Nitric oxide (NO) generation from the spin-trapping agents, phenyl-tert-butylnitrone (PBN), α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), under UV irradiation in the presence of dissolved oxygen and by oxidation with the Fenton reagent was examined by using ESR spin-trapping and spectrophotometric methods. A triplet signal at g=2.041 was observed after the ferrous complex of dithiocarbamate [Fe(MGD)2] had been added to a solution of these trapping agents treated with UV irradiation and the Fenton reagent, showing that NO was trapped with Fe(MGD)2. The concentration of nitrite induced from NO was determined via the Griess reaction to increase with the time of the treatment. It is speculated by reference to the ESR signal observed at the position around g=2.006 that the C=N double bond might have been cleaved by oxidation, resulting in the formation of a nitroso compound, and that NO was then generated by the fission of the C-N bond of the nitroso compound. NO generated in this way activated guanylate cyclase, from which it can be expected that a spin-trapping agent acts as an NO generator in vivo as well as a free radical scavenger.
The biosynthesis of 2H-labeled phenylalanine was done by converse of low molecular weight substrates ([U-2H]methanol and 2H2O) in a new RuMP facultative methylotrophic mutant Brevibacterium methylicum. To make the process work, adapted cells with improved growth characteristics were used on minimal medium M9 with the maximum content of 2H-labeled substrates. Alanine, valine, and leucine/isoleucine were produced and accumulated exogeneously in addition to the main product of biosynthesis. Electron impact mass spectrometry of methyl esters of the N-Dns-amino acid mixture obtained after the chemical derivatization of growth medium with dansyl chloride and diazomethane, was done to calculate the deuterium enrichment of the amino acids synthesized. The experimental data testified to the character of labeling of amino acid molecules as heterogeneous; however, high levels of deuterium enrichment were detected in all presented molecules-for phenylalanine the enrichment was six, leucine/isoleucine-five, valine-five, and alanine-three deuterium atoms.
Starting from dihydrodihydroxyphthalic acid (DDP), (1R, 2S, 5R, 6S)-(-)-3,4-bis(benzyloxymethyl)-5-hydroxy-8,8-dimethyl-6,8-dioxabicyclo[4.3.0]non-3-en-2-yl chloroacetate (>95%e.e.), a highly oxygen-functionalized derivative, was prepared by a combination of chemical and enzymatic reactions. The key step for asymmetrization was hydrolysis of the corresponding meso bis-chloroacetate with pig pancreatic lipase.
An overexpression system for squalene-hopene cyclase (SHC) was constructed by using the pET3a vector, which is responsible for high expression with help from the strong T7 promoter when incorporated into E. coli BL21(DE3). Site-directed mutagenesis experiments prove that two amino acid residues of tryptophan and aspartic acid inside the QW-motif 5 resided as active sites.
Antioxidative activity of carp blood plasma was estimated by measuring hydroperoxides formed by liposome peroxidation during the exposure of liposomes to AAPH. Ascorbic acid of high concentration, uric acid of low content, and tocopherol formed special protective system against lipid poroxidation in fish plasma. The decrease of uric acid, ascorbic acid, and tocopherol showed synergism of ascorbic acid and tocopherol, uric acid, and tocopherol. Carp blood plasma with a low concentration of protein (about 2%) and SH groups (88 μM) had a great effect on the antioxidative activity, as the effects of ascorbic acid, uric acid, and tocopherol were dramatically extended. Dialysed carp protein also displayed a very strong antioxidative activity on lipid peroxidation of a multilayer liposome system.
The antioxidative activities of several water-soluble marine polysaccharides, alginate (ALG), alginate sulfate (SALG), propylene glucolalginate sodium sulfate (PSS), propylene glucol mannuronate sulfate (PGMS), the oligosaccharide of chitosan (OLC), N,O-carboxymethyl chitosan (NOCC) and hydroxypropylated chitosan (HPC), were examined in a phosphatidylcholine (PC)-liposomal suspension containing the water-soluble radical emitter, 2,2′-azobis (2-amidinopropane) dihydrochloride. In the suspensions containing OLC and SALG, the initial rates of PC-OOH accumulation were 2.78×10-8 Ms-1 and 2.88×10-8 Ms-1, respectively, while all the polysaccharides tested showed antioxidative activity. Liposoluble marine polysaccharides, hexanoyl chitin (HCH) and an N-benzoylhexanoyl chitosan (NBHC) solution, also retarded the hydroperoxide accumulation of methyl linoleate by effectively trapping peroxide radicals in organic solvents when the radical chain reaction had been initiated by 2,2′-azobis (2,4-dimethylvaleronitrile). The kinetic data presented indicate that the alginate and chitin derivatives can be expected to play a role in the antioxidative mechanism of biological systems.
To supply α2,6-sialyltransferase for the large-scale synthesis of sialoside, we investigated culture conditions for the production of sialyltransferase 0160. The addition of galactose and beef extract, and control of the pH of the culture medium were effective on the production of sialyltransferase 0160. The maximal enzyme productivity reached 550 units/L. Using a crude extract of Photobacterium damsela JT0160 cells as an enzyme source, enzymatic syntheses were performed with mono- and di-saccharides as the sialyl acceptors. It was clarified that a crude extract of P. damsela JT0160 cells can be used as an synthetic catalyst for the enzymatic synthesis of sialyloligosaccharides. Furthermore, the enzyme assay showed that sialyltransferase 0160 could transfer NeuAc to not only N-linked but also O-linked carbohydrate chains. These results indicated that an abundant supply of sialyltransferase 0160 and its broad specificity make possible the synthesis of sialoside on a large scale.
The amino acid sequences of destrin and cofilin are very similar (84% homology) throughout the entire range of proteins, but they have different functions. In this study, we constructed a new cofilin expression plasmid, which had high expression frequency, and the structures of destrin and cofilin were analyzed by limited proteolysis and circular dichroism (CD). When destrin was digested by trypsin, two fragments of 17.0 kDa and 9.2 kDa were obtained, whereas only one 8.4 kDa fragment was obtained from cofilin. In spite of the overall sequence homology, an N-terminal amino acid sequence analyses of the fragments revealed the cleavage sites on destrin and cofilin to be different. These results suggest that destrin and cofilin differ in their overall tertiary folds. Cofilin showed activity similar to destrin at high pH values, although no pH-dependent structural change in cofilin was confirmed by using limited proteolysis and CD.
A new transglucosylated derivative of thiamin could be synthesized by the actions of cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus and glucoamylase from Rhizopus sp., in this order, on a mixture of dextrin and thiamin. The derivative was isolated in crystalline form and identified as 5′-O-(α-D-glucopyranosyl)thiamin by spectroscopy (FAB-MS, UV, 1H-NMR, and 13C-NMR), thiochrome formation with K3 [Fe(CN)6]-NaOH reagent, and the hydrolysis products by α- and β-glucosidases. O-α-Glucosylthiamin was odorless and mildly sweet with no tongue-pricking taste, and was more stable than thiamin hydrochloride in aqueous solutions at pHs 7.0 and 9.3.
Cecropins are a family of antibacterial peptide synthesized in insects as a response to bacterial infection. To study the regulation of the immune genes in insects, two cecropin A genes were cloned and sequenced from the silkworm, Bombyx mori. The two genes, CecA1 and CecA2, encoded identical preprocecropin A, having one intron of 609 bp and 929 bp, respectively. The 5′-upstream regions of the genes contained a NF-κB like element and IL-6-RE Type I element. Electrophoretic mobility shift assay revealed that a nuclear protein of fat body which specifically bound to the κΒ-like element was activated by injection of the larvae with peptidoglycan.
An alanine racemase (EC 220.127.116.11) from an acidophilic heterotrophic bacterium, Acidiphilium organovorum 13H, was purified and characterized. The enzyme had a dimeric structure with identical subunits of Mr 33,000 each. Although A. organovorum 13H is an acidophile, the enzyme had its maximum velocity at pH 9, corresponding to its location in the cytoplasm. Activity was maximum between 50 and 60°C. For an enzyme from a mesophile, it was stable to heat, showing no loss of activity after a 30-min incubation at 65°C. The enzyme needed pyridoxal 5′-phosphate (PLP) as a cofactor for its activity, as seen from the loss of activity upon dialysis against PLP-free buffer containing hydroxylamine and its absorption maximum at 420 nm. Activity was ihhibited by common inhibitors of PLP-dependent enzymes. PLP content studies found that 1 mole of enzyme contained 2 moles of PLP. The enzyme catalyzed the symmetric reversible racemization of alanine exclusively.
An endo-β-N-acetylglucosaminidase has been purified to homogeneity from mature seeds of Ginkgo biloba. The molecular mass of the endo-β-N-acetylglucosaminidase, named Endo-GB, was estimated to be around 63 kDa by SDS-PAGE and around 62 kDa by Hiprep S-200 chromatography, respectively. The substrate specificity has been explored with regard to the pyridylaminated N-glycans. Several high mannose-type sugar chains bearing α-1,2-mannosyl residue(s), Man9-6GlcNAc2-PA, were the most favored substrates followed by Man5GlcNAc2-PA and a typical hybrid-type structure (GlcNAc1Man5GlcNAc2-PA) which does not bear an α-1,2-mannosyl residue. On the contrary, endo-GB could hardly hydrolyze the common core pentasaccharide of N-glycan (Man3GlcNAc2-PA) and the xylose-containing sugar chains (Man4-3Xyl1GlcNAc2-PA, Man3Fuc1Xyl1GlcNAc2-PA) being widely distributed in plant glycoproteins. Furthermore, we analyzed the structures of N-glycans conjugated to storage glycoproteins in the mature Ginkgo seeds to see the occurrence of endogenous substrates for Endo-GB. The structural analysis showed, however, only xylose-containing type N-glycans (Man3Fuc1Xyl1GlcNAc2 (95%) and Man3Xyl1GlcNAc2 (5%)), which can not be substrate for Endo-GB, predominantly occur in the storage glycoproteins.
Subtilisin-like serine protease, which is associated with the dormant spores of Bacillus cereus, was solubilized by washing the spores with 2 M KCl and purified to homogeneity by carbobenzoxy-D-phenylalanine-liganded affinity column chromatography and hydrophobic interaction column chromatography. Enzyme activity was completely inhibited by reagents for sulfhydryl groups such as HgCl2 as well as by conventional subtilisin inhibitors, suggesting the enzyme to be cysteine-dependent. The enzyme retained activity in 5 M urea at 4°C for at least 2 months, and the specific activity was 50 times that of subtilisin BPN′ when measured for a common chromogenic substrate, carbobenzoxy-glycyl-glycyl-L-leucine p-nitroanilide. The gene encoding this protease was cloned in Escherichia coli, and its nucleotide sequence was analyzed. The deduced amino acid sequence suggested that the protease is produced as a precursor comprising three portions; a signal sequence (28 amino acid residues), a prosequence (80 amino acid residues) and a mature enzyme (289 amino acid residues). The mature region of the enzyme had high similarity with a thermitase from Thermoactinomyces vulgaris (72% identity) and a thermostable alkaline protease from Thermoactinomyces sp. E79 (66% identity), which have the N-terminal sequence showing scarcely noticeable similarity with corresponding stretches of subtilisins and mercuric ion-sensitive free cysteine in the equivalent position of the primary structure.
The evgAS operon of Escherichia coli encodes the EvgA response regulator and the EvgS sensory kinase, which are members of one of the two-component signal transduction systems of Escherichia coli. In this study, we identified the evg promoter and the EvgA-responsive element. Primer extension analysis found two evg transcritional initiation sites, designated P1 (+1) and P2 (-10), and placed them 114 bp and 124 bp upstream of evgA, respectively. A gel retardation assay demonstrated that EvgA specifically bound to an inverted repeat located between -102 and -128 counting from P1. We also did a β-galactosidase induction experiment using a promoter-probing vector and found that the EvgA-binding sequence was important to stimulate the evg promoter. These results suggest that the expression of evgAS is positively regulated by its own product, EvgA.
The gastrolith of the crayfish Procambarus clarkii contains a small amount of an organic matrix that is mainly chitin and proteins, together with a large amount of calcium carbonate. As the first step to understand the mechanism of calcification, we tried to characterize matrix proteins in the gastrolith. An insoluble matrix protein, referred to as gastrolith matrix protein, was made soluble with 1% SDS containing 10 mM dithiothreitol, and was purified by reverse-phase high-performance liquid chromatography. The protein had a molecular weight of about 50,500 and a blocked amino terminus. By enzymatic digestion and microsequencing, five partial amino acid sequences with a total of 225 amino acid residues were identified and found to include a repetitive sequence not reported previously.
Non-thermal effect of a ceramics radiation on glucose-6-phosphate dehydrogenase has been investigated using the enzyme, glucose-6-phosphate and NADP+ separately irradiated at 10°C by a ceracompo R plate and a ceramics un-sewed cloth (sheet). The Km for glucose-6-phosphate was increased 20% after 6 h of irradiation by the plate, but the Vmax/Km was decreased 24%. After 3 h of irradiation by the sheet, the Km was increased 17%, but after 6 h of irradiation it was decreased 11%. The 3 h of irradiation by the sheet slightly increased both enthalpy and entropy changes of the reaction, but the 6 h of irradiation significantly decreased them. Both thermodynamic parameters in the activated state were increased by the sheet irradiation. The promotion energy for both formations of the enzyme-substrate and their activated complex depended on enthalpy. The different effects of two ceramics radiators on G6PDH activity were discussed.
Thermoanaerobic bacteria are of considerable interest as producers of thermostable amylolytic enzymes. The soluble amylolytic enzyme system of Thermoanaerobacterium thermosaccharolyticum DSM 571 was fractionated into a pullulanase, a glucoamylase, and an α-glucosidase. The enzymes were purified to homogeneity and their physical and catalytic properties were studied. The pullulanase, which cleaved both α-1,4- and α-1,6-glucosidic bonds, was an amylopullulanase closely related to similar enzymes from other thermoanaerobic bacteria. Partial amino acid sequences of the glucoamylase were identical with the corresponding sequences deduced from the cga gene encoding the glucoamylase from Clostridium sp. strain G0005. The α-glucosidase was identified as an isomaltase belonging to a group of structurally related exo-α-1,4-glucosidases and oligo-1,6-glucosidases from bacilli. Comparison of enzyme activities indicated that the glucoamylase had the major amylolytic activity of T. thermosaccharolyticum, with amylopullulanase and α-glucosidase assisting in the cleavage of α-1,6-glucosidic bonds.
A recombinant strain of Aspergillus oryzae has been constructed in which 1,2-α-mannosidase, an intracellular glycochain processing enzyme with specificity toward 1,2-α-mannosidic linkages, has been overexpressed. For the construction, the N-terminal signal-encoding sequence of the 1,2-α-mannosidase gene (msdC) from Penicillium citrinum was replaced with that of the aspergillopepsin I signal, and the fused gene was inserted between amyB promoter-terminator elements in the expression plasmid pTAPM1. A transformant of A. oryzae (the strain PM-1) secreted a great deal of heterogeneous 1,2-α-mannosidase into the culture media, which was purified by CM ion-exchange chromatography. Approximately 21 mg of the purified enzyme was obtained per liter of culture. N-terminal amino acid analysis indicated that the signal peptide was removed from the secreted enzyme. The Penicillium 1,2-α-mannosidase expressed in A. oryzae did not show any notable difference from the enzyme from P. citrinum in such properties as Mr, specific activity, CD spectra, or kinetic parameters. Man7GlcNAc2 accumulated temporarily during the degradation of Man9GlcNAc2 to Man5GlcNAc2 by fungal 1,2-α-mannosidase.
Soft-shelled turtle egg-white lysozyme was purified and sequenced. Lysozyme was reduced and carboxymethylated to fragment it with trypsin, V8 protease and CNBr. The peptides yielded were purified by RP-HPLC and sequenced. Every trypsin peptide was overlapped by V8 protease peptides and CNBr fragment. The amino acid sequence was compared with other lysozymes. This lysozyme has an extra Gly residue at N-terminus, which was found in pheasant lysozyme. Further, this lysozyme has an insertion of a Gly residue between 47 and 48 residues when compared with chicken lysozyme, as found in human lysozyme, therefore it proved that this lysozyme has the largest number of amino acids (131 aa) in chicken type lysozymes. The amino acid substitutions were found at subsites E and F. Namely Phe34, Arg45, Thr47, and Arg114 were replaced by His, Tyr, Arg, and Tyr, respectively. The time course using N-acetylglucosamine pentamer as a substrate showed a reduction of the rate constant for glycosidic cleavage and increase of binding free energy for subsites E and F, which proved the contribution of amino acids mentioned above for substrate binding at subsites E and F.
Diisopropylfluorophosphate (DFP), a general serine protease inhibitor, inhibited the DNA fragmentation and cell death in MDCK cells treated with ricin, modeccin, Pseudomonas toxin, or diphtheria toxin. A trypsin-like serine protease inhibitor, N-tosyl-L-lysine chloromethyl ketone (TLCK) also prevented ricin-induced DNA fragmentation and cell death, albeit less effectively than DFP. Microscopic observation showed that the morphological changes of MDCK cells induced by ricin were prevented by DFP. DFP did not affect the binding, internalization, or subsequent excretion of ricin, but reduced the degradation of ricin in MDCK cells, suggesting that DFP inhibits at least the cellular protease that may be involved in the degradation of internalized ricin. In addition, SDS-PAGE analysis of cytosolic proteins suggested that DFP-sensitive endogenous proteases are activated in the ricin-treated cells. In the cells treated with DFP, the protein synthesis inhibitory activity of ricin was increased rather than inhibited. The activities of modeccin and Pseudomonas toxin were also slightly increased by DFP, but no effect of DFP on the activity of diphtheria toxin was observed. Therefore, these results suggest that protein toxins have a DFP-sensitive common pathway leading to apoptosis that is distinct from the pathway leading to the inhibition of cellular protein synthesis.
The photosynthetic bacterium Rhodobacter sphaeroides f. sp. denitrificans IL106 can grow in high osmolarity. The accumulation of intracellular inorganic ions and organic solutes in cells grown in a synthetic medium containing different concentrations of NaCl was examined. Together with potassium ion, trehalose was the major organic osmoprotectant and its accumulation depended on the external salt concentration. Intracellular levels of glycine betaine and other osmoprotectants such as proline did not change when osmolarity increased.
The pH dependence of the reaction of purified recombinant human renin with purified recombinant sheep angiotensinogen was not a typical bell-shape but had two peaks, pH 6.4 and 8.9. The pH dependence of the reaction of human renin with human, hog, and rat angiotensinogens partially purified from plasma had a peak with a shoulder containing two peaks close together. These findings indicate that the reaction of renin with angiotensinogen involves at least two amino acid residues other than the two aspartic acid residues known to be involved and occurs at acidic pH and basic pH by two different pairs of these amino acid residues.
A gene encoding a novel intracellular sorbitol oxidase of a soil bacterium, Streptomyces sp. H-7775, was cloned and sequenced. The gene consists of an open reading frame of 1,260-bp encoding a protein of 420 amino acids with a molecular weight of 45,148. Deduced amino acid sequence of the gene has 25.3% identity and 68.1% similarity to that of rat L-gulonolactone oxidase at the overall amino acids. Nucleotide-binding motifs were not found in the deduced amino acid sequence of SOX protein. We succeeded in expressing recombinant sorbitol oxidase with covalently bound FAD in E. coli at about a 4,000-fold higher total enzyme activity than that of the Streptomyces sp. H-7775. The enzymatic properties of the recombinant SOX were similar to those of the enzyme from Streptomyces sp. H-7775. This is the first report of the cloning and expression of a newly categorized enzyme, sorbitol oxidase, from Streptomyces sp.
Two mutant forms, which had truncated N-terminals, of lipase activator protein (LipB) from Pseudomonas aeruginosa TE3285 were prepared, and their molecular properties and activity were compared with those of the full-length form. A truncated LipB lacking its hydrophobic N-terminal 21 residues was dispersed homogeneously in solution, and could reactivate the stoichiometric amount of denatured lipase. In contrast, full-length LipB formed soluble aggregates, and reactivated less than an equimolar amount of the lipase even under the most suitable conditions. These findings suggest that some or all of the N-terminal 21 residues caused aggregation of the protein molecules, and prevented LipB from fully stoichiometric reactivation. A truncated LipB lacking the N-terminal 61 residues also reactivated denatured lipase, suggesting that the N-terminal 61-residue region of LipB is not involved in reactivation.
The activity of protein disulfide isomerase, in the extracts of several dormant seeds including soybean, rice, wheat, and maize was assayed. The activity was higher in the extracts of beans than in those of the other seeds. A correlation was significant (R=0.95 and 0.93; p<0.01) between the PDI activity and the concentration of protein soluble in a salt solution.
We constructed an overexpression system in Escherichia coli of the leuB gene coding for 3-isopropylmalate dehydrogenase in Thiobacillus ferrooxidans. E. coli harboring the plasmid we constructed, pKK leuB1, produced 17-fold the enzyme protein of the expression system previously used for purification. The substrate specificity of the enzyme was analyzed with synthetic (2R, 3S)-3-alkylmalates. The 3-isopropylmalate dehydrogenase of Thiobacillus ferrooxidans had broad specificity toward the alkylmalates.
During the photosynthetic carbon reduction (PCR) cycle of Synechococcus PCC 7942 and Synechocystis PCC 6803, fructose-1,6-bisphosphatase, NADP+-glyceraldehyde-3-phosphate dehydrogenase, and ribulose-5-phosphate kinase were not sensitive to treatment with dithiothreitol (DTT), a reducing agent, in vitro and were not regulated by light in vivo, unlike the chloroplastic enzymes of higher plants. These results indicate that the PCR cycle in the cyanobacterial cells may not be actually regulated by light in vivo even if the ferredoxin/thioredoxin system is present.
Extract of Raji cells treated with sodium n-butyrate (1 mM) and a tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA, 40 ng/ml), was analyzed by immunoblotting using ten human sera with different antibody titers against Epstein-Barr virus early antigens. Two human sera reacted with one induced polypeptide of 48 kDa and its induction was inhibited by curcumin (4 μg/ml), an antitumor promoter from turmeric. A mouse antiserum against P3HR-1 cells treated with TPA and sodium n-butyrate also detected the 48-kDa polypeptide in Raji cells treated with TPA at concentrations of 2.5 to 80 ng/ml. These results indicate that the immunoblotting analysis can be used in a confirmation test for detection of antitumor promoting activity.
Carboxyl groups of rye seed chitinase-c (RSC-c) were modified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl ester (GEE) at pH 5.5 and 5°C in the presence and absence of (GlcNAc)4. In the absence of (GlcNAc)4, 5.2 carboxyl groups were modified by 90 min-reaction and the chitinase activity was reduced to 2.0%, while in the presence of (GlcNAc)4, 4.6 carboxyl groups were modified and 72% of the activity was retained. To identify the carboxyl group protected by (GlcNAc)4 from the modification, RSC-c was first modified with EDC and GEE in the presence of (GlcNAc)4 and then radiolabeled with EDC and [14C]GEE in the absence of (GlcNAc)4. Analyses of the radioactive peptides from the tryptic and chymotryptic digests of radiolabeled RSC-c showed that the main radiolabeled carboxyl group is that of Asp95, suggesting that Asp95 is located at or near substrate-binding site of RSC-c.
The complete amino acid sequence of gladiolus bulb chitinase-a (GBC-a) was determined. First the tryptic peptides from GBC-a after it was reduced and S-carboxymethylated were sequenced and then the peptides were further studied by chemical cleavage of the enzyme. GBC-a consisted of 274 amino acid residues and had a molecular mass of 30,714 Da. Two consensus sequences essential for chitinase activity by plant class III chitinases were conserved in GBC-a, although its sequence similarity with plant class III chitinases was less than 20%. Sequence comparison of GBC-a with sequences of other proteins in a protein identification resource (PIR) showed that the GBC-a sequence was 33% similar to that of narbonin, a seed storage 2S globulin from narbon beans.
We have isolated from Thermus aquaticus an insertion-sequence-like genetic element (ISLtaq1) that induces thermotolerance and has a high sequence similarity to IS150 belonging to the IS3 family. An open reading frame on ISLtaq1, termed ORF1, encodes the ORF1 protein, which carries a DNA-binding motif. In this study, we found an imperfect inverted repeat in ISLtaq1. We next overproduced and purified a His-tagged ORF1 protein. Gel retardation analysis demonstrated that this protein specifically bound to an DNA fragment containing the inverted repeat in ISLtaq1. These results suggest that ISLtaq1 and the ORF1 protein are an insertion sequence and part of the transposase encoded by ISLtaq1, respectively.
Three β-N-acetylglucosaminidases, GlcNAcase A, B, and C, were purified from the culture fluid of Lactobacillus casei ATCC 27092, and the molecular weights of these enzymes were estimated to be 54,000, 51,000, and 44,000, respectively, by SDS-PAGE. The production of these GlcNAcases was accelerated by the addition of N-acetylglucosamine to the culture. These enzymes had pIs of about 5.2, an optimum pH of 5.0-5.5, and an optimum temperature of 37-40°C. The Km values of GlcNAcase A, B, and C for p-nitrophenyl-β-N-acetylglucosamine were 0.85, 1.30, and 1.04 mM and those for p-nitrophenyl-β-N-acetylgalactosamine were 39.6, 57.7, and 60.8 mM, respectively.
We report here the isolation and characterization of a peptide-N4-(acetyl-β-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the β-aspartylglycosylamine linkage (GlcNAcβ1→Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.
The formation and dissociation of complexes composed of potato starch and sucrose-lipid monoesters (SE: monocaprate, monolaurate, monomyristate, monopalmitate, and monostearate) were studied by differential scanning calorimetry (DSC). The formation and dissociation temperatures of each complex increased as the number of carbon atoms in the alkyl chain of SE increased, and as the content of starch increased, overlapping with the gelatinization temperature. Therefore, the DSC curves for starch gelatinization differed according to the added SE and water content. The completion temperature for the dissociation of each starch-SE complex depended on the water content, similar to that for the gelatinization of starch. The heat of fusion of the starch-SE (monopalmitate) complex obtained from the completion temperature was nearly twice that of the original starch, 140 kJ/mol glucose unit. It is suggested that a stable conformation of each complex was not formed during the gelatinization of the starch granules, but during cooling from a temperature higher than the dissociation temperature of the complex which had formed during the gelatinization process.
Enzymatic hydrolysis of pectate was carried out continuously to produce pectate oligosaccharides by immobilized endo-polygalacturonase in a continuous stirred tank reactor (CSTR) with high efficiency. The enzyme was immobilized on to chitosan beads by the absorption method, and the reaction was performed with an initial pectate concentration of 10 gl-1 at 35°C and pH 4.0 at a dilution rate of 0.87-2.8 h-1. The hydrolysis products mainly consisted of mono-, di-, tri-, tetra-, penta-, hexa- and heptasaccharides, with the highest conversion being 0.78. A higher volumetric production rate of the total hydrolyzate, which was dependent on the dilution rate, was obtained than that by a batch reaction. The hydrolysis process was mathematically modeled from the basic material balance and rate equations, and showed agreement between the simulated and experimental results. This reactor system was found to be effective for obtaining pectate oligosaccharides with a high production rate.
The calcium-binding properties of soybean globulins that have been deamidated or enzymatically hydrolyzed after the removal of phytate were physicochemically investigated. The level of calcium was reduced from 0.32% to 0.013% and that of phosphorus was reduced from 1.1% to 0.050% by treating with cation- and anion-exchange resins. The calcium-binding properties of soybean globulins were described by the Langmuir equation, the maximum amount of bound calcium (N) and the affinity parameter for calcium (K) being obtained for each sample. The value of N was decreased by the removal of phytate, while the deamidation caused the value of N to increase. As hydrolysis proceeded, the value of N increased to a degree of hydrolysis of 32%, and then decreased. Based on this result, there seems to be an optimum molecular weight of hydrolyzed soybean globulins for the amount of bound calcium. In addition, the value of K for every soybean globulin sample was much lower than that of phytic acid, indicating that the globulins had proper calcium-binding properties for calcium absorption in the small intestine.
Two PCR-amplified genomic DNA fragments encoding apple (cv. Fuji) polyphenol oxidase (PPO) were cloned and sequenced. A comparison of genomic DNA with cDNAs revealed that the PPOs lacked introns. Both PPO DNAs appear to encode a 66-kDa precursor protein consisting of a 56-kDa mature protein and a N-terminal transit peptide of 10-kDa N-terminal transit peptide. Apple PPO DNA was expressed in Escherichia coli, and the gene product (56 kDa) without a transit peptide was immunochemically detected and was the same size (ca. 65 kDa) as the main PPO of apple fruit by SDS-PAGE.
Cruciferae plants, banana and coriander each showed antibacterial activity. The highest activity among the foodstuffs tested was found in the stems of wasabi. An ethereal extract from wasabi stems had potent antibacterial activity and we isolated the active compound from the extract. Instrumental analysis identified the compound as 6-methylsulfinylhexyl isothiocyanate. Some homologues of 6-methylsulfinylhexyl isothiocyanate were also active against Escherichia coli and Staphylococcus aureus.
Germinated barley foodstuff (GBF) derived from the aleurone and scutellum fractions of germinated barley was rich in glutamine and low-lignified hemicellulose. The diarrhea caused by ceco-colectomy could be prevented by feeding GBF to rats. GBF could also increase the protein content and sucrase activity of small intestinal mucosa in this model. This diarrhea-preventive effect of GBF would be based on the water-holding capacity and bulging force under alkaline conditions, e.g. in the small intestine.
Effects of binding heterocyclic amines to cells of lactic acid bacteria on theirs absorption were investigated. Cells of Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 bind both 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). The binding of strain 1131 cells to Trp-P-1 was maximum in the pHs from 4 to 8, but strain 2038 cells bound Trp-P-1 and MeIQx only slightly at pH 7. We investigated the absorption of heterocyclic amines by the small intestine of F344 rats in the presence of these bacterial cells, using an in situ loop technique. The absorption of Trp-P-1 by the small intestine was significantly lower in the presence of strain 1131 cells than in the absence of the cells, but the presence of strain 2038 cells had no effect on Trp-P-1 absorption. Perhaps strain 1131 cells bind to Trp-P-1 at the same pH as that of the small intestine (pH 6-7) and thus decrease its absorption.
Qualitative analyses of lipid component parts (core lipids, phospholipid-polar head groups, and glycolipid-sugar moieties) without separation of individual lipids were done for further 14 strains of methanogens. The results confirmed the conclusion of our previous paper (System. Appl. Microbiol. 16, 342 1993) that the distribution of lipid component parts was characteristic to taxonomic groups of methanogens at a family or genus level. Our previous and present analyses of lipid component part distribution of methanogens supported the division of the order Methanomicrobiales into two new orders Methanomicrobiales and “Methanosarcinales” proposed by Boone et al. based on 16S rRNA analyses (Methanogenesis: Ecology, Physiology, Biochemistry, & Genetics, 1993, pp 35-80). The whole results also phenotypically supported the establishment of new families “Methanocaldococcaceae” and “Methanosaetaceae” and new genera “Methanothermococcus”, “Methanocaldococcus”, “Methanoignis”, and “Methanosalsus” proposed by Boone et al.
A NADPH-dependent carbonyl reductase was purified to homogeneity from Candida magnoliae AKU4643 through four steps, including Blue Sepharose affinity chromatography. The enzyme catalyzed the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to the corresponding (S)-alcohol with a 100% enantiomeric excess, which is a useful chiral building block for the chemical synthesis of pharmaceuticals. The relative molecular mass of the enzyme was estimated to be 76,000 on high performance gel filtration chromatography and 32,000 on SDS polyacrylamide gel electrophoresis. The enzyme reduced α-, β-keto esters and conjugated diketones in addition to ethyl 4-chloro-3-oxobutanoate. The enzyme activity was inhibited by quercetin and HgCl2, but not by EDTA. The N-terminal amino acid sequence of the enzyme showed no apparent similarity with those of other oxidoreductases.
The P1 protein is liberated from the N-terminal region of the potyviral polyprotein by cleavage depending on its own autoproteolytic activity. Existence of the 32-kDa P1 protein in tobacco plants infected with potato virus Y ordinary strain (PVY-O) was detected by an antiserum against a recombinant PVY-O P1 protein. In vivo analysis using tobacco protoplasts confirmed that the Phe284-Ser285 was the cleavage site separating the P1 protein from the PVY-O polyprotein. Phe284 was indispensable for proteolysis and Ser285 was needed for optimal cleavage susceptibility.
We investigated the mechanism of multidrug resistance conferred by overexpression of bfr2+/pad1+/sks1+ or pap1+ genes of Schizosaccharomyces pombe. Overexpression of bfr2+ did not confer multidrug resistance on a pap1-disrupted strain. In a mutant with bfr1+ (a putative membrane transporter which belongs to the ATP-binding cassette superfamily) disrupted, overexpression of either bfr2+ or pap1+ did not confer multidrug resistance. These findings suggest that bfr1+ acts as the most downstream effector of the multidrug resistance conferred by bfr2+ and pap1+ genes.
p-NP-α-D-Glucoside-hydrolyzing activity in the culture filtrate of Bacillus circulans KA-304, a producer of Schizophyllum commune cell-wall lytic enzyme, increased remarkably when the bacterium was grown on dextran as a carbon source. It was suggested that the increase of the activity was caused by increases of two major species, α-D-glucosidase I and α-D-glucosidase II. α-D-Glucosidase I, which showed a certain reactivity toward dextran, was isolated from the filtrate (MW 70 kDa, 35-fold, 10% recovery). The enzyme was stable around pH 6.5-7.5 and showed its highest activity at pH 6.5. The enzyme preparation inactivated with p-chloromerucuribenzoic acid recovered its activity by incubating with ditiothereitol. Its substrate specificity suggested that the enzyme was an exo-type enzyme with certain affinity toward α-1,6-glucosidic linkage.