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Highlights • Lactic acid bacteria were screened from traditional fermented foods. • High γ-aminobutyric acid productivity was detected in ten strains. • They comprised Lactobacillus buchneri, Lactobacillus brevis, and Weissella hellenica. • Angiotensin-converting enzyme inhibitory activities (IC50) were high in milk protein media. • Three out of ten strains had high GABA-producing activities at low pH (pH 3)., Many traditional fermented products are onsumed in Ishikawa Prefecture, Japan, such as kaburazushi, narezushi, konkazuke, and ishiru. Various kinds of lactic acid bacteria (LAB) are associated with their fermentation, however, characterization of LAB has not yet been elucidated in detail. In this study, we evaluated 53 isolates of LAB from various traditional fermented foods by taxonomic classification at the species level by analyzing the 16S ribosomal RNA gene (rDNA) sequences and carbohydrate assimilation abilities. We screened isolates that exhibited high angiotensin-converting enzyme (ACE) inhibitory activities in skim milk or soy protein media and produced high γ-aminobutyric acid (GABA) concentrations in culture supernatants when grown in de Man Rogosa Sharpe broth in the presence of 1% (w/v) glutamic acid. The results revealed that 10 isolates, i.e., Lactobacillus buchneri (2 isolates), Lactobacillus brevis (6 isolates), and Weissella hellenica (2 isolates) had a high GABA-producing ability of >500 mg/100 ml after 72 h of incubation at 35 °C. The ACE inhibitory activity of the whey cultured with milk protein by using L. brevis (3 isolates), L. buchneri (2 isolates), and W. hellenica (2 isolates) was stronger than that of all whey cultured with soy protein media, and these IC50 were

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1–3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase+ strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1–3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc), and sialyllacto-N-tetraose a (Neu5Acα2–3Galβ1–3GlcNAcβ1–3Galβ1–4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1–3GlcNAcβ-pNP) and GalNAcβ1–3GlcNAcβ-pNP. GalNAcβ1–3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches. Background: Phenotypically lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase. Results: Hypothetical proteins BLLJ_1505 and BLLJ_1506 encode lacto-N-biosidase and its designated chaperone, respectively. Conclusion: The enzyme showed unique and unexpected substrate specificity. Significance: The enzyme is important for understanding how B. longum consumes human milk oligosaccharides and also may serve as a new tool in glycobiology.

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.

γ-Glutamyltranspeptidase (GGT) catalyzes the cleavage of such γ-glutamyl compounds as glutathione, and the transfer of their γ-glutamyl group to water or to other amino acids and peptides. GGT is involved in a number of biological phenomena such as drug resistance and metastasis of cancer cells by detoxification of xenobiotics. Azaserine and acivicin are classical and irreversible inhibitors of GGT, but their binding sites and the inhibition mechanisms remain to be defined. We have determined the crystal structures of GGT from Escherichia coli in complex with azaserine and acivicin at 1.65 A resolution. Both inhibitors are bound to GGT at its substrate-binding pocket in a manner similar to that observed previously with the γ-glutamyl-enzyme intermediate. They form a covalent bond with the O γ atom of Thr391, the catalytic residue of GGT. Their α-carboxy and α-amino groups are recognized by extensive hydrogen bonding and charge interactions with the residues that are conserved among GGT orthologs. The two amido nitrogen atoms of Gly483 and Gly484, which form the oxyanion hole, interact with the inhibitors directly or via a water molecule. Notably, in the azaserine complex the carbon atom that forms a covalent bond with Thr391 is sp 3 -hybridized, suggesting that the carbonyl of azaserine is attacked by Thr391 to form a tetrahedral intermediate, which is stabilized by the oxyanion hole. Furthermore, when acivicin is bound to GGT, a migration of the single and double bonds occurs in its dihydroisoxazole ring. The structural characteristics presented here imply that the unprecedented binding modes of azaserine and acivicin are conserved in all GGTs from bacteria to mammals and give a new insight into the inhibition mechanism of glutamine amidotransferases by these glutamine antagonists.

Phospholipids are major components of cellular membranes that participate in a range of cellular processes. Phosphatidic acid (PA) is a key molecule in the phospholipid biosynthetic pathway. In Saccharomyces cerevisiae, SLC1 has been identified as the gene encoding lysophosphatidic acid acyltransferase, which catalyzes PA synthesis. However, despite the importance of PA, disruption of SLC1 does not affect cell viability (Nagiec, M. M., Wells, G. B., Lester, R. L., and Dickson, R. C. (1993) J. Biol. Chem. 268, 22156-22163). We originally aimed to identify the acetyl-CoA:lyso platelet-activating factor acetyltransferase (lysoPAF AT) gene in yeast. Screening of a complete set of yeast deletion clones (4741 homozygous diploid clones) revealed a single mutant strain, YOR175c, with a defect in lysoPAF AT activity. YOR175c has been predicted to be a member of the membrane-bound O-acyltransferase superfamily, and we designated the gene LPT1. An Lpt1-green fluorescent protein fusion protein localized at the endoplasmic reticulum. Other than lysoPAF AT activity, Lpt1 catalyzed acyltransferase activity with a wide variety of lysophospholipids as acceptors, including lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, and lysophosphatidylserine. A liquid chromatography-mass spectrometry analysis indicated that lysophosphatidylcholine and lysophosphatidylethanolamine accumulated in the Deltalpt1 mutant strain. Although the Deltalpt1 mutant strain did not show other detectable defects, the Deltalpt1 Deltaslc1 double mutant strain had a synthetic lethal phenotype. These results indicate that, in concert with Slc1, Lpt1 plays a central role in PA biosynthesis, which is essential for cell viability.

Kluyveromyces marxianus NBRC1777 was identified as a thermotolerant yeast and was developed as a host for the expression of thermostable cellulase genes. The previously isolated genes for thermostable endo-beta-1,4-glucanase, cellobiohydrolase, and beta-glucosidase were introduced into the chromosome of K. marxianus and successfully expressed under the control of high-expression promoters. The recombinant K. marxianus expressing cellulase genes became able to grow in synthetic medium containing cellobiose or carboxymethyl-cellulose as the single carbon source. Moreover, the recombinant strain produced 43.4 g/L ethanol from 10% cellobiose. These results suggest that K. marxianus may afford a useful host system, which may be applicable to the simultaneous saccharification and fermentation and the foundation of cellulose consolidated bioprocessing.

γ-Glutamyltranspeptidase (GGT) is an extracellular enzyme that plays a key role in glutathione metabolism. The mature GGT is a heterodimer consisting of L- and S-subunits that is generated by posttranslational cleavage of the peptide bond between Gln-390 and Thr-391 in the precursor protein. Thr-391, which becomes the N-terminal residue of the S-subunit, acts as the active residue in the catalytic reaction. The crystal structure of a mutant GGT, T391A, that is unable to undergo autocatalytic processing, has been determined at 2.55-A resolution. Structural comparison of the precursor protein and mature GGT demonstrates that the structures of the core regions in the two proteins are unchanged, but marked differences are found near the active site. In particular, in the precursor, the segment corresponding to the C-terminal region of the L-subunit occupies the site where the loop (residues 438–449) forms the lid of the γ-glutamyl group-binding pocket in the mature GGT. This result demonstrates that, upon cleavage of the N-terminal peptide bond of Thr-391, the newly produced C terminus (residues 375–390) flips out, allowing the 438–449 segment to form the γ-glutamyl group-binding pocket. The electron density map for the T391A protein also identified a water molecule near the carbonyl carbon atom of Gln-390. The spatial arrangement around the water and Thr-391 relative to the scissile peptide bond appears suitable for the initiation of autocatalytic processing, as in other members of the N-terminal nucleophile hydrolase superfamily.

Gamma-glutamyl transpeptidase (GGT, EC 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione and plays a central role in glutathione homeostasis. We report here the synthesis and evaluation of a series of hydrolytically stable gamma-(monophenyl)phosphono glutamate analogues with varying electron-withdrawing para substituents on the leaving group phenols as mechanism-based and transition-state analogue inhibitors of Escherichia coli and human GGTs. The monophenyl phosphonates caused time-dependent and irreversible inhibition of both the E. coli and human enzymes probably by phosphonylating the catalytic Thr residue of the enzyme. The inactivation rate of E. coli GGT was highly dependent on the leaving group ability of phenols with electron-withdrawing groups substantially accelerating the rate (Brønsted betalg = -1.4), whereas the inactivation of human GGT was rather slow and almost independent on the nature of the leaving group. The inhibition potency and profiles of the phosphonate analogues were compared to those of acivicin, a classical inhibitor of GGT, suggesting that the phosphonate-based glutamate analogues served as a promising candidate for potent and selective GGT inhibitors.

We found endo-alpha-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590-1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Galbeta1,3GalNAc disaccharide from Galbeta1,3GalNAcalpha1pNP and asialofetuin, but did not release GalNAc, Galbeta1,3(GlcNAcbeta1,6)GalNAc, GlcNAcbeta1,3GalNAc, and Galbeta1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by (1)H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-alpha-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).

A novel bacterial putrescine utilization pathway was discovered. Seven genes, the functions of whose products were not known, are involved in this novel pathway. Five of them encode enzymes that catabolize putrescine; one encodes a putrescine importer, and the other encodes a transcriptional regulator. This novel pathway involves six sequential steps as follows: 1) import of putrescine; 2) ATP-dependent gamma-glutamylation of putrescine; 3) oxidization of gamma-glutamylputrescine; 4) dehydrogenation of gamma-glutamyl-gamma-aminobutyraldehyde; 5) hydrolysis of the gamma-glutamyl linkage of gamma-glutamyl-gamma-aminobutyrate; and 6) transamination of gamma-aminobutyrate to form the final product of this pathway, succinate semialdehyde, which is the precursor of succinate.

We found for the first time that Zygomycetes species showed resistance to Aureobasidin A, an antifungal agent. A novel family of neutral glycosphingolipids (GSLs) was found in these fungi and isolated from Mucor hiemalis, which is a typical Zygomycetes species. Their structures were completely determined by compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry, and (1)H NMR spectroscopy. They were as follows: Gal beta 1-6Gal beta 1-1Cer (CDS), Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTS), Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTeS), and Gal alpha 1-6Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CPS). The ceramide moieties of these GSLs consist of 24:0, 25:0, and 26:0 2-hydroxy acids as major fatty acids and 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid. However, the glycosylinositolphosphoceramide families that are the major GSLs components in fungi were not detected in Zygomycetes at all. This seems to be the reason that Aureobasidin A is not effective for Zygomycetes as an antifungal agent. Our results indicate that the biosynthetic pathway for GSLs in Zygomycetes is significantly different from those in other fungi and suggest that any inhibitor of this pathway may be effective for mucormycosis, which is a serious pathogenic disease for humans.

γ-Glutamyltranspeptidase (GGT) from Bacillus subtilis is an extracellular enzyme that exhibits glutaminase activity and is thus suitable for the fermentation of foods. As GGT of B. subtilis is synthesized only during the mid-stationary phase, which is inconvenient for industrial use, a strain overexpressing GGT for a sufficiently long period was generated to obtain large quantities of GGT. A plasmid vector, pHY300PLK, containing the ggt gene cloned from chromosomal DNA was introduced into a spo0A abrB double mutant strain, in which the level of GGT activity is high after the mid-stationary phase. The level of GGT activity in this strain increased steadily after the exponential phase, becoming 15-fold higher than that in the parental strain. The recombinant GGT was purified by 252-fold with a yield of 30.4%. The enzyme is a heterodimer consisting of one large subunit (45 kDa) and one small subunit (21 kDa). The enzyme is highly salt-tolerant and converts glutamine to glutamic acid effectively even in the presence of 18% NaCl. This is the first report of salt-tolerant GGT.

gamma-Glutamyltranspeptidase is the key enzyme in glutathione metabolism, and we previously presented evidence suggesting that it belongs to the N-terminal nucleophile hydrolase superfamily. Enzymatically active gamma-glutamyltranspeptidase, which consists of one large subunit and one small subunit, is generated from an inactive common precursor through post-translational proteolytic processing. The processing mechanism for gamma-glutamyltranspeptidase of Escherichia coli K-12 has been analyzed by means of in vitro studies using purified precursors. Here we show that the processing of a precursor of gamma-glutamyltranspeptidase is an intramolecular autocatalytic event and that the catalytic nucleophile for the processing reaction is the oxygen atom of the side chain of Thr-391 (N-terminal residue of the small (beta) subunit), which is also the nucleophile for the enzymatic reaction.

Theanine (γ-glutamylethylamide) is the major “umami” (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of theanine involving bacterial γ-glutamyltranspeptidase (GGT) was developed. The optimum reaction conditions were 200 mM Gln, 1.5 M ethylamine, and 0.4 units/ml GGT, pH 10. After 2-h incubation at 37 °C, 120 mM theanine was obtained, the conversion rate against Gln being 60%. Theanine was purified on Dowex 50 W×8 and Dowex 1×8 columns, and then identified by 1 H -NMR. This is the first report that a GGT, regardless of its source, can utilize an alkyl amine as an acceptor of the γ-glutamyl moiety in its transpeptidation reaction.

γ-L-Glutamyltaurine is a naturally occurring peptide and known to have several physiological functions in mammals. This paper describes a new method for the enzymatic production of γ-L-glutamyltaurine from L-glutamine and taurine through the transpeptidation reaction of γ-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12. The optimum conditions for the production of γ-L-glutamyltaurine were 200 mM L-glutamine, 200 mM taurine and 0.2 U/ml γ-glutamyltranspeptidase, pH 10, and 1-h incubation at 37°C. Forty-five mM γ-L-glutamyltaurine was obtained, the yield being 22.5%. γ-L-Glutamyltaurine was purified on Dowex 1 × 8 and C 18 columns, and identified by means of NMR and a polarimeter.

Cell growth and differentiation are regulated by nutrient availability in the yeast Saccharomyces cerevisiae. Under conditions of nitrogen limitation, diploid cells of S. cerevisiae differentiate to a filamentous growth known as a pseudohyphal growth, while haploid cells produce invasive filaments which penetrate the agar in nutrient-rich medium. We have found that GPR1, which encodes a putative G-protein-coupled receptor, is required for both pseudohyphal and invasive growth. Pseudohyphal growth was defective in Deltagpr1/Deltagpr1 mutant strain and this defect was reversed by addition of cAMP. Also, haploid Deltagpr1 mutant strain was defective in invasive growth. Northern blot analysis revealed that the transcriptional level of FLO11, which encodes a recently identified cell surface flocculin required for pseudohyphal growth, was reduced in Deltagpr1 mutant strain. These results indicate that GPR1 regulates both pseudohyphal and invasive growth by a cAMP-dependent mechanism.

Glutathione is the most abundant low-molecular-weight thiol compound in aerobic bacterial cells. Although its biosynthetic pathway in Escherichia coli is known, its degradative pathway is not clear. We have studied its degradative pathway using E. coli K-12 as a model bacterium. Glutathione synthesized during the exponential phase of growth is excreted into the medium. During the stationary phase, extra cellular glutathione penetrates into the periplasm where its γ-glutamyl residue is cleaved off by γ-glutamyltranspeptidase localized in the periplasm. The released cysteinylglycine is taken up into the cytoplasm through peptide transport systems and the peptide linkage of cysteinylglycine is cooperatively cleaved by enzymes with cysteinylglycinase activity. The resultant cysteine and glycine are used as cysteine and glycine sources, respectively. This cycle acts as a salvage system for cysteine (glycine) in the cells. γ-Glutamyltranspeptidase, the key enzyme of this cycle, was studied extensively not only from a physiological point of view, but also with the aim of applying this enzyme as a catalyst for the synthesis of useful γ-glutamyl compounds.

Eel calcitonin derivatives containing various N -linked oligosaccharides were chemo-enzymatically synthesized by the transglycosylation reaction of Mucor hiemalis endo- β - N -acetylglucosaminidase ( Endo - M ) to a glycosylated calcitonin derivative [ Asn ( GlcNAc 3 ]-CT in which N -acetyl-D-glucosamine (GlcNAc) is attached to the L-asparagine (Asn) residue of the peptide.

Penicillium oxalicum β-N-acetylhexosaminidase was found to have transglycosylation activity in addition to hydrolytic activity. The enzyme effectively transferred N-acetylglucosamine to various alcohols in the presence of N-acetylchitooligomers as donors. The enzyme also transferred N-acetylglucosamine to diol-derivatives. Using this transglycosylation activity, a drug carrier, 6-benzyloxyhexyl-β-N-acetylglucosaminide, was synthesized by incubating the enzyme with 6-benzyloxyhexan-1-ol and N-acetylchitotriose. The product was isolated by silica gel column chromatography and was analyzed by mass-spectrometry and 1H-NMR.

The β- d -glucosidase I (EC 3.2.1.21) activity of Bifidobacterium breve 203 isolated from feces of adult humans was found to be very low, but when B. breve 203 was acclimated to cellobiose, the activity of the enzyme increased in the acclimated cell, B. breve 203 clb. With the aim of elucidating the mechanism, responsible for this increased activity the translation and transcription of the β- d -glucosidase I gene were investigated using an antibody against β- d -glucosidase I and a probe specific to the gene, respectively. Western and Northern blots showed marked differences in the expression of the enzyme and the level of its mRNA between B. breve 203 and B. breve 203 clb. However, genomic Southern blots revealed only insignificant differences in the copy numbers of the open reading frames in the two strains. The 5′ flanking regions of the β- d -glucosidase I gene of B. breve 203 and that of B. breve 203 clb were therefore analyzed. The transcriptional start point, located 94 bp upstream from the first methionine codon, was identified by primer extension. A sequence, -TTGGAA-(15 bp)-TAATCT-, located 8 bp upstream from the transcriptional start point, was assigned as the promoter of the gene. A sequence highly homologous to the lac operator region of Escherichia coli was found downstream of the transcriptional start point.

Lactobacillus, a representative useful bacterium, in the intestinal tract was found to bind to some specific glycosphingolipids, like the pathogenic intestinal bacteria. Thin layer chromatography overlay assays using rabbit antiserum against Lactobacillus casei revealed that the bacteria bound to G A1 and trihexosylceramide strongly, but not to any gangliosides. The bacteria generally bound to glycosphingolipids having short sugar chains and galactosyl moiety in the non-reducing terminal. L.casei did not bind to G M1 , but bound to the product after sialidase treatment, G A1 . This indicated that sialic acid inhibited the adhesion of L.casei to tissues. L.casei actually bound nonacid glycosphingolipids but not acid glycosphingolipids extracted from the small intestinal mucosa of rats.

The sensitivity of HeLa cells to cholera toxin decreased by Corynebacterium sp. endoglycoceramidase treatment. This endo-enzyme destroyed the cholera toxin receptor, ganglioside G(M1), on the cell surface membrane by liberating intact oligosaccharide from it, which was confirmed by the decrease of intracellular cAMP accumulation and the results of the analysis of released oligosaccharide with a combination of pyridylamination method and HPLC. Fluorescence microscopy using the immunofluorescence method revealed that the amount of cholera toxin attached to the cells decreased in endoglycoceramidase-treated cells. The enzyme acted on cellular glycosphingolipids without addition of any activator protein which is required by other similar enzymes. Corynebacterium endoglycoceramidase is a useful tool to elucidate the function of glycosphingolipids on the cell surface in situ.

pepB gene which encodes peptidase B was mapped at 54 min on the Escherichia coli K-12 linkage map. pepB was cloned and its nucleotide sequence was determined. pepB gene has an open reading frame which encodes a protein of 420 amino acid residues the molecular weight of which is estimated to be 45,666. Peptidase B was suggested to be a homohexamer. The deduced amino acid sequence of peptidase B has an absolutely conserved octapeptide, NTDAEGRL, known as a zinc/manganese binding site and a so-called cytosol aminopeptidase signature in its central region. Peptidase B was suggested to belong to the peptidase family M17.

An endo-β-1,4-glucanase (EC 3.2.1.4) was purified from a culture filtrate of Aspergillus niger IFO31125 by column chromatography through TSK-gel DEAE-3SW and TSK-gel DEAE-5PW, and by gel filtration through TSK-gel G2000SW by high performance liquid chromatography. The enzyme was estimated to have a molecular weight of about 40 kDa by both gel filtration and SDS-polyacrylamide gel electrophoresis, and appeared to consist of a monomeric protein. It contained 8.9% carbohydrate. The optimal pH for activity was 6.0–7.0, and the stable pH range was 5.0–10.0. The optimum temperature at pH 6.0 was around 70°C. The enzyme was very thermally stable and no loss of original activity was found on incubation at 60°C for 2 h. The enzyme efficiently hydrolyzed carboxymethylcellulose and lichenan, but crystalline forms of cellulose, curdlan, laminarin, cellobiose, p-nitrophenyl-β- d -glucopyranoside and p-nitrophenyl-β- d -cellobioside were barely hydrolyzed. The activity of the enzyme was inhibited by Hg2+ and Cu2+ but was not affected by other inhibitors of thiol enzymes such as p-chloromercuribenzoate and N-ethylmaleimide. N-Bromosuccinimide showed a strong inhibitory effect, suggesting that a tryptophan residue is essential for the activity of the enzyme. The N-terminal amino acid sequence of the enzyme showed considerable homology to those of endo-β-1,4-glucanases from some other microorganisms, including Sclerotinia sclerotiorum and Schizophyllum commune. The enzyme had very strong protease-resistance, and showed no loss of activity when incubated with proteases such as Savinase at 40°C, even for 2 weeks.

Various yeast strains were screened for platelet-activating factor (PAF) production. High PAF production was found mainly in the strains of Saccharomyces genus. Yeast PAF showed a typical platelet aggregation pattern, which was inhibited by specific PAF antagonist, such as CV-3988, CV-6209 and L-652731. The main molecular species of yeast PAF were identified as 1-palmitoleoyl, -palmitoyl-, -oleoyl- and -stearoyl-2-acetyl- sn -glycero-3-phosphocholines (16:1, 16:0, 18:1 and 18:0 acylPAFs) and 1-hexadecenyl- and hexadecyl-2-acetyl- sn -glycero-3-phosphocholines (16:1 and 16:0 PAFs), by mass spectrometry. PAF formation in yeast cells increased at the middle stationary phase of growth.

We found that the structural gene for monoamine oxidase was located at 30.9 min on the Escherichia coli chromosome. Deletion analysis showed that two amine oxidase genes are located in this region. The nucleotide sequence of one of the two genes was determined. The peptide sequence of the first 40 amino acids from the N terminus of monoamine oxidase purified from E. coli agrees with that deduced from the nucleotide sequence of the gene. The leader peptide extends over 30 amino acids. The nucleotide sequence of the gene and amino acid sequence of the predicted mature enzyme (M.W. 81,295) were highly homologous to those of the maoA K gene and monoamine oxidase from Klebsiella aerogenes , respectively. From these results and analysis of the enzyme activity, we concluded that the gene encodes for monoamine oxidase ( maoA E ). The tyrosyl residue, which may be converted to topa quinone in the E. coli enzyme, was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidases.

γ-Glutamyltranspeptidase (EC 2.3.2.2) from Escherichia coli K-12 has been purified and crystallized by means of vapor diffusion in hanging drops. Two kinds of crystals on cell dimensions were found for X-ray diffraction analysis, one from ammonium sulfate and the other from polyethylene glycol 6000 as precipitants. The crystals of the orthorhombic form grown in the presence of 15% polyethylene glycol and 20 mM sodium acetate buffer were chosen for further analysis. The crystals belonged to space group P 2 1 2 1 2 1 , with cell dimensions of a =128·1, b =129·9 and c = 79·2 A, and two molecules constitute an asymmetric unit. These crystals diffracted to 2·0 A resolution and were suitable for X-ray crystallographic studies.

Endoglycoceramidase (EGCase) catalyzes the hydrolysis of the linkage between oligosaccharides and ceramides of various glycosphingolipids (GSLs). We found that the EGCase from Corynebacterium sp. had transglycosylation activity. Digesting GSLs with the enzyme in the presence of a suitable acceptor gave a mixture of hydrolytic and transglycosylic products. When GM1 was used as the substrate (donor), 1-hexanol was found to be the best acceptor of transglycosylation activity. Hexyl-II 3NeuAcGgOse4 produced was confirmed by fast-atom bombardment-mass spectrometry analysis and exoglycosidase digestion. The enzyme also transferred oligosaccharides of various GSLs to 1-hexanol.

The gene that codes for tyrosine phenol-lyase (EC 4.1.99.2) of Erwinia herbicola AJ2982 was cloned and its nucleotide sequence determined. The deduced protein product was 456 residues long with a molecular weight of 51,364 daltons. The amino acid sequences of Er. herbicola and Escherichia intermedia were calculated to be 90.6% identical.

Arginyl residues 513 and 571 of Escherichia coli K-12 gamma-glutamyl-transpeptidase (EC 2.3.2.2) were substituted with alanyl and glycyl residues, respectively, by oligonucleotide-directed in vitro mutagenesis. Both mutants were devoid of the enzymatic activity. On Western blot analysis, we found that both mutants accumulated a gamma-glutamyltranspeptidase precursor which was not processed into large and small subunits in the periplasmic space of Escherichia coli.

Aspartate racemase from Streptococcus thermophilus contains no pyridoxal 5'-phosphate or other cofactors such as FAD, NAD+, and metal ions. It was affected by neither carbonyl reagents such as hydroxylamine nor sodium borohydride but was strongly inhibited by iodoacetamide and other thiol reagents. Aspartate, cysteate, and cysteine sulfinate were the only substrates. The Km values for L- and D-aspartate were 35 and 8.7 mM, respectively. The enzyme catalyzed the exchange of alpha-hydrogen of the substrate with the solvent hydrogen. Racemization of L-aspartate in 2H2O showed an overshooting in the optical rotation of aspartate before the substrate was fully racemized. This shows that the removal of alpha-hydrogen of the substrate is at least partially rate-determining. When L- or D-aspartate was incubated with aspartate racemase in tritiated water, tritium was incorporated preferentially into the product enantiomer. The results strongly suggest that aspartate racemase contains two hydrogen acceptors.

An enzymic method for producing a disaccharide was developed using the endo-α-N-acetylgalactosaminidase from Alcaligenes sp. From 0.5 g asialofetuin, 2.66 mg of pure disaccharide was obtained by procedures including enzymic hydrolysis, alcohol fractionation, gel filtration on Sephadex G-10 and normal-phase high-performance liquid chromatography on Cosmosil 5 NH2. The structure of the disaccharide was determined as Ga1β1→3GalNAc by thin-layer chromatography, gas-liquid chromatography and proton nuclear magnetic resonance spectroscopy. The molecular extinction coefficients of the disaccharide at 210 nm, 200 nm and 190 nm were found to be 1.17 × 103 M−1, 4.36 × 103 M−1 and 8.00 × 103 M−1, respectively, and the molecular extinction coefficient of its derivative at 585 nm was determined as 15.6 × 103 M−1 by using the method of Reissig et al (J. Biol. Chem., 217, 959–966, 1955). This method of producing the disaccharide is considered to be applicable for practical use.

The generation of basic sites on a TiO{sub 2} surface by reduction is discussed on the basis of the results of catalytic reactions, temperature-programmed desorption of adsorbed CO{sub 2}, and measurements of the numbers of surface hydrogen acceptors and donors (DPPH{sub 2} and DPPH methods). The results of catalytic reactions show that both acidic and basic active sites are present on TiO{sub 2} surfaces and reduction of TiO{sub 2} increases its basic character. The TPD profile of adsorbed CO{sub 2} suggests that basic sites increase following the reduction of TiO{sub 2} but their strengths are not high. Reduction of TiO{sub 2} not only suppresses the surface acidity but also increases the number of surface proton acceptors. This is also supported by DPPH-DPPH{sub 2} experiments. These results lead to the conclusion that basic sites generated by the reduction of TiO{sub 2} are surface oxygen species which can abstract protons from adsorbed species.

Glutathione S-transferase Y-2 mRNA synthesis was induced in yeast Issatchenkia orientalis approximately 37 - fold by cultivation with o -dinitrobenzene. A cDNA library complementary to poly (A) +RNA of I. orientalis grown with o -dinitrobenzene was screened by colony hybridization. Twenty positive clones were obtained from 6,000 clones and seven of twenty positive clones expressed glutathione S-transferase activity in E. coli . One of the essing clones harboring plasmid pHT108 had 28 times more glutathione S-transferase activity induced by Isopropyl-β-D-thio-galactopyranoside than a brain harboring plasmid pUC118. Expressed glutathione S-transferase Y-2 protein comigrated with yeast glutathione S-transferase Y-2 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as detected by immunoblot analysis.

The endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. could release T-antigen (Ga1 beta 1----3GalNAc), a carcinoma-associated marker from asialo glycophorin and human cells. The released T-antigen from human asialo erythrocytes was determined by thin layer chromatography and gas liquid chromatography. The released T-antigen from human gastric carcinoma cell Kato III was identified by high performance liquid chromatographies with a reverse-phase column and a size fractionation column. Released T-antigen could be analyzed quantitatively by a sensitive method including pyridylamino derivatization and following high performance liquid chromatography. This suggests that the enzyme is useful for detection and determination of T-antigen from cells.

The effect of copper concentration of the culture medium on the production of copper-containing monoamine oxidase by Escherichia coli K-12 was studied. As previously reported, the addition of CuSO4 to the culture medium resulted in an increase in the total enzymatic activity. The added copper had been thought to enhance the activity of amine oxidases by affecting protein conformation. However, western blot analysis reveals that the addition of CuSO4 also results in an increase in the monoamine oxidase content of E. coli cells. Furthermore, northern blot analysis results indicate that an increase in monoamine oxidase activity also occurs at the transcriptional level. Increased transcription was observed in accordance with the addition of CuSO4 up to 50 μM final concentration.

Vanillin is a main flavor ingredient of vanilla beans. It is widely used as a food flavor, and is an important material in various industrial fields. The production of vanillin from vanillylamine was investigated using amine oxidase (AO) from Aspergillus niger and monoamine oxidase (MAO) from Escherichia coli, which have previously been isolated, characterized, and molecularly cloned in our laboratory. Oxidation of vanillylamine was observed with both AO and MAO. Kinetic analyses of the enzyme reactions revealed that AO was a more efficient producer of vanillin than MAO. With AO, 1 mM of vanillylamine was oxidized almost completely in about 30 min by 2.7 μg/ml of the enzyme. The product of the enzyme reaction was confirmed to be vanillin by HPLC and GC-MS. Continuous production of vanillin with immobilized AO was also investigated. The results suggest that AO from A. niger will be useful for the industrial synthesis of vanillin.

Six gram negative bacterial strains producing lactate oxidase were screened from 50 bacterial strains isolated from soil. The strains were capable of producing lactate oxidase both in shaking and static culture. Lactate oxidase activity was highest in strain KY6 and this strain was chosen as the enzyme source. The crude extract of strain KY6 containing 1.29 g of protein per liter converted dl -lactate (90 mM) to pyruvate yielding 61.8 % in 12 h, which corresponds to a space-time yield of 86.5 mol l−1 d−1 g−1 of cell-free protein. Morphological and physiological studies according to Bergey's Manual of Systematic Bacteriology revealed that the bacterial strain KY6 belongs to the Edwardsiella tarda Biogroup 1.

tolA strains of Escherichia coli harboring plasmids carrying the ggt gene excreted about 70% of their γ-glutamyltranspeptidase (GGT) (EC 2.3.2.2) into the medium. The excreted GGT was purified to electrophoretic homogeneity by a simple two-step method with a yield of 51%.

Summary Synthesis of S-methyl-L-cysteine from pyruvate, ammonia and methylmercaptan is catalyzed by highly purified cysteine desulfhydrase from Aerobacter aerogenes . The synthetic reaction proceeds optimally at pH 10, as a function of enzyme concentration and incubation time. Methylmercaptan is replaced by ethylmercaptan and propylmercaptan to synthesize S-ethyl-L-cysteine and S-propyl-L-cysteine, respectively. The enzymatically synthesized S-methyl-L-cysteine and S-ethyl-L-cysteine were purified and identified by physicochemical meanings.

Glutaminase activity was found in a water extract of a wheat bran koji (extracellular fraction) of Aspergillus oryzae strains Lee-1, H-16 and MA-27-IM isolated from a commercial koji ssed for soy sauce fermentation, as well as in thier mycelia (intracellular fraction). Both the intracellular and the extracellular glutaminases were purified from strain MA-27-IM. Polyacrylamide gel electrophoresis of each purified preparation gave a single band with identical electrophoretic mobility. The molecular weight of the intracellular and the extracellular glutaminases were estimated to be approximately 113, 000. Both preparations hydrolyzed various γ-glutamyl compounds besides l -glutamine but did not exhibit asparaginase activity. Further investigation of these preparations inidicated that these glutaminases possessed almost the same properties, suggesting their similarity.

Omega-Aminohexyl-Sepharose 4B served as an excellent biospecific adsorbent for affinity chromatography of amine oxidase (monoamine:O2 oxidoreductase (deaminating), EC 1.4.3.4) from Aspergillus niger. The enzyme was completely adsorbed on this affinity resin when applied to a column in 0.1 M potassium phosphate buffer (pH 7.2). Although a small part of the enzyme was retained on the column through ionic interaction and eluted with 1.0 M potassium phosphate buffer (pH 7.2), most of the enzyme adsorbed was eluted with 0.5 M potassium phosphate buffer (pH 7.2) containing 10 mM butylamine. Essentially no retention of the enzyme on a column of epsilon-aminopentyl-Sepharose or delta-aminobutyl-Sepharose occurred under the same conditions, indicating that an appropriate length (more than approx. 12 A) of a hydrocarbon extension between the agarose matrix and the terminal amino group would be necessary for efficient adsorption of amine oxidase. The modification of the enzyme with 3-methyl-2-benzothiazolinone hydrazone (carbonyl inhibitor) or dithionite (reducing agent) resulted in loss of the ability to bind to omega-aminohexyl-Sepharose. It was also demonstrated that the affinity chromatography on omega-aminohexyl-Sepharose can be used as a powerful means of purifying this enzyme from crude extracts of Aspergillus niger. All of the three adsorbents were effective as a substrate in the amine oxidase reaction, but their substrate activities were as low as the corresponding free diamines.

Based on the results of mapping of ggt , eight strains were selected from a gene library of E. coli . One of the strains harboring pLC9-12 was found to show 14 times higher γ-glutamyltranspeptidase activity per cell than the wild type strain. The ggt was subcloned to the Bam HI site of pUC18 and the recombinant plasmid sSH101 was obtained. Ggt− phenotype of γ-glutamyltranspeptidase-deficient mutants was complemented by pSH101. The specific activity of the enzyme in cells harboring pSH101 was 37-fold higher than that in the wild type cells. γ-Glutamyltranspeptidase was isolated from the periplasmic fraction of the cells by simple two steps and crystallized.

A rapid enzymatic assay method for ammonia was developed by using glutamine synthetase from glutamate-producing bacteria together with pyruvate kinase, lactate dehydrogenase, and NADH. The time required for determination of 25 nmol of ammonia was 5 min with 1 unit of glutamine synthetase, as opposed to 14-30 min with 1 unit of glutamate dehydrogenases from various sources. The present method was used to determine ammonia in serum, microbiol-culture broth, and waste water. The method can be modified for spectrophotometry in the visible region by substituting pyruvate oxidase, peroxidase, and appropriate chromogens for lactate dehydrogenase and NADH. With 4-aminoantipyrine (4AA) and phenol, and with 4AA and N-ethyl-N-2-hydroxyethyl-m-toluidine as chromogens, the sensitivity of ammonia determination was 0.65 and 1.7 times that with glutamate dehydrogenase, respectively. The present method was also applicable to the continuous detection of the activity of some ammonia-forming enzymes such as guanase, adenosine deaminase, and urease and to the determination of 0.5-30 microM ATP-ADP after some modification of the mixture.

A highly sensitive assay for gamma-glutamyltranspeptidase activity involving high-performance liquid chromatography (HPLC) with electrochemical detection was devised. gamma-Glutamyl-DOPA, a new synthetic dipeptide, which consists of naturally occurring amino acids, was found to be a good substrate for gamma-glutamyltranspeptidase purified from Proteus mirabilis. Enzymatically formed DOPA was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid and determined by HPLC with electrochemical detection. The sensitivity limit of this method was 0.5 pmol of DOPA formed. Some properties of gamma-glutamyltranspeptidase purified from P. mirabilis were investigated using gamma-glutamyl-DOPA as a substrate. In the presence of 0.15 M glycylglycine, the KM value of the enzyme for gamma-glutamyl-DOPA was 0.013 mM, and the maximum velocity was 247 nmol/min per mg protein. This method was applied to the assay of the enzymatic activity in human serum.

A trihydroxy derivative of phenylalanine was synthesized from S-methyl-L-cysteine and pyrogallol by the crystalline tyrosine phenollyase (L-tyrosine phenol-lyase (deaminating) EC 4. I. 99.2 formerly known as β-tyrosinase) from Escherichia intermedia. The product was isolated as its N-acetyl-triacetoxymethylester and identified as 2,3,4-trihydroxy-L-phenylalanine by the analyses of NMR, MS spectra and optical rotation.

Bifidobacterial strains showed various mortalities during storage at 30°C in boiled yogurt prepared with Streptococcus sp. and Lactobacillus sp. Bifidobacterium breve 203 was most stable, maintaining its initial cell number for more than 5 days. It was also stable at 4 or 10°C in fresh yogurt. B. longum 401 rapidly lost viability in the boiled yogurt and was unstable in the fresh yogurt at any temperature. Lactobacillus sp. remained fully viable for one week or more at 4–30°C while Streptococcus sp. lost viability at 20°C or above. The difference in mortality between B. breve 203 and B. longum 401 was mainly due to their sensitivity to the acidic environment, with temperature during storage having a secondary effect. Effects of lysozyme, pepsin and bile acids on the two strains were also investigated.

γ-Glutamylglycylglycine (γ-GluGlyGly) was formed through the γ-glutamyltranspeptidase (GGT) reaction catalyzed by glutaminase in a water extract of wheat bran koji obtained with Aspergillus oryzae MA-27-IM. The yield of γ-GluGlyGly was about 18% from l -glutamine in a reaction mixture containing 50 mM l -glutamine, 50 mM glycylglycine, and the extract (0.1 unit ml as GGT activity) in a 100 mM Tris-HCl buffer solution (pH 7.2), which was incubated for 7 h at 30°C. The γ-GluGlyGly formed was purified by ion exchange chromatographies, and the identified by chemical and enzymatic methods as well as by infrared and PMR spectroscopic analyses.

Photoinactivation of the β2 subunit of tryptophan synthetase is dependent on the presence of the coenzyme, pyridoxal 5′-phosphate, and results in the specific destruction of 1 histidyl residue per β monomer. The rate of photoinactivation is strikingly increased by the addition of a substrate, l -serine. Inactivation of the β2 subunit can also be achieved by the modification of 2 to 3 histidyl residues per monomer with diethylpyrocarbonate. β2 subunit modified by either procedure can still form a complex with pyridoxal 5′-phosphate. The chemically modified β2 subunit can form some Schiff base intermediate between pyridoxal 5′-phosphate and l -serine but appears to be blocked in the further conversion of this intermediate. These results suggest that 1 of the chemically modified histidyl residues may serve a role in the abstraction of the α-proton of l -serine. This suggestion is supported by the finding that the Vmax for the conversion of l -serine to pyruvate is dependent on an ionizing group with a pK of 6.7.