals derived from AsA was also observed during the formation of heat-induced fish gel (kamaboko).

View full abstract

A cell-wall preparation from the cells of Elsinoe leucospila, which produces elsinan extracellularly when grown on sucrose or glucose-potato extract medium, was fractionated systematically. The heteropolysaccharide that was released by treatment with Actinase E digestion, comprised D-mannose, D-galactose, and D-glucose (molar ratio, 1.5 : 1.0 : 0.1). Methylation, mild acid hydrolysis, and ¹3C-NMR studies suggested that the polysaccharide contains a backbone of α-(1→6)-linded D-mannose residues having two kinds of side chains, one attached at the O-4 with single or short β-(1→6)-linked D-galactofuranosyl residues, and the other attached at O-2 with short side chains, most probably, of α-(1→3)-linked D-mannopyranosyl residues. A moderately branched D-glucan fraction, obtained from the cold alkali extract, was fractionated to give an antitumor-active purified β-(1→3)-glucan having branches of single β-D-glucosyl groups, one out of eight D-glucose residues being substituted at the O-6.

View full abstract

Some polysaccharides extracted from ningyotake, the fruiting body of Polyporous confluens, with hot water (100°C, FI), 1% ammonium oxalate solution (100°C, FII), and 5% sodium hydroxide solution (80°C FIII) in that order. These polysaccharide fractions were further fractionated by ethanol precipitation and gel filtration on the column of Toyopeart HW-65F with 0.3M sodium hydroxide solution. A strong antitumor activity were found in five xyloglucan-protein complexes, FI-2-a, -b, -c ; FII-2-a, and FIII-2-a. Analyses of physico-chemical properties and IR- and NMR-spectra of these active fractions showed that their main components were (1→3)- ; (1→6)-β-D-glucopyranans containing a small amount of xylose residues and 2-3% of protein. A mucilaginous polysaccharide (N-PS) prepared from the liquid cultured mycelial broth of the fungus, consisted of (1→6)- ; (1→3)-β-D-glucopyranan. Antitumor activities were also found in a subfraction that was purified from N-PS by gel filtration. Also, a strong antitmor activity was found in a polyol polysaccharide and in a formolysis product from N-PS.

View full abstract

The biosynthesis of alterporriol A, a metabolite of Alternaria porri (Ellis) Ciferri, was elucidated by incorporation experiments with single- and double-labelled sodium acetates, Na[2-¹³C], [1-¹³C, and [1, 2-¹³C₂] acteate, Experimental tests revealed that macrosporin (1) and altersolanol A (4), other metabolic pigments of Alternaria porri, were formed through the octaketide pathways, and that alterporriol A (2) was formed by the oxidative coupling of 1 and 4. It is also assumed that alterporriol B (3), an atropisomer of alterporriol A, can be biosynthesized by the same process as that for alterporriol A.

View full abstract

The culture conditions for red pigment formation were investigated in the cultured cells of Carthamus tinctorius. In suspension culture, magnesium sulfate strongly inhibited the red pigment formation. In suspension culture of C.tinctorius, the cells in before-25th subcultures did not require phytohormones for the red pigment formation, however, the phytohormones (NAA and kinetin) were necessary for red pigment formation by cells in the subcultures following them. Red pigment formed by cultured cells of C.tinctorius consisted of several compounds. HPLC and UV spectra analysis indicated that these compounds are different from carthamin.

View full abstract

A facultative alkaliphilic strain of Bacillus sp. C-125 grown at alkaline pH had many sinuous peritrichous flagella and was highly motile. However, most of the cells grown initially at pH 7 were non-motile and possessed few straight flagella. The amount of flagellin was when low when the organism was grown at pH 7, suggesting that non-motility is due to poor synthesis of flagellin. The molecular mass of the flagellin was 37kDa and the isoelectric point was pH 5.0. The amino acid composition of the flagellin was similar to that found in the flagellin from neutrophilic Bacillus subtilis 168.

View full abstract

Pyrophosphate-dependent phosphofructokinase (PFP : EC 2.7.1.90) from mature green tomato fruit was purified by a DEAE-Sephacel column and preparative gel electrophoresis. Tomato PFP was composed of oligomeric forms and the tetramer of 243-262kDa molecular mass was predominant. Treatment of PFP with 20mM pyrophosphate effectively dissociated oligomers into monomers as shown by the gel filtration patterns of a Sepharose 6B column. Kinetic analysis indicated that tomato PFP had a K_m of 0.3mM for glucose-6-phosphate and a K_i of 1.3mM for pyrophosphate. About 11-fold activation was attained by fructose-2, 6-bisphosphate, though there was no activation in the direction of the reverse, gluconeogenesis reaction. Various effectors had a role to control the PFP activity in the direction of either the forward or reverse reaction.

View full abstract

The mechanism for the formation of vinegar-cured, unheated surimi-gel, so called shime-kamaboko in Japan, from Alaska pollack frozen surimi was investigated. In the SDS-PAGE pattern, a band due to the myosin heavy chain disappeared after dialyzing a sol of the salted fish flesh, shio-surimi, against Walpole buffer (pH0.5-6.5). The breaking force and deformation of shime-kamaboko were maximized at around pH4.0, but remained constant in the pH range of 1.0-4.2 if the muscle proteins of the surimi were succinylated. These values were increased by adding salt, but decreased by adding urea or guanidine-HCl. The fluorescence intensity of shio-surimi measured in the presence of ANS-Na was scarcely affected by reducing the pH value.

View full abstract

A novel alkaline pullulanase (pullulan 6-glucanohydrolase, EC3.2.1.41) was purified to homogeneity from the culture filtrate of the alkalophilic Bacillus sp.KSM-1876. The pullulanase had an optimum pH for activity of around 10.0-10.5, which is the highest pH for optimum activity of any pullulanases reported to date. The optimum temperature at pH 10 was around 50°C. The enzyme had a molecular mass of 120 kDa and an isoelectric point of pH 5.2. The enzyme acted specifically on pullulan to generate maltotriose as the major end product ; the K_m for pullulan was 1.8mg/ml. N-Bromosuccinimide abolished the enzymatic activity, and pullulan protected the enzyme from inactivation by this tryptophan-specific oxidant, suggesting that a tryptophan residue (s) is involved in the mechanism of action of the pullulanase from Bacillus sp.KSM-1876.

View full abstract

An adipokinetic hormone (AKH) was isolated from the adult heads of the silkworm, Bombyx mori. Fast atom bombardment mass spectrometry (FAB-MS) of the intact AKH and sequence analysis of the AKH after deblocked with pyroglutamate aminopeptidase revealed that the structure of Bombyx AKH is pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-Gly-NH₂, which is identical with those of Manduca sexta and Heliothis zea AKH. Bombyx AKH is relezsed just after adult eclosion and elevates the lipid level but not the carbohydrate level in the hemolymph.

View full abstract

Diferric ovotransferrin was hydrolyzed by thermolysin, a thermostable protease, at elevated temperatures. At 65°C, the amino(N)-terminal lobe was completely digested into small peptides, while the carboxyl(C)-terminal lobe was significantly resistant to the protease. This permitted the isolation of an iron-bound C-terminal half-molecule consisting of a glycosylated single polypeptide in an excellent yield (about 90%). The fragment comprises the residoes from 336 to the C-terminus of ovotransferrin. The results for the visible absorpdon spectrum of the copper-bound fragment, the stability of the iron-bound fragment in high concentration of urea, and the CD spectra of the fragment in the far and near UV regions indicated that it retains the metal binding activity and conformation of the C-terminal lobe of intact ovotransferrin.

View full abstract

The gene coding for the maltopentaose-(G5)-forming amylase of Pseudomonas sp.KO-8940 was cloned into Escherichia coli and its nucleotides were sequenced. It was expected that a long open reading frame composed of 1, 842-bp that encoded 614 amino acid residues for secretory precursor polypeptide including the typical signd sequence with an NH₂-terminal was the gene. An extract of Escherichia coli carrying the cloned G5-forming amylase gene had amylolytic activity with which produced only G5 from starch, the same as that of the donor strain enzyme. In the deduced primary structure of this enzyme, the four conserved regions of many α-amylases were found, and the COOH-terminal portion of this enzyme showed high homology with other raw starch digesting amylases.

View full abstract

Gibberellins A₁(GA₁) and GA₂0, in addition to GA₁9, which had earlier been isolated from bamboo shoots of Phyllostachys edulis, were identified from shoots of three bamboo species, P.edulis, Phyllostachys bambusoides and Sasa kurilensis by GC/MS, ELISA and RIA after HPLC of purified extracts. This endogenous GA pattern suggests the early C-13 hydroxylation GA biosynthedc pathway in vegetadve tissue of bamboo, which is similar to that in rice belonging to the same family as bamboo. The identification of three GAs from three species in subfamily Bambusoideae suggests the common occurrence of GAs in this subfamily.

View full abstract

Thermostabk β-tyrosinase (tyrosine phenol-lyase, E.C.4.1.99.2) was extracted from an obligately symbiotic and thermophilic bacterium, Symbiobacterium thermophilum, which grows only in co-cultivation with a specific thermophilic Bacillus sp., strain S.The enzyme was purified 300-fold to homogeneity with 4.2% recovery by ammonium sulfate fractionation and several steps of chromatography with anion-exchange, hydroxylapatite, and hydrophobic interaction columns. The enzyme has a molecular weight of approximately 200, 000, as estimated by gel filtration column chromatography, and 48, 000, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which indicates that the native enzyme is composed of four homologous subunits. The enzyme was stable up to 80°C and the optimum temperature for the activity was 80°C. The enzyme had an optimum pH at 7 and the isoelectric point of pH 4.8. The addition of K⁺ and NH⁺₄ accelerated the enzyme activity. In contrast, Na⁺ and Mg²⁺ showed no effect. The enzyme showed a broad range of substrate specificity, including L-cysteine, D-tyrosine, L-serine, S-methyl-L-cysteine, and β-chloro-L-alanine. Among them, S-methyl-L-cysteine and β-chloro-L-alanine were degraded to form pyruvate with higher rates than L-cyrosine. The K_m values for L-tyrosine, S-methyl-L-cysteine, and β-chloro-L-alanine were estimated to be 0.054, 1.67, and 10.2mM, respecdvely. Efficient synthesis of 3, 4-dihydroxyphenyl-L-alanine (L-DOPA) from pyrocatechol and pyruvate was achieved in the presence of a high concentration of ammonia through the reverse reaction of α, β-elimination by the enzyme.

View full abstract

The effect of alginates, a dietary fiber, on calcium phosphate formation was investigated, and compared with that of dae well-known inhibitors, poly-L-glutamate and citrate. First, the amount of calcium phosphate precipitate was measured at various alginate concentrations. An alginate delayed the formation of the calcium phosphate precipitate as well as poly-L-glutamate could. Second, the induction time (i.t.), when the transition of amorphous calcium phosphate to the crystalline form would occur, was measured in base titration experiments. Alginates delayed i.t., again as well as poly-L-glutamate could. These results indicate that alginates were an inhibitor of calcium phosphate formation. The plot of i.t. vs. alginate concentration was sigmoidal, which is similar to that of i.t.vs. poly-L-glutamate concentration and different from that of i.t.vs. citrate concentration. The induction time increase was slight at molar ratios of caldum to uronate above approximately 1/2. This indicates that the interaction between the uronate residue and calcium ion played an important role in the inhibition of calcium phosphate formation by alginates.

View full abstract

We obtained a purified antibiotic protein from the submerged cultivation broth of Rhizopus oligosporus IFO 8631 by using CM-Cellulofine chromatography and HPLC.The antibiotic did not show a broad spectrum of activity, but it was very active against some of the Bacillus species, especially against Bacillus subtillis (B.natto) at a very low concentration (less than 1 ppm). It also showed ctivity against other gram-positive bacteria, including Staphylococcus aureus and Streptococcus cremoris. The purified antibiotic was a simple protein of about 5, 500 in molecular weight, the amino acid component being characteristically high in cystine content. This high cystine content contributed to the stability of the antibiotic over a wide pH range and against strong heating (50% of the activity remained after boiling for 1 hr).

View full abstract

The components responsible for undesirable taste in soybeans have been investigated and intensities of undesirable taste were measured by electrophysiological methods. Bitterness and astringency in soybean were shown to be caused by soybean glycosides such as saponins and isoflavones, and the soyben sapponin A group contributes most strongly to the undesirable taste. In the three electrophysiological methods to measure intensity of undesirable tastes, soybean saponins did not induce the membrane potendal change of neuroblastoma cells (N-18 clone) and the ekctrical response of the chonla tympany nerve of rats, and only the electrical response of the glossopharyngeal nerve of the frog was induced by soybean saponins. These results showed that the mechanism of the undesirable tastes caused by soybean saponins was likely to be different from those of basic tastes (sweet, salty, sour, and bitter).

View full abstract

The synthesis of HS-toxin A aglycone (4) starting from (-)-carvone via two routes is describet. Ketol 5 was converted to enone 13 through an eight-step reaction sequence, which was successively submitted to α-hydroxylation, protection, LiAlH₄ reduction, xanthate fomation and reduction with (n-Bu)₃ SnH to give protected HS-toxin A aglycone 19. The two-step deprotection of 19 led to natural Hs-toxin A aglycone 4. Another short-step approach involved the regioselective functionalization of an isopropenyl substituent. Transformation of enone 21 via a five-step sequence of reactions yielded 26, which, upon treatment with HOCl followed by KOAc, afforded 28. Finally, alkaline hydrolysis of 28 gave rise to 4.

View full abstract

For the classical solution synthesis of human epidermal growth factor (h-EGF), four protected peptide derivatives, Boc-Lys (Cl-Z)-Trp-Trp-Glu (OcHex)-Leu-Arg (Tos)-OBzl (5), Boc-Glu (OcHex)-Arg (Tos)-Cys (MeBzl)-Gln-Tyr (Br-Z)-Arg (Tos)-Asp (OcHex)-Leu-OH (13), Boc-Tyr (Br-Z)-Ile-Gly-OH (16), and Boc-Cys (MeBzl)-Asn-Cys (MeBzl)-Val-Val-Gly-OH (22) were synthesized to build up the sequence corresponding to 31-53.

View full abstract

Oryzalic acid B and its related compounds (A, B and C) were isolated from healthy flag leaves of a bacterial leaf blight-resistant cultivar of rice as a group of novel antibacterial compounds. Their structures were determined to be ent-kaurane or ent-kaurene analogues by chemical and spectroscopic studies ; i.e., oryzalic acid B, ent-15-hydroxy-2, 3-secokauren-2, 3-dioic acid (Fig.1-I) ; compound A, ent-15, 16-epoxy-2, 3-dihydroxy-kaurane (II) ; compound B, ent-2, 3, 15-trihydroxy-kaurene (III) ; compound C, ent-15, 16-epoxy-kauran-3-one (IV).

View full abstract

Solubilization of a lipophilic compound in highly concentrated saccharide solutions containing protein was studied by measuring the amount of the lipophilic compound solubillzed, the surface hydrophobicity of protein, the line width of the water signal in ¹H-NMR spectra, and the unfreezable water content using a differential scanning calorimeter (DSC). The solubilizing ability, which was shown by the amount of solubilized p-dimethylaminoazobenzene (DMAB), increased with increasing saccharide concentration in the aqueous system. The effects of different saccharides on the solubilizing ability of a saccharide and bovine serum albumin (BSA) mixture decreased in the following order : sucrose>mdtose>fructose>glucose. The solubilizing ability of a saccharide and BSA mixture was higher about 4-5 times than that of saccharide only. A good correlation was observed between the solubilizing ability of a saccharide and BSA mixture and the surface hydrophobicity of BSA. The line width of the water signd in ¹H-NMR spectrum and the unfreezable water content using DSC, that is, the bound water content in saccharide solution containing BSA increased with increasing saccharide concentration. From these results, a large amount of DMAB solubilized in a highly concentrated saccharide solution containing BSA would be attributed to the hydrophobicity interaction between BSA and DMAB due to the surface hydrophobicity of BSA which increased with increasing bound water content.

View full abstract

Lipase-catalyzed enantioselective lactonization of racemic methyl 6-phenyl-4-hydroxy-5-hexynoate worked well to afford (R)-6-phenyl-5-hexyn-4-olide (42% yield, 81% e.e.). The e.e. of the product was enhanced to 97% by the repetition of enzymatic reaction. From this lactone, (4R, 5R)-isomer of 5-hydroxy-6-phenyl-4-hexanolide, a lateal root inducing substance from Erwinia quercina was synthesized via a selective epoxidation of the intermediate and subsequent hydrogenolysis as the keystep.

View full abstract

Sexual aggregation involved in conjugative transfer of Enterococcus faecalis plasmid pAD1 is enhanced by the sex pheromone cAD1, which is excreted from recipient cells. A membrane-anchored 137kDa protein is a pAD1-encoded aggregation substance designated asal, which is responsible for cell-cell contact and leads to the aggregation of cells. An AD74 protein is a proteolytic product corresponding to the N-terminal half of asal. The C-terminal of AD74 was identified as lysine at position 510 (K-510) by liquid chromatography/mass spectrometry (LC/MS) : it indicates that asal is cleaved specifically between K-510 and G-511.

View full abstract

A cosmid library and physical maps of mitochondrial DNA (mtDNA) from a liverwort, Marchantia polymorpha, were constructed using the cosmid clones. Electrophoresis profile and the physical maps indicated that the liverwort mtDNA was approximately 183kb long, the smallest among plant mtDNAs, and that it consisted of a single circular molecule. Southern hybridization analysis showed that genes typical to the mitochondrial genome existed in a single copy, and also that there was no incorporation of chloroplast DNA fragments into the mitochondrial genome.

View full abstract