Analysis of the Hyperthermophilic Chitinase from Pyrococcus furiosus: Activity toward Crystalline Chitin

Chitinase [EC 3.2.1.14] is an enzyme that can hydrolyze the β-1,4 linkage between N-acetyl-D-glucosamine in chitin. In the genome database of the hyperthermophilic archaeon Pyrococcus furiosus, we found two adjacent genes (PF1233 and PF1234) homologous to those of the chitinase of Thermococcus kodakaraensis. In the cultured medium of P. furiosus, however, no chitinase activity was detected. On analysis of the structural gene of P. furiosus, it appears that one nucleotide insertion in PF1234 caused a frame shift and separated a gene. By deletion of one nucleotide in PF1234, the best match was achieved between chitinases of T. kodakaraenesis and P. furiosus. We succeeded in constructing an artificial recombinant chitinase exhibiting hydrolytic activity toward not only colloidal but also crystalline chitins at high temperature. Furthermore, by analyzing the characteristics of the domains, a recombinant enzyme comprising two domains exhibiting high activity toward crystalline chitin was prepared.     

Purification and Characterization of a Magnesium Ion Requiring N-Acetyl-D-glucosamine Specific Lectin from Seeds of Quercus ilex L.

A new magnesium ion requiring N-acetyl-D-glucosamine specific lectin QIL was purified to electrophoretic homogeneity from seeds of Quercus ilex L. through successive steps of (i) lectin extraction, (ii) ammonium sulphate (30–50%) fractionation, (iii) diethylaminoethyl (DEAE)-cellulose chromatography, (iv) carboxymethyl (CM)-cellulose chromatography, and (v) Sephadex G-75 chromatography. The lectin, having specific activity of 25,600 hemagglutination units (HAU)/mg of protein, was found to be a monomeric protein with a native molecular weight of 13.2 kDa. N-Acetyl-D-glucosamine was found to exhibit most potent inhibitory action on the lectin activity among all the sugars tested. The lectin was also found to exhibit specificity for human blood groups A, B, and AB. It was converted to the corresponding apo-lectin by ethylenediaminetetraacetic acid (EDTA) treatment followed by buffer dialysis. The apo-lectin exhibited a specific and characteristic requirement for magnesium ions for the expression of its activity.     

Structural Analysis of the Fundamental Polymer of the Sheath Constructed by Sphaerotilus natans

The sheath of Sphaerotilus natans is composed of cysteine-rich peptide and polysaccharide moieties. The polysaccharide was prepared by treating the sheath with hydrazine, and was determined to be a mucopolysaccharide containing β-D-GlcA, β-D-Glc, α-D-GalN, and β-D-GalN. To elucidate the structure of the peptide, the sheath was labeled with a thiol-selective fluorogenic reagent, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. Enantiomeric determination of the S-derivatized Cys in the fluorescent sheath suggested that it contained L-Cys mainly. Fluorescent cysteinylglycine was detected in the partial acid hydrolysate of the fluorescent sheath. The sheath-degrading enzyme secreted by Paenibacillus koleovorans produced a fluorescent disaccharide-dipeptide composed of GalN, Gly, and N-acetylated Cys from the fluorescent sheath. The disaccharide and dipeptide moieties were found to be connected by an amide bond. Based on these results, the sheath was deduced to be formed by association of a mucopolysaccharide modified with N-acetyl-L-cysteinylglycine.     

ESR Studies on Lipid-protein Interaction in Gluten

The interaction of protein with lipid in wheat gluten has been studied by electron spin resonance (ESR). The gluten in the flour suspension was spin-labeled with a fatty acid spin label (N-oxyl-4,4'-dimethyloxazolidine derivative of 5-ketostearic acid) and washed out from the flour. The ESR spectra of the spin label incorporated in gluten exhibited clearly separated parallel and perpendicular hyperfine splittings. The orientation of the gluten lipid and its fluidity showed temperature dependence. Phase transition was observed at 25°C. Compared with gluten, vesicles of the lipids extracted from flour were found to be in a less oriented, highly fluid state, and with much lower activation energy for rotational viscosity, while the reconstituted gluten, which was prepared by mixing purified gluten protein and the extracted lipids, had a lipid environment similar to that of gluten. The results indicate that the lipid was immobilized in the gluten matrix by strong interaction with protein.     

Tridec-1-ene-3,5,7,9,11-pentayne,an Ovicidal Substance from Xanthium canadense

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC. 1.4.3.5) has been purified from dry baker’s yeast to an apparent homogeneity on a polyacrylamide disc gel electrophoresis in the presence of 10 µm of phenylmethylsulfonyl fluoride throughout purification.

1) The purified enzyme, obtained as holo-flavoprotein, has a specific activity of 27µmol/mg/hr for pyridoxamine 5′-phosphate at 37°C, and a ratio of pyridoxine 5′-phosphate oxidase to pyridoxamine 5′-phosphate oxidase is approximately 0.25 at a substrate concentration of 285 µm. Km values for both substrates are 18 µm for pyridoxamine 5′-phosphate and 2.7 µm for pyridoxine 5′-phosphate, respectively.

2) The enzyme can easily oxidize pyridoxamine 5′-phosphate, but when pyridoxamine and pyridoxine 5′-phosphate are coexisted in a reaction mixture the enzyme activity is markedly suppressed much beyond the values expected from its high affinity (low Km) and low V_max for the latter substrate.

3) Optimum temperature for both substrates is approximately 45°C, and optimum pH is near 9 for pyridoxamine 5′-phosphate and 8 for pyridoxine 5′-phosphate.

4) From the data obtained, the mechanism of regulation of this enzyme in production of pyridoxal 5′-phosphate and a reasonable substrate for the enzyme in vivo are discussed.     

Culture Conditions of l-Isoleucine Fermentation from Acetic Acid

Culture conditions were studied for l-isoleucine production from acetic acid. Acetate and ammonium concentration in culture liquid exerted a great influence on the fermentation, and optimum concentration was 2–5 g/liter and 2–3 g/liter respectively. To maintain these conditions throughout the culture, it was necessary to supply intermittently a small amount of feeding solution which consisted of ammonium acetate and acetic acid. Molecular ratio of the former to the latter was 0.175, and total concentration of acetic acid was 700 g/liter.

Carbon dioxide showed an inhibitory influence on l-isoleucine production and adequate ventilation was necessary for satisfactory result. Maximum amount of l-isoleucine was 33.5 g/liter after 77-hr cultivation at 28°C and at pH 7.7. Production yield of l-isoleucine was 10% by weight from acetic acid.     

Purification,characterization, and gene cloning of a novel aminoacylase from Burkholderia sp. strain LP5_18B that efficiently catalyzes the synthesis of N-lauroyl-l-amino acids

ABSTRACT

An N-lauroyl-l-phenylalanine-producing bacterium, identified as Burkholderia sp. strain LP5_18B, was isolated from a soil sample. The enzyme was purified from the cell-free extract of the strain and shown to catalyze degradation and synthesis activities toward various N-acyl-amino acids. N-lauroyl-l-phenylalanine and N-lauroyl-l-arginine were obtained with especially high yields (51% and 89%, respectively) from lauric acid and l-phenylalanine or l-arginine by the purified enzyme in an aqueous system. The gene encoding the novel aminoacylase was cloned from Burkholderia sp. strain LP5_18B and expressed in Escherichia coli. The gene contains an open reading frame of 1,323 nucleotides. The deduced protein sequence encoded by the gene has approximately 80% amino acid identity to several hydratase of Burkholderia. The addition of zinc sulfate increased the aminoacylase activity of the recombinant E. coli strain.     

Physicochemical Studies on Sulfated Polysaccharides Extracted from Seaweeds at Various Temperatures

Sulfated polysaccharides (SPS) were extracted from three species of seaweeds of Ulvacea (Ulva pertusa, Ulva conglobata and Entromorpha prolifera) for 4 hr at various temperatures and their physicochemical properties were studied using viscometric and equilibrium sedimentation measurements in order to determine the optimum extracting condition.

Sulfated polysaccharides extracted at various temperatures from the seaweed of U. pertusa had the same physicochemical properties, while the larger molecular components of SPS was not extracted from U. conglobata and E. prolifera, at the low temperature of 30~40°C. This was confirmed by analyses of their viscosity and molecular weight and by gel filtration chromatography, in which each SPS showed two or three peaks.

The larger molecular component of SPS could be extracted at the high temperature of 80~90°C in the thermostable form.     

Preparation of Enzymes Required for Enzymatic Quantification of 5-Keto-D-gluconate and 2-Keto-D-gluconate

For easy measurement of 5-keto D-gluconate (5KGA) and 2-keto D-gluconate (2KGA), two enzymes, 5KGA reductase (5KGR) and 2KGA reductase (2KGR) are useful. The gene for 5KGR has been reported, and a corresponding gene was found in the genome of Gluconobacter oxydans 621H and was identified as GOX2187. On the other hand, the gene for 2KGR was identified in this study as GOX0417 from the N-terminal amino acid sequence of the partially purified enzyme. Several plasmids were constructed to express GOX2187 and GOX0417, and the final constructed plasmids showed good expression of 5KGR and 2KGR in Escherichia coli. From the two E. coli transformants, large amounts of each enzyme were easily prepared after one column chromatography, and the preparation was ready to use for quantification of 5KGA or 2KGA.     

Partial Purification and Characterization of Acid and Neutral α-Glucosidases from Preclimacteric Banana Pulp Tissues

An acid α-glucosidase (AAG) with an optimum pH of 4.5 and two isoforms of neutral α-glucosidase (NAG I and II) with an optimum pH of 6.5 were partially purified from preclimacteric banana pulp tissues by monitoring the 4-methylumbelliferyl α-D-glucoside (4MUαG) hydrolyzing activity. The molecular weights of the AAG and the two NAG were 70,000 and 42,000, respectively, by gel filtration. By kinetic studies, the AAG was found to be a typical maltase that required substrates such as maltose, maltotriose, maltotetraose, and maltopentaose rather than soluble starch. On the other hand, the two NAGs preferred 4MUαG to maltose as substrate and their maltase activities were about 50 times lower than that of the AAG. The NAGs, as well as the AAG, did not hydrolyze isomaltose, trehalose, sucrose, or glycogen at all. Sucrose was a competitive inhibitor of the AAG but not NAGs toward 4MUαG. Glucose and maltose were also competitive inhibitors of both AAG and NAGs.     

Regioselectivity in β-Galactosidase-catalyzed Transglycosylation for the Enzymatic Assembly of D-Galactosyl-D-mannose

The regioselectivity of β-galactosidase derived from Bacillus circulans ATCC 31382 (β-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for β-1,3-galactosidase but also for β-galactosidases of different origins. The relative hydrolysis rate of Galβ-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of β-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of β-1,3-, β-1,4- and β-1,6-linked D-galactosyl-D-mannose was subsequently established.     

Identity of Brusatol and Yatansin,An Antidysentric Agent

In order to develop a fermentation process for lactase (β-d-galactosidase) production, we selected an excellent lactase producer, Kluyveromyces lactis. KY5466, from our yeast culture collection. Some of its mutant derivatives which formed a blue pigment from 5-bromo-4-chloro-3-indolyl-β-d-galactoside in the presence of glucose and those which assimilated phenyl-β-d-galactoside as a carbon source produced 2 to 2.7 times as much lactase as the parent strain. In the late stage of cultivation, the lactase activity decreased to zero for all strains tested soon after the complete consumption of sugar. This phenomenon was found to be correlated with a decrease in the efficiency of protein extraction from the cells. The maximal amount of lactase produced reached 155 units per ml at 48 hr in a 5-1 jar fermentor culture with sugar feeding.     

Substrate Specificity of Thermostable D-Alanine-D-alanine ligase from Thermotoga maritima ATCC 43589

D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 °C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, D-cysteine, and D-serine, in that order.     

Syntheses of s-Triazolo [4,3-a] Pyrimidine Derivatives

The metabolism in rats of dihydrocapsaicin, a pungent principle of hot pepper, was investigated in vivo and in vitro by thin-layer chromatography, high-performance liquid chromatography and combined gas chromatography-mass spectrometry. Within 48 hr of oral administration of dihydrocapsaicin (20 mg/kg body weight) to male adult rats, unchanged dihydrocapsaicin and eight of its metabolites were identified in urine; i.e., dihydrocapsaicin (8.7% of total dose), vanillylamine (4.7%), vanillin (4.6%), vanillyl alcohol (37.6%) and vanillic acid (19.2%) as free forms and/or their glucuronides. The proportions of free and glucuronide metabolites in urine were 14.5% and 60.5% of the total dose. Part of the unchanged dihydrocapsaicin (10% of total dose) was excreted into the feces within 48 hr. Cell-free extracts of rat liver catalyzed the hydrolysis of dihydrocapsaicin to vanillylamine and 8-methyl nonanoic acid. The former compound was further transformed to vanillin in situ. Dihydrocapsaicin-hydrolyzing enzyme activity was found in various organs of rat. The activity was located mainly in the liver. On the basis of the present data, the metabolic pathway of dihydrocapsaicin in rats was proposed.     

Synthesis of l-Tryptophan from Indole and dl-Serine by Tryptophan Synthetase Entrapped in Fibres

Optimal culture conditions for microbial production of tryptophan synthetase were studied. It was found that on cultivation of Escherichia coli 476, a tryptophan auxotroph, in a medium containing 5g/liter glycerol as C source, supplemented with 1 g/liter of acid-treated peptone, cells with high tryptophan synthetase activity could be obtained.

The enzyme was extracted from cells and 3-fold purified by heat treatment and ammonium sulfate precipitation. The overall yield of the isolation procedure was 60%.

The partially purified tryptophan synthetase was entrapped in cellulose triacetate fibres. Under storage conditions, in refrigerator, the entrapped enzyme was stable at least for 6 months. The activity of the entrapped enzyme was about 75% with respect to the free enzyme.

Similar behaviour for the free and entrapped enzyme was observed as to the effect of temperature and pH on the enzymic activity. The operational stability of the entrapped tryptophan synthetase was very good (activity unchanged after 50 days) provided the accumulation of indole on the fibres was avoided.     

Inducibility of Rat Liver Drug-Metabolizing Enzymes as an Index of Food-Screeing for Safety Assessment

D-Lactate dehydrogenase (D-LDH) from Pediococcus pentosaceus ATCC 25745 was found to produce D-3-phenyllactic acid from phenylpyruvate. The optimum pH and temperature for enzyme activity were pH 5.5 and 45 °C. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat?K _m) values for the substrate phenylpyruvate were estimated to be 1.73 mmol/L, 173 s^?1, and 100 (mmol/L)^?1 s^?1 respectively.     

Main Polyol Dehydrogenase of Gluconobacter suboxydans IFO 3255, Membrane-bound D-Sorbitol Dehydrogenase,That Needs Product of Upstream Gene,sldB, for Activity

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.     

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O₂ or a cofactor for the conversion, but was stimulated by Mn²⁺. N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.     

Relation of Cheddar Cheese Ripening to Bacterial Lipolysis

This investigation was undertaken to find the relationship between fat hydrolysis and lipolytic activities of lactic acid bacteria participated in Cheddar cheese ripening. Increases in titratable acidities due to lactic fermentation were completed at early stage of ripening. Ripening indices (ratio of water-soluble nitrogen to total nitrogen) increased rapidly until 90 days and thereafter gradually up to 150 days. Considerable amounts of free fatty acids were released from cheese fat throughout the ripening period. Cheese bacteria were enumerated on the media of tomato-glucose-agar and acetate-agar. About 70% of bacteria isolated from cheese at age of 150 days were classified into Lactobacillus casei and L. plantarum. Lipolytic activities of lactobacilli isolated were detected definitely on double-layered agar plates containing Victoria blue-stained olive oil. Lipase activities were determined in cheese extracts during ripening.     

Practical Synthesis of the Disaccharide Epitope,D-Galactopyranosyl-α-1,3-D- galactopyranose,by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose as the Glycosyl Acceptor

D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.