Efficient biochemical processes were developed for the synthesis of several chiral alcohol and acid intermediates of insecticides by a combination of strictly stereoselective hydrolytic enzyme-catalyzed reactions and subsequent chemical transformations with inversion or racemization of the chiral center of the undesired antipodes. The whole amounts of starting racemic mixtures are converted to desired stereoisomers in the processes, which are generally applicable to the industrial productions of various chiral secondary alcohols and α-substituted acids once a highly stereospecific enzyme is obtained for the target compounds. The alcohols reported here are: 1-(4-phenoxyphenoxy)-2-propanol, 1; 4-hydroxy-3-methyl-2-(2’-propynyl)-2-cyclopentenone, 2; and α-cyano-3-phenoxybenzyl alcohol, 3. The acids are 2, 2-dimethyl-3-(2-methyl-1-propenyl)-cyclopropanecarboxylic acid (chrysanthemic acid), 4; and 2-(4-chlorophenyl)-3-methylbutyric acid, 5. In addition, the mechanism of action of Pseudomonas cepacia lipase (PCL), the most effective enzyme for the resolution of 1, and the recombinant Arthrobacter globiformis esterase (AES) for 4 was studied from the reaction kinetics. The site-directed mutagenesis techniques were also used for AES. The results indicated that the stereoselectivity of PCL is caused by the position and direction of a medium-sized substituent at the chiral center of the alcohol moiety in the rate-determining breakdown of a tetrahedral intermediate in the acylation of the enzyme and that the catalytic site of AES has the characteristics of the penicillin-recognizing enzymes in which Ser 59 in the concensus motif Ser-X-X-Lys plays a vital role as a nucleophile during acylation and Lys 62 acts as a general base.
A simple method is described for detecting glycoproteins which had been dot-blotted or electro-blotted on to a polyvinylidene difluoride (PVDF) membrane after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. This method is based on the periodate oxidation of glycoproteins on a PVDF membrane with subsequent staining by the chromophoric hydrazide, 4-N,N-dimethylamino-4′-azobenzene sulfonyl hydrazide. The glycoproteins could be visualized as red-colored spots or bands in the range of 0.16-0.31 μγ.
Chlorogenic acid, a dietary antioxidant, effectively inhibited the iron-induced lipid peroxidation of bovine liver microsomes in a concentration-dependent manner. In the Fenton-type reaction, chlorogenic acid inhibited the production of the hydroxyl radical by iron-EDTA or iron-ADP, while iron plus chlorogenic acid did not generate the hydroxyl radical. The formation of an iron complex with chlorogenic acid was demonstrated by UV/vis absorbance spectroscopic, ESR and 1H-NMR studies. The ferric complex with chlorogenic acid was in the ferric high-spin state near rhombicity, and had no radical scavenging activity. The results indicate that chlorogenic acid prevented the formation of the hydroxyl radical by forming a chelate with iron whose complex cannot catalyze the Fenton-type reaction.
Fourteen phenylphenalenone-type phytoalexins (1-14), including three new compounds, were isolated from the peel of unripe Musa acuminata [AAA] cv. Buñgulan fruit which had been injured and then inoculated with conidia of Colletotrichum musae. These new phytoalexins were identified as (+)-cis-2,3-dihydro-2,3-dihydroxy-4-(4′-hydroxyphenyl)phenalen-1-one (12), 9-(3′,4′-dimethoxyphenyl)-2-methoxyphenalen-1-one (13) and 9-(4′-hydroxyphenyl)-2-methoxyphenalen-1-one (14). The ratios of the relative intensities of the [M]+/[M-H]+ ions or [M-H2O]+/[M-H2O-H]+ ions in the EI mass spectra were applied to discriminate between 4- and 9-phenylphenalenones. An antifungal test on the phytoalexins showed that a phenolic hydroxyl group was essential for the activity.
Baker’s yeast reduction of methyl and ethyl (2-oxocyclohexyl)acetates proceeded with enantio- and diastereo-selectivity, affording the corresponding (2S)-trans-alcohols (major), (2S)-cis-alcohols (minor), and the unaltered (1S)-ketones with high optical purity.
Levoglucosenone reacted with furfural in the presence of an aqueous base to give a product in high yield with high stereoselectivity. The structure, including the stereochemistry of the product, was elucidated by NMR analyses.
A protease was purified from the growing point of asparagus, Asparagus officinalis, using a cystatin-Sepharose column. The asparagus protease is the first protease isolated from the growing point of a plant tissue and from Liliaceae. Its molecular mass was estimated to be 28 kDa by SDS-PAGE. The optimum pH of the enzyme was 7 at 30°C using casein as a substrate. The enzyme was strongly inhibited by monoiodoacetic acid, but not by diisopropylfluorophosphate, suggesting that it is a cysteine protease. Asparagus protease had broad specificity on the hydrolysis with oxidized B-chain of insulin as a substrate. However, for the P2 position of the cleavage site, the large hydrophobic side chains of amino acid residues such as Phe, Val, and Leu were considerably preferred. Asparagus protease was similar to papain about specificity at the P2 position. The N-terminal sequence of the first 12 residues was identified and 8 residues among them agreed with that of papain, accompanying the addition of one residue (Ala) to that of papain.
Further studies of mimosine toxicity in broiler chicks were done to clarify a possibility of osteopathy. The mineral content and density of femur and the strength, ductility, and toughness for the index of mechanical properties significantly decreased in the 1% mimosine group, compared with those in the control and restricted groups. The stiffness had a decreasing tendency in the 1% mimosine group. Consequently, it was concluded that chicks fed ad libitum a 1% mimosine diet for 12 days developed osteopathy. The bone mineral density and the strength of the restricted group were lower than those of the control group, and those of the 1% mimosine group were still lower than those of the restricted group. Contents of pyridinoline and deoxypyridinoline in the excrement were significantly higher in the restricted group than those in the control group, but the contents in the 1% mimosine group were significantly lowest among the groups. Osteopathy in chicks fed mimosine, therefore, seemed to be done by loss of appetite and changing to a low turnover of bone caused by mimosine.
Vase solution containing 2% sucrose prevented the deterioration of chrysanthemum (Dendranthema grandiflorum Kitamura) cut flowers induced by gamma-rays at 750 Gy. Glucose, fructose, and sucrose in florets and leaves of irradiated chrysanthemums decreased more rapidly than those of unirradiated ones, when the cut chrysanthemums were held in a vase solution without sucrose. The sugar contents of florets and leaves and the respiratory rate of irradiated chrysanthemums held with sucrose remained at higher levels than those of unirradiated ones. Incorporation of 14C from [14C]sucrose into CO2 was increased by irradiation. Incorporation of [α-32P]dTTP into trichloroacetic acid (TCA) insoluble substances in florets was increased by irradiation and by exogenous sucrose supply. These results suggest that sucrose in a vase solution was used as a respiratory substrate and facilitated the repair of radiation-induced damage, resulting in the extension of longevity of irradiated chrysanthemums.
Analysis of quiescent seed extracts of Phaseolus vulgaris cultivars found high activities of chitinase, N,N′-diacetylchitobiase and β-N-acetylhexosaminidase in whole seeds and their dissected organs (cotyledons, axis, and seed coat). Activities of these enzymes were compared in seeds of two cultivars phenotypically distinguishable by soft white (cv. Surattowonder) and hard brown (cv. Maisugata) seed coats. In both cultivars, chitinase activity was found high in all organs, chitobiase in the seed coat, and β-N-acetylhexosaminidase mainly in the axis. In terms of specific activity, all three enzymes were extraordinarily higher in extracts of seed coat than the others, specially referring to the cultivar Surattowonder. Although the cultivars showed in general similar distribution patterns of activities among their seed organs, the discrepancies found between them seem to be expressing intrinsic attributes of their seed coats. The relationship between the two cultivars, the enzyme activities measured and defense mechanisms are discussed.
The gene coding for an extracellular lipase of Bacillus stearothermophilus L1 was cloned in Escherichia coli. Sequence analysis showed an open reading frame of 1254 bp, which encodes a polypeptide of 417 amino acid residues. The polypeptide was composed of a signal sequence (29 amino acids) and a mature protein of 388 amino acids. An alanine replaces the first glycine in the conserved pentapeptide (Gly-X-Ser-X-Gly) around the active site serine. The expressed lipase was purified by hydrophobic interaction and ion exchange chromatography using buffers containing 0.02% (v/v) Triton X-100. The lipase was most active at 60-65°C and in alkaline conditions around pH 9-10. The lipase had highest activity toward p-nitrophenyl caprylate among the synthetic substrates and tripropionin among the triglycerides. It hydrolyzed beef tallow and palm oil more rapidly than olive oil at 50°C.
Starfish oocytes are naturally arrested at the prophase stage of the first meiotic division and resume meiosis in response to the maturation-inducing hormone 1-methyladenine. Five analogs of 1-methyladenine including three novel ones were synthesized and tested for biological activities as 1-methyladenine agonists or antagonists in triggering reinitiation of meiosis of starfish Asterina pectinifera oocytes, as well as for competition in binding to putative 1-methyladenine receptors with respect to 1-methyladenine. 1-Ethyladenine was an effective agonist, but 1-propyladenine served as a weak antagonist to 1-methyladenine, indicating strict specificity for a relatively small N-1 substituent. Analogs in which carboxymethyl or methyl group substitutes for a hydrogen of 6-amino group still retained oocyte maturation-inducing activity, but to a much lesser degree. The results of the competitive binding assay with cortices of oocytes demonstrated that these agonists or antagonist inhibited the binding of [3H]1-methyladenine to receptors. 8-methylamino-1-methyladenine competed only weakly with [3H]1-methyladenine for the binding to cortices, although it behaved as a potent antagonist.
EvgA and EvgS constitute one two-component signal transduction system in Escherichia coli. Although probable signaling domains of these proteins have been estimated, the molecular mechanism of their inteaction remains to be elucidated. Here, we investigated protein to protein interactions between EvgA and EvgS and also between the EvgAS system and other related signaling pathways by means of surface plasmon resonance. EvgA and EvgS interacted directly and inhibition of phosphorylation of their functional domains abolished formation of the EvgAS complex. No interaction was observed either between EvgA and Bordetella BvgS or BvgA and EvgS. OmpR, a response regulator for the osmoregulative gene expression of E. coli, had similar but not identical behavior towards EvgS to that of EvgA. These results indicate that interaction between the signaling proteins is closely related to phosphorylation of the functional domain of the proteins.
Squid (Todarodes pacificus) liver RNase (RNase Tp) was purified. RNase Tp was a base non-specific and acid RNase. Upon hydrolysis of RNA, RNase Tp released four mononucleotides in the order of G>A>U>C. RNase Tp consisted of two peptides with 198 and 23 amino acid residues. The amino acid sequences of these peptides were analyzed. The large peptide had two unique segments containing most of the active site amino acid residues of RNase T2 family enzymes. From the comparison of the sequence of short peptide with the sequences of the other RNase belonging to RNase T2 family RNases, it was found that the amino acid sequence of the short peptide was very similar to that of the C-terminal portion of RNases of the RNase T2 family. Thus, we concluded that the short peptide was a C-terminal part of RNase Tp. The molecular mass of the protein moiety of RNase Tp was 25,582 daltons. The amino acid sequence of RNase Tp most resembles that of oyster RNase (91 amino acid residues identical) in the RNase T2 family RNases. However, the N-terminal portion of RNase Tp was unusually similar to those of plant RNases, rather than the other animal RNases.
Treatment with KI and its subsequent removal induced disassembly of Bacillus stearothermophilus pyruvate dehydrogenase complex (PDC) and association of disassembly products, respectively. The disassembly yielded neither completely dissociated components nor aggregate, but did yield a few molecular forms smaller than PDC. Depending on the KI concentration, these changes were of three phases: K-1, below 0.6 M; K-2, 1.0-1.5 M; K-3, above 1.8 M. PDC was disassembled in K-1 to C1 comprising pyruvate decarboxylase and lipoate acetyltransferase mainly and C2 comprising the decarboxylase and dihydrolipoamide dehydrogenase. In K-1, the removal of KI resulted in an apparent reconstitution of PDC. The mixing of C1 with an excess of C2 yielded an assembly larger than PDC and restored enzyme activities, but specific activities were different from those of PDC. In K-2 and K-3 phases, complexes smaller than PDC were yielded from disassembly products, and activities except for that of the acetyltransferase were not restored.
An isomaltotriose-producing endo-dextranase was simply purified from cell-free culture broth of a Fusarium sp. by ethanol fractionation and consecutive column chromatographies using DEAE-Toyopearl and Bio-Gel P-100. The purified enzyme was judged to be homogeneous on PAGE and SDS-PAGE as well as isoelectric focusing. The molecular mass of the enzyme was estimated to be about 69 kDa by SDS-PAGE. The enzyme is an acidic protein with a pI of 4.6. The optimum pH and temperature were pH 6.5 and 35°C, respectively. The enzyme was completely stable over the range of pH 4.5-11.8 at 4°C for 24 h and at temperatures below 45°C. Inactivation of the enzyme was found to be partial with 5 mM Cu2+, being about 70% inhibition and complete with 5 mM of Fe3+, Hg2+, Ag+ or NBS. The enzyme split dextran in an endo-lytic action to produce a large amount of isomaltotriose and a slight amount of isomaltose and glucose. The anomeric configurations of the reaction products formed by the enzyme were α-form, indicating that the α-glycoside linkages in the substrate are retained. The final yield of isomaltotriose from dextran T-2000 was about 62%.
We isolated glucan-binding peptides of a dextransucrase from Leuconostoc mesenteroides B-512F. The dextransucrase was bound to DEAE-Sephadex A-50, Sephadex G-100, and mutan from Streptococcus mutans. Mild trypsin digestion dissociated the enzyme and glucan binding. In the presence of ammonium sulfate, several peptides were bound to glucan after trypsin digestion. Four main mutan-binding peptides were obtained by this method, and those amino acid sequences were analyzed. One of them was identical with the dextran-binding peptide that contains lysine, which was previously isolated by differential chemical modification with o-phthalaldehyde. We also found mutan-binding peptides in sucrose- and dextran-binding regions and a lysine-rich region. Also, there was a peptide similar in sequence to glucan-binding A-repeat of streptococcal glucosyltransferases.
A chitin binding protein (CBP21) 21 kDa in size, is a major protein in the culture supernatant when Serratia marcescens 2170 is grown in the presence of chitin. The gene (cbp) for CBP21 was found to be located in a region 1.5 kb downstream of the chiB gene encoding chitinase B. The cbp gene encodes a polypeptide of 197 amino acids with a calculated size of 21.6 kDa containing a putative signal sequence of 27 amino acids. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins showed that CBP21 is similar (45.3% amino acid identity) to CHB1 of Streptomyces olivaceoviridis. Purified CBP21 prepared from the periplasmic fraction of Escherichia coli carrying the cloned cbp gene showed its highest binding activity to squid chitin (β-chitin) followed by colloidal chitin and regenerated chitin. Binding of CBP21 to regenerated chitin was affected by pH, in particular, low pH reduced binding activity markedly. The presence of similar chitin binding proteins in the distantly related microorganisms, Streptomyces and Serratia, suggests a wide distribution of this type of chitin binding protein in chitinolytic microorganisms.
Calpastatin, an endogeneous inhibitor protein acting on calpain (Ca2+-dependent cysteine proteinase), is widely distributed in animal tissues and cells. Two different expression systems, baculovirus-infected Spodoptera frugiperda (Sf9) insect cells and Escherichia coli, were used for overexpression of the human calpastatin tagged with N-terminal hexahistidine peptide. Recombinant calpastatin was purified to homogeneity by nickel ion affinity chromatography and gel filtration separation. Purified recombinant proteins from both systems have similar inhibitory activity for calpain.
To examine the mechanism of starch degradation in legume cotyledons and the physiological role of α-glucosidase, mung bean seeds were germinated in the presence of Bay m 1099, an α-glucosidase inhibitor. Bay m 1099 (10 μg/ml medium), which minimized the growth deterioration of the mung bean seedlings, caused no changes in the overall rate of starch degradation and of soluble carbohydrate production in the cotyledons, although α-glucosidase activity had been completely suppressed. Total amylase and phosphorylase activities were not influenced by Bay m 1099. These results suggest that the mung bean α-glucosidase is less responsible for starch degradation, unlike wheat α-glucosidase [Konishi et al., Biosci. Biotech. Biochem., 58, 135-139 (1994)].
A cDNA encoding glutamine-dependent asparagine synthetase was isolated from dark-adapted Glycine max cell culture. The deduced amino acid sequence showed 76-89% identity with other plant sequences. The gene for asparagine synthetase is expressed predominantly in shoots as compared to roots of etiolated plants and the level of expression decreases following light treatment, suggesting that the gene expression is down-regulated by light.
A genomic clone containing part of the coding region and upstream sequences of a phosphoenolpyruvate carboxylase (PEPC) gene was isolated from a soybean genomic library. The first intron of this gene is located in the 5′ untranslated region. This intron-spanning fragment is capable of increasing GUS activity in plant cells.
The mode of action of poly(1,4-α-L-guluronide) lyase from Enterobacter cloacae M-1 on unsaturated oligoguluronic acids was studied using fluorophore-assisted carbohydrate electrophoresis. The polyguluronate lyase degraded unsaturated penta-, hexa-, and heptaguluronic acids, but not unsaturated oligoguluronic acids with DPs less than 4. On comparison with the aspect of enzymatic degradation of unsaturated oligoguluronic acid and saturated oligoguluronic acid having the same DP, the former was degraded faster than the latter, and also the cleavage pattern of the polyguluronate lyase on unsaturated oligoguluronic acids was considerably different from that on saturated oligoguluronic acids. From the results described above, we suggest that the affinity of the first subsite from the non-reducing end side of the enzyme to Δ residues is lower than that to GulA residues.
A new pyriculol-related phytotoxin, designated as pyricuol (1), was isolated from a liquid culture of Magnaporthe grisea, the causal fungus of rice blast disease, together with two known metabolites, pyriculol (2) and dihydropyriculol. Its structure was determined on the basis of physicochemical and spectroscopic data to be 2-(3-hydroxymethyl-1,4-hexadienyl)-6-hydroxybenzaldehyde.
Glutaraldehyde-crosslinked α-casein polymers with molecular masses of 1-5×105 were found to inhibit adhesive insoluble glucan formation catalyzed by glucan synthases from Streptococcus sobrinus B13, a cariogenic oral bacterium. Of the three subcomponents of the glucan synthases tested, primer-dependent insoluble glucan synthase was inhibited specifically by the Mr~2.7×105 polymer at a concentration of 1.4-8.5 μg/ml. The polymer was also active in inhibiting the artificial plaque formation by S. sobrinus B13.
To study the effects of lactic acid bacteria on the mucosal defence against dietary protein antigens, we compared the mucosal IgA responses to β-lactoglobulin (β-LG) of two groups of mice fed a whey protein diet with and without a culture condensate of Bifidobacterium longum. Both total IgA and anti-β-LG IgA levels in tissue extracts of the small intestinal wall were significantly higher in mice fed the B. longum diet for 2 weeks than in control ones. Peyer’s patch (PP) cells from B. longum-fed mice had a much larger increase in in vitro IgA production than ones from control mice. Furthermore, the in vitro IgA response to β-LG was detected only when PP cells from B. longum-fed mice were assayed. These results suggest that orally ingested lactic acid bacteria may protect a host from invasion of the intestinal mucosa by dietary antigens that have escaped enzymatic digestion in the intestine.
When milk is ingested, casein micelles will be successively digested by pepsin in the stomach and trypsin in the intestine. Therefore, we digested casein micelles successively with pepsin at pH 4.0 and trypsin at pH 7.0, and recovered casein phosphopeptides (CPP) as CPP-calcium phosphate (CP) complexes. The CPP-CP complexes contained 248 mg of calcium/g peptides and 175 mg of inorganic phosphorus/g peptides, which were higher than those of CPP-CP complexes driven from acid-precipitated casein and casein micelles by tryptic digestion only. It contained more αS1-CN-5P(f59-79) than the other CPP preparations did.
The atherogenic index was found to be significantly better in rats fed a high-cholesterol diet supplemented with black tea extract than in the ones not given the extract. It was also evident that black tea inhibited the proliferation of smooth muscle cells involved in the development and progression of atherosclerosis, and suppressed the production of oxidized low-density lipoprotein, a cause of lipid accumulation. It thus seems likely that black tea has an antiatherosclerotic action.
Using Caco-2 cell monolayers and MTT assay, the relationship between cell viability (a) and transport-enhancement effect of 1,2-dicaproin (C6DG), monocaprin (C10MG), and capric acid sodium salt (C10FANa) was examined. Transport enhancement effect was assessed by apparent permeability (Papp) of penicillin V. There was a linear relationship between (Papp-aSa) and (1-a) values, where Sa was the apparent permeability for the viable cells. The apparent permeability for the damaged cells (Sd) was evaluated from the slope of the line. Each of the enhancer compounds gave a different Sd value 2.00×10-4, 0.82×10-4, and 0.10×10-4 cm/s for C6DG, C10MG, and C10FANa, respectively, but the value was independent of its concentration for C10MG and C10FANa. C6DG would be the safest enhancer among the three compounds because of its high Sd value at the low level of cell damage. Sd could be used as a criterion for estimating the safety of enhancers.
Transmittance near infra red (NIR) spectra (500-1500 nm) of individual peanut was measured to detect the internally moldy nuts. The moldy nuts the appearance of which had little difference from the sound nuts by visual observations could be distinguished from each other by comparing the transmittance ratio of 700 nm to 1100 nm by NIR spectrometry. The fungal hydrolysis of the triglycerides that were contained in the nut seemed to account for these differences on the NIR spectra. Because of the higher incidence of aflatoxin (AF) contamination on these moldy nuts, taking out the internally moldy nuts detected by NIR, could drastically reduce the AF content of the overall lot of peanuts.
This study provided the first evidence for an antioxidant action of the silk protein sericin by showing that sericin suppressed in vitro lipid peroxidation. Furthermore, sericin was found to inhibit tyrosinase activity. These results suggest that sericin may be a valuable natural ingredient for food and cosmetic industries.
Some typical antioxidants, sesamol, α-tocopherol, BHT and ascorbic acid, were examined for their effects on preventing the thermal decomposition of phosphatidylcholine hydroperoxide (PC-OOH), sesamol alone showing some activity. The others, however, had no significant effect, which is in sharp contrast to our previous studies on their remarkable activity toward the thermal decomposition of fatty acid hydroperoxide. Ascorbic acid, which accelerated the decomposition of fatty acid hydroperoxide, was found to have no effect on PC-OOH in its decomposition and prevention.
Synthetic phosphatidylcholine hydroperoxide (PC-OOH), phosphatidylethanolamine hydroperoxide (PE-OOH) and phosphatidylglycerol hydroperoxide (PG-OOH) could be analyzed by ion spray ionization mass spectrometry, clearly affording the molecular ion peaks as protonated ion species without any fragmentation. Linearity between PC-OOH concentration and peak intensity, and the minimum detectable concentration of PC-OOH were obtained.
The antioxidative activity was studied for 25 kinds of tea and catechins by a new evaluation method using an oxygen electrode. The concentration of catechins in 6 types of green tea was analyzed by HPLC. The result indicates that the antioxidative activity of green tea depends to some extent on the amount of catechins present.
Hydrolyzing activities toward p-nitrophenyl (p-NP)-β-D-glucoside and laminarin in a culture filtrate of Bacillus circulans KA-304, which has been observed to form protoplasts from Schizophyllum commune mycelia, increased when the bacterium was grown on a cell-wall preparation (CWP) of S. commune or laminarin as a carbon source. An analysis of the filtrate with the CWP suggested occurrence of two major p-NP-β-D-glucoside-hydrolyzing enzymes (β-D-glucosidases I and II) and a laminarin-hydrolyzing enzyme. After separation by DEAE-cellulose column chromatography, β-D-glucosidases I and II were isolated (β-D-glucosidase I: 13-fold purification with 34% yield; β-D-glucosidase II: 26-fold with 8%). The enzymes resembled each other in their properties except for their molecular weight, subunit structure (β-D-glucosidase I: 200,000, tetramer; II: 100,000, dimer), and susceptibility to such substances as p-chloromercuribenzoic acid and Ag+ ion. β-D-Glucosidases I and II hydrolyzed gentiobiose (β-1,6 glucosidic linkage; Km=3.6 mM, β-D-glucosidase I; 4.6 mM, β-D-glucosidase II) and laminaribiose (β-1,3 glucosidic linkage; Km=6.1 mM, β-D-glucosidase I; 6.7 mD β-D-glucosidase II), and showed a certain reactivity toward laminarin as well.
We cloned a chitinase-encoding gene from Aspergillus nidulans by polymerase chain reaction using degenerated oligonucleotide primers designed from the conserved amino acid sequences among chitinases from yeasts and Rhizopus spp. The cloned gene, named chiA, encoded a polypeptide consisting of 660 amino acids. Disruption of chiA had no effect on hyphal or conidiophore morphology, but germination frequency and hyphal growth rate decreased substantially. Expression of chiA was investigated using Escherichia coli β-galactosidase as a reporter enzyme. The β-galactosidase activity was present during hyphal growth and increased twice as the conidiophores developed. In situ staining of β-galactosidase activity found high expression in metulae, phialides, and conidia during conidiophore development, indicating that the expression of chiA is developmentally regulated. This is the first report to isolate a chitinase gene from A. nidulans and investigate its functions using the gene disruption technique and gene fusion methods in filamentous fungi.
2-Ketoaldonate reductase, which is involved in ketogluconate catabolism, was purified to homogeneity from Brevibacterium ketosoreductum ATCC21914. The enzyme was found to catalyze the reduction of 2,5-diketo-D-gluconate to 5-keto-D-gluconate, and to a lesser extent, 2-keto-D-gluconate to D-gluconate, and 2-keto-L-gulonate to L-idonate. The molecular mass of the reductase was 35 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 72 kDa by gel filtration, indicating that the native enzyme may exist as a dimer. The reductase was optimally active at pH 6.0 with NADPH as a preferred electron donor. The pI of 4.7 was measured for the enzyme. The apparent Km for 2,5-diketo-D-gluconate and NADPH were 5 μM and 10 μM, respectively. The amino-terminal amino acid sequence was NH2-Ala-Ser-Ile-Ser-Val-Ser-Val-Pro-Ser-Ala-Arg-Leu-Ala-Glu-Asp-Leu-Ser-Asp-Ile-Glu.
Escherichia coli JM109 (pGDA2) overexpressing the glucose dehydrogenase (GDH) gene from Bacillus megaterium IWG3 was examined for use as a cofactor regenerator. In the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by E. coli JM109 (pKAR) which is an aldehyde reductase-overproducing transformant, E. coli JM109 (pGDA2) can act as an NADPH regenerator with NADP+ and glucose, similarly to commercially available GDH.
The interesterification reaction by immobilized lipase between triolein and behenic acid (BA) or ethyl behenate (EB) was investigated in supercritical carbon dioxide (SCCO2) to produce 1,3-dibehenoyl-2-oleoyl glycerol (BOB). The incorporation rate of behenoyl group to triolein was much higher with EB as a substrate than with BA. The solubility in SCCO2 was found to be significantly higher for EB than BA, which seemed to cause the higher formation rate of the behenoyl-enzyme complex in the former case to give the higher production rate of the final interesterification product, BOB.
A taxonomic study was done for the isolates obtained as cellulose high producers from sucrose. These strains were found to have a common characteristic unique for Acetobacter; they did not oxidize acetate and lactate. Therefore, we concluded that these isolates are classified as a new subspecies and proposed Acetobacter xylinum subsp. nonacetoxidans subsp. nov. BPR 2002 (=JCM 10150) was designated as a type strain of the new subspecies.
D-Glutamate, an indispensable component of peptidoglycans of bacteria, is provided by glutamate racemase in E. coli cells. Compensation for D-glutamate auxotrophy of E. coli WM335 cells lacking the glutamate racemase gene, murI, with the D-amino acid aminotransferase gene suggests the presence of a threshold concentration for the D-glutamate required by E. coli cells, as well as a regulation system for murI expression.