The isotope ratio of monoterpene hydrocarbons in citrus essential oils of different origins was measured by ordinary high-resolution gas chromatography-mass spectrometry (HRGC-MS). The isotope ratio (Ir) was determined by the ratio of the isotope peak intensity (m/z 137) to the molecular mass peak intensity (m/z 136) of the monoterpene hydrocarbons. The accuracy of Ir was examined by measuring monoterpene hydrocarbon standards and 13C-labeled compounds. The isotope fingerprints based on the values of monoterpene hydrocarbons from lemon, lime and yuzu essential oils were determined. These citrus essential oils were also discriminated by a principal component analysis of their Ir data. The characteristic vectors showed that α-terpinene, β-pinene and β-phellandrene were important components for distinguishing between the citrus species. It is suggested that this technique will be applicable to evaluate the quality, genuineness and origin of citrus fruits and their products.
In our search for new fusicoccins of unique diterpene glucosides from Phomopsis amygdali, we found that a fragrant substance was formed in the early stage of fusicoccin fermentation. This fragrant constituent was isolated and identified as (+)-menthol, which is a novel fungal metabolite as the enantiomer of well-known peppermint (−)-menthol. (+)-7-Hydroxymenthol and new (+)-(6S)-hydroxymenthol were also isolated and identified as fungal metabolites. In addition, p-menthanetriol, which has been reported as the first fungal monoterpene from the fungus, was also isolated. The possible biosynthetic relationship of these metabolites is discussed.
A tyrosinase inhibitor was isolated from the seeds of Euphorbia lathyris L. by bioassay-guided fractionation and purification, using silica gel column chromatography. It was identified as esculetin by comparing its physical properties and spectral data with those of an authentic sample. The IC50 value of esculetin in the mushroom tyrosinase activity test was 43 μM. The kinetic study indicates that esculetin exhibited competitive inhibition against the oxidation of 3-(3,4-dihydroxyphenyl)-alanine by mushroom tyrosinase. The structure-activity relationships among five esculetin analogs suggest that hydroxyl groups at the C6 and C7 positions of the coumarin skeleton played an important role in the expression of tyrosinase inhibitory activity.
The effect of the host-specific phytotoxins, AM-toxins, on the photosynthetic activity of leaves from susceptible apple cultivars was investigated by using an oxygen electrode. The photosynthetic O2 evolution was inhibited by AM-toxin I in a host-specific manner. The inhibitory activity of several AM-toxin analogs against photosynthesis was also evaluated and the findings were correlated with their necrosis-inducing activity.
The new inhibitors of 3α-hydroxysteroid dehydrogenase, 0231A 1 and 0231B 2, have a unique benz[c,d]indol-3(1H)-one structure in their molecules. In our advanced studies on indole chemistry, we have developed an efficient synthetic method for benz[c,d]indol-3(1H)-one derivatives. We report here its application to the synthesis of 0231B in 10 steps (8.1% overall yield) from 6-methylindole 8 by introducing an acyl group into the 3-position of the indole nucleus, cyclization of the side chain at the 3-position to the 4-position and subsequent elimination of the phenyl group, and conjugate addition of the substituted phenyl group.
A lot of reports of antifreeze protein (AFP) from fish have been published, but no report has mentioned of commercialized mid-latitude fresh water fish which producing AFP in its body fluid. We found that the AFP in the body fluid of Japanese smelt (Hypomesus nipponensis) from mid-latitude fresh water was purified and characterized. The N-terminal amino acid sequence of the Japanese smelt AFP was 75.0% identical to Type II AFP from herring. Results of EDTA treatment and ruthenium red staining suggested that the Japanese smelt AFP had at least one Ca2+-binding domain. Interestingly, the antifreeze activity of the Japanese smelt AFP did not completely disappear when Ca2+ ions were removed. The molecular mass of the Japanese smelt AFP was calculated to be 16,756.8 by the TOF-mass analysis. The Open reading flame of the gene coding for the Japanese smelt AFP was 444 bp long and was 85.0% identical with the entire herring AFP gene. The cDNA and amino acid sequence of the Japanese smelt AFP were the same length as those of herring AFP.
The immunosuppressant, FTY720 causes apoptosis of lymphocytes, reduces numbers of lymphocytes in peripheral blood, and prevents infiltration of lymphocytes into allografts, which may be one of the mechanisms involved in its effects. Here we compared caspase activation and expression of cell-cycle regulators during apoptosis caused by FTY720, and Fas-stimulation in a mouse lymphoma transfected with human Fas antigen. FTY720 activated caspases-3, -8, and -9 as rapidly as did Fas-mediated apoptosis. The activation was blocked by a peptide inhibitor for caspase-3, DEVD-CHO. Fas-induced activation of caspases-8 and -9 was unaffected by the inhibitor. FTY720 eliminated proliferating cell nuclear antigen, retinoblastoma family members, differentiation regulated transcription factor polypetide-1, and cyclin H. These cell-cycle regulators were not eliminated when the peptide inhibitor was used. Dysfunction of cell-cycle regulators may play a critical role in the signal transduction pathway for activation of FTY720-mediated apoptosis.
Aspartate aminotransferase (AspAT) was purified to homogeneity from cell extracts of the non-N2-fixing cyanobacterium Phormidium lapideum. The NH2-terminal sequence of 25 amino acid residues was different from the sequences of the subfamily Iα of AspATs from eukaryotes and Escherichia coli, but it was similar to sequences of the subfamily Iγ of AspATs from archaebacteria and eubacteria. The enzyme was most active at 80°C and was stable at up to 75°C. Thermal inactivation (60-85°C) of the enzyme followed first-order kinetics, with 2-oxoglutarate causing a shift of the thermal inactivation curves to higher temperatures. However, at 25°C the kcat of P. lapideum AspAT was nearly equal to the values of AspATs from mesophilic organisms. The enzyme used L-aspartate and L-cysteine sulfinate as amino donors and 2-oxoglutarate as an amino acceptor. The Km values were 5.0 mM for L-aspartate, 5.7 mM for L-glutamate, 0.2 mM for 2-oxoglutarate, and 0.032 mM for oxaloacetate.
Simmondsin was modified with acarviosine-glucose using the transglycosylation activity of Thermus maltogenic amylase to synthesize a novel compound with both antiobesity and hypoglycemic activity. The LC/MS and 13C NMR analyses confirmed that the structure of the major transglycosylation product was acarviosine-simmondsin (Acv-simmondsin), in which acarviosine was attached to the glucose moiety of simmondsin by an α-(1,6)-glycosidic linkage. It was found that Acv-simmondsin was a potent competitive inhibitor of α-glucosidase with the Ki value of 0.69 μM and a mixed type inhibitor of α-amylase with the Ki and KI of 20.78 μM and 26.31 μM, respectively. The administration of Acv-simmondsin (0.1 g/100 g diet/day) to mice for 5 days significantly reduced food intake by 35%, compared to 25% with simmondsin in control obese mice. Acv-simmondsin (50 mg/kg BW) suppressed the postprandial blood glucose response to sucrose (1 g/kg BW) by 74%, compared to 71% with acarbose, in normal rats.
The courses of the reaction catalyzed by guinea hen egg-white lysozyme (GHL), in which Asn113 and Arg114 at subsites E and F in hen egg-white lysozyme (HEL) are replaced by Lys and His, respectively, was studied with the substrate N-acetylglucosamine pentamer, (GlcNAc)5. Although GHL was found to retain the main-chain folding similar to HEL as judged from CD spectroscopy, the courses of GHL showed increased production of (GlcNAc)4 and reduced production of (GlcNAc)2 when compared with HEL. To identify critical residue(s) involved in the alteration in the courses of GHL, two mutant enzymes as to subsites E and F in HEL, N113K and R114H, were prepared by site-directed mutagenesis. Kinetic analysis of these mutants revealed that the mutation of Asn113 to Lys had little effect on the courses of HEL, while the Arg114 to His mutation completely reproduced the courses of GHL, demonstrating that His114 in GHL is the key residue responsible for the characteristic courses of GHL. Computer simulation of the reaction courses of the R114H mutant revealed that this substitution decreased not only the binding free energies for subsites E and F, but also the rate constant of transglycosylation. The Arg residue at position 114 may play an important role in the transglycosylation activity of HEL.
The histidine (His)-to-Aspartate (Asp) phosphorelay is a paradigm of intracellular signaling systems through protein phosphorylation in both prokaryotes and eukaryotes. The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), together with two response regulators (Mcs4 and Prr1). The results of recent extensive studies suggested that these His-to-Asp phosphorelay components are involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. It was also suggested that they were somehow implicated in control of both the mitotic and meiotic cell proliferations. Among these S. pombe His-to-Asp phosphorelay components, however, the function of Prr1 is less clear. We here characterized a mutant, named prr1-D418N, specifying an altered Prr1 protein that presumably acts as a gain-of-function (or constitutive-active) mutant, with special reference to sexual development. The mutant cells showed a striking phenotype in that they underwent mating even in a nitrogen-sufficient medium, under which conditions the wild-type cells hardly did so. Furthermore, the mutant cells underwent mating very rapidly in a nitrogen-deficient medium. Under anaerobic (or micro-aerobic) growth conditions, the wild-type cells were not capable of undergoing sexual development even in a nitrogen-deficient medium. The prr1-D418N cells underwent mating efficiently under such anaerobic growth conditions. Taken these together, it was suggested that the function of Prr1 is closely linked to the well-characterized signaling pathways for induction of the sexual development, in a way that this response regulator regulates a critical step of the initiation of meiosis through activating the transcription of ste11+, mam2+, and mei2+, in S. pombe.
cDNA of a monosaccharide transporter in rice, OsMST5 (Oryza sativamonosaccharide transporter 5) was cloned and its sugar transport activity was characterized by heterologous expression analysis. The amino acid sequence and topology were similar to the sequences and topology of other plant monosaccharide transporters. Yeast cells co-expressed with OsMST5 cDNA transported some monosaccharide substrates. The transport rate increased when ethanol as an electron donor was added, so the transporter was an energy-dependent active one. Most of the OsMST5 was expressed in panicles before pollination, indicating that it is associated with pollen development in rice.
In order to clarify the position where paratropomyosin binds to connectin in the A-I junction region of a sarcomere, chicken β-connectin was digested by Staphylococcus aureus V8 protease under denaturing conditions and the digested peptides were electrophoretically separated. Five peptides, 150-kDa, 100-kDa, 70-kDa, and 43-kDa fragments, were simultaneously detected by biotinylated paratropomyosin and an anti-connectin monoclonal antibody. The N-terminal sequence of the 43-kDa fragment was found to be YQFRVYAVNK, similar to the sequence of 7556-7565 amino acids in the I51 fibronectin type 3 domain that was located at the A-I junction region of human cardiac titin/connectin. Therefore, we propose that paratropomyosin binds to the 43-kDa fragment from β-connectin at the A-I junction region in both living muscle and in muscle immediately postmortem, and the N-terminus of the 43-kDa fragment is localized in the I51 domain.
In order to investigate the effects of mutation of Gln32, a component of a base recognition site (B2 site) of a base-nonspecific RNase from Rhizopus niveus, we prepared several enzymes mutant at this position, Q32F, Q32L, Q32V, Q32T, Q32D, Q32N, and Q32E, and their enymatic activities toward RNA and 16 dinucleoside phosphates were measured. Enzymatic activities of the mutant enzymes towards RNA were between 10-125% of the native enzyme. From the rates of hydrolysis of 16 dinucleoside phosphates by mutant enzymes, we estimated the base specificity of both B1 and B2 sites. The results indicated that mutation of Gln32 to Asp, Asn, and Glu caused the B2 site to prefer cytosine more and to a less extent, to prefer uracil (Q32N), and that Q32F made the enzyme more guanine-base preferential. The results suggested that we are able to construct an enzyme that preferentially cleaves internucleotidic linkages, at the 5′-side of cytosine residues (Q32D, Q32N, and Q32E) and guanine residues (Q32F and Q32T), thus, cleaves purine-C(Q32D, Q32N, Q32E) and GpG and ApG (Q32F, and Q32T) most easily. The results seemed to suggest converting a base-non-specific RNase to a base-specific one.
The roles of N-linked glycosylation in the intracellular transport and biological activities of the Sendai virus hemagglutinin-neuraminidase (HN) protein were studied. The protein contains four potential N-glycosylation sites: N77, N448, N499, and N511. By site-directed mutagenesis of these positions, the mature protein contained three N-linked oligosaccharides attached to N77, N499, and N511. The role of each added oligosaccharide in the structure and functions of the protein was identified by characterization of surface expression, hemadsorption, and neuraminidase activities of the corresponding mutant proteins. Elimination of the sites of N499 and N511 had the most detrimental effect, decreasing surface expression and hemadsorption. Elimination of the sites of N77 and N448 had similar but weaker effects. Mutants missing the sites of N499 and N511 were not able to induce syncytia formation in cells expressing mutant HN proteins and the fusion protein. Therefore, the N-linked oligosaccharides attached to N499 and N511 were important for intracellular transport and for the fusion promotion.
We have isolated a β-ketoacyl CoA synthase (KCS) gene, MpFAE2, from a liverwort, Marchantia polymorpha, and identified its substrate specificity using the technique of dsRNA-mediated gene silencing and overexpression. KCS catalyzes an essential reaction in the fatty acid elongation process, i.e., condensation of malonyl-CoA with acyl-CoA. By introducing a construct with a hairpin structure containing a partial MpFAE2 gene, the level of the MpFAE2 gene expression was suppressed constitutively. The transgenic plants showed a specific accumulation of fatty acid 18:0. In contrast, in transgenic M. polymorpha plants overexpressing the MpFAE2 gene, fatty acid 22:0 is accumulated. These results indicate that the MpFAE2 gene product catalyzes the elongation steps of 18:0 to 20:0 and possibly also of 20:0 to 22:0.
To characterize its enzymic property we cloned and sequenced the gene of Deinococcus radiodurans encoding UV-endonuclease β, an alternative enzyme to UvrABC repairing damaged DNA. Amino acid substitutions were found in UV-sensitive mutants. The putative amino acid sequence had some similarity with those of eukaryotic UV-endonucleases and with a sequence found in a protein data base of Bacillus subtilis.
To gain some insights into the structure of the S-locus and the mechanisms that have kept its diversity, a 75-kb genomic fragment containing the self-incompatibility (S) locus region was isolated from the S12-haplotype of Brassica rapa and compared with those of other S-haplotypes. The region around the S determinant genes was highly polymorphic and filled with S-haplotype-specific intergenic sequences. The diverse genomic structure must contribute to the suppression of recombination at the S-locus.
A β-galactosidase was purified 1300-fold by lactosyl-Sepharose 4B and Sephacryl S-200 column chromatographies from the cultured medium of a rice-cell suspension. The purified enzyme appeared as 47 kD and 40 kD polypeptides on SDS-PAGE and had a specific activity of 65.1 units/mg. Optimum activity was observed at pH 3.5 and 60°C. The enzyme released galactose from galactoxyloglucan and pectic galactans.
Polypeptide synthesis directed by DNA as the messenger in a cell-free system of Thermus thermophilus was investigated. Polypeptides were synthesized with the addition of neomycin in the presence of DNA catalyzed by the cell extract. The stability of messenger DNA was greater than that of messenger RNA. Continuous cell-free translation with messenger DNA produced polypeptides at the rate of more than 8 μg/h in the presence of spermine.
Two new compounds of 5-bromouridine, 3′-O-(β-D- galactopyranosyl)-5-bromouridine and 3′-O-[β-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl]-5-bromouridine, were found to be selectively formed in a high yield in a culture filtrate of Sporobolomyces singularis, when grown on a medium containing lactose and 5-bromouridine.
We isolated a neuronal calcium sensor 1/frequenin-like gene, Mg-NCS-1, from Magnaporthe grisea and evaluated the phenotypes of null-mutants of the gene. The putative Mg-NCS-1 protein showed high similarity to the other NCS-1 proteins. The null-mutants had normal growth and pathogenicity similar to the parental strain, but their growth was suppressed in high concentrations of Ca2+ or acidic conditions.
YY1 is a ubiquitously expressed multifunctional transcription factor that is involved in both positive and negative regulation of gene expression as well as initiation of transcription. Here, we isolated cDNA encoding a full-length open reading frame (ORF) of rat YY1. Rat YY1 is composed of 411 amino acid residues and its amino acid sequence is 97.6% identical to that of mouse YY1 and 97.8% identical to that of human YY1. The transactivating abilities of wild-type rat YY1 and four truncated mutant forms of YY1 were examined by transient reporter assays. When residues 114-193, which sequence includes a portion of the activation region and most of the Gly/Lys-rich region, were lacking, transactivation activity decreased somewhat, but the further deletion in the activation region (of residues 56-113) did not cause further decrease of the activity. On the other hand, N-terminus of the activation region (1-78/100-106) did not have transactivation activity by itself as well as synergistic activity with an erythroid specific transcription factor GATA-1.
Gnetol (2,3′,5′,6-tetrahydroxy-trans-stilbene), a naturally occurring compound particularly found in the genus Gnetum, had a strong inhibitory effect on murine tyrosinase activity. Gnetol (IC50, 4.5 μM) was stronger than kojic acid (IC50, 139 μM) as a standard inhibitor for murine tyrosinase activity. Moreover, gnetol significantly suppressed melanin biosynthesis in murine B16 melanoma cells.
This study was done to modify erythritol to change its physicochemical and sensory properties. Erythritol, a four-carbon sugar alcohol, was transglycosylated by Bacillus stearothermophilus maltogenic amylase with maltotriose as a donor molecule. The presence of various transglycosylation products of erythritol was confirmed by TLC and high performance ion exchange chromatography (HPIC). The major transfer product was purified by gel filtration chromatography on Bio-Gel P-2. Examination by LC-MS, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), and 13C NMR showed that the major transfer product was maltosyl-erythritol. Results of 13C NMR of maltosyl-erythritol suggested that linkage was formed between the C1 carbon of glucose unit in maltose and either one of the two carbon atoms of the terminal hydroxyl groups of erythritol, so that a mixture of 1-O- and 4-O-α-maltosyl-erythritol was produced. The sweetness of maltosyl-erythritol was about 40% that of sucrose, and its negative sensory properties were less than those of erythritol.
We recently investigated the effects of the major proteins in cow's milk on gastric mucosal injuries in rat ulcer models. We found that α-lactalbumin (α-LA) has marked preventive effects against gastric mucosal injuries and that prostaglandin (PG) synthesis may contribute to these effects [Matsumoto et al., Biosci. Biotechnol. Biochem., 65, 1104-1111, 2001]. In this study, we investigated the effects of α-LA on several defense mechanisms of gastric mucosa by evaluating gastric PGE2 content, gastric mucin content, gastric luminal pH, gastric fluid volume, and gastric emptying in naive rats. Oral administration of α-LA (200, 500, and 1000 mg/kg) elevated endogenous PGE2 levels in gastric tissue and increased the gastric mucin contents of both the gastric fluid and the adherent mucus gel layer. In addition to these PG-related responses, α-LA also caused PG-independent responses such as elevation of gastric luminal pH, increase in gastric fluid volume, and delay in gastric emptying. These responses were observed to be dose-dependent (200-1000 mg/kg of α-LA). Thus, we demonstrated that α-LA enhances both PG-dependent and PG-independent gastric defense mechanisms in naive rats. Both of these mechanisms are probably involved in its gastroprotective action.
This study investigates whether some dietary fibers can the toxicity due to cystine added to the diet. Wistar rats were investigated for the effects of adding pectin, sugar beet fiber or konjac mannan to a cystine diet on the growth rate and on the activities of liver antioxidant enzymes and serum enzymes. The addition of pectin, sugar beet fiber or konjac mannan to the cystine diet resulted in a significant increase in both the food intake and body weight gain. Feeding the cystine diet caused lower activities of total and Cu, Zn-superoxide dismutase, and of catalase in the liver. The addition of pectin to the cystine diet counteracted the activities of the total and Cu,Zn-superoxide dismutase, and of catalase in liver. Of the dietary fibers tested, konjac mannan prevented the elevation of the two enzyme activities in the serum induced by feeding the cystine diet, indicating that this fiber might have the ability to alleviate hepatic damage due to dietary cystine.
Quorum sensing of Gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. We used PCR to study a gene cassette encoding this three-component regulatory system. Degenerate primers were designed from consensus amino acid sequences in the HPK10 subfamily, mostly involved in quorum sensing. Products amplified from genomic DNA of Lactobacillus, Enterococcus, and Clostridium species were cloned and sequenced; their deduced amino acid sequences were similar to those of members of the HPK10 subfamily. Complete genes for the putative gene cassette were cloned by inverse PCR from L. paracasei E93490 and L. plantarum WCFS6. Phylogenetic analysis grouped the cloned putative HPKs into the HPK10 subfamily. These results indicated the usefulness of this high-throughput gene screening and suggested that the three-component regulatory gene cassette are widely present.
Microorganisms from culture collections and isolates from nature were screened for the ability to catalyze the regioselective glucosylation of pyridoxine (PN) to produce pyridoxine 5′-α-D-glucoside (PN-5′-α-G) or pyridoxine 4′-α-D-glucoside (PN-4′-α-G). Transglucosylation activity specific to 5′-position of PN was found in fungi belonging to genera such as Coriolus and Verticillium, and activity at the 4′-position of PN was found in bacteria belonging to genera such as Bacillus and Serratia. From 100 mM PN, intact cells of Verticillium dahliae TPU 4900 produced 42 mM (13.9 mg/mL) PN-5′-α-G after 70 h of reaction. Intact cells of Bacillus cereus TPU 5504 produced 33 mM (10.9 mg/mL) PN-4′-α-G after 19 h of reaction. The selectivities for 5′- and 4′-positions were 80% and 90%, respectively.
Optimization of culture and reaction conditions for 5′-position-selective transglucosylation to pyridoxine by Verticillium dahliae TPU 4900 was investigated. V. dahliae TPU 4900 had high transglucosylation activity when grown with soluble starch as a carbon source and organic nitrogens such as Esusan meat as a nitrogen source at 15–20°C. Both the yield of pyridoxine 5′-α-D-glucoside (PN-5′-α-G) and the 5′-position-selectivity reached a maximum when an intact-cell reaction was done at 50–60°C and pH 7 with additions of dextrin. The transglucosylation activity in culture broth was 71 times with the optimization of culture conditions that under the conditions used for screening. The productivity of PN-5′-α-G synthesis was 6.9 times that under the initial conditions when the reaction conditions of intact cells were optimized. From 1000 mM (206 g/L) pyridoxine hydrochloride, PN-5′-α-G was synthesized to the concentration of 300 mM (98.4 g/L as PN-5′-α-G) with 5′-selectivity of 85% in 53 h by intact cells of V. dahliae TPU 4900.
Screening for morphological mutants of a haploid strain of Saccharomyces cerevisiae was done on the basis of their cell-shape on a solid medium containing isoamyl alcohol, which causes cell elongation, to obtain information on the morphogenesis. Mutant J19, which had pseudohyphae in liquid medium even in the absence of isoamyl alcohol, had many elongated cells. Few reports exist of haploid cells growing as pseudohyphae in liquid culture. Cell-wall analysis showed that J19 had ordinary amounts of alkali-insoluble glucan and chitin, but that isoamyl alcohol in the medium caused structural changes in the cell wall. Addition of a DNA fragment that included the wild-type SCL1 gene to J19 complemented its morphological phenotype. Sequencing of J19 SCL1 showed that the glycine at position 226 in the Scl1 protein had been replaced by asparatic acid, suggesting that this mutation in the protein, a subunit of proteasomes, may be involved in the morphological change.
Xylitol dehydrogenase (XDH) was purified from the cytoplasmic fraction of Gluconobacter oxydans ATCC 621. The purified enzyme reduced D-xylulose to xylitol in the presence of NADH with an optimum pH of around 5.0. Based on the determined NH2-terminal amino acid sequence, the gene encoding xdh was cloned, and its identity was confirmed by expression in Escherichia coli. The xdh gene encodes a polypeptide composed of 262 amino acid residues, with an estimated molecular mass of 27.8 kDa. The deduced amino acid sequence suggested that the enzyme belongs to the short-chain dehydrogenase/reductase family. Expression plasmids for the xdh gene were constructed and used to produce recombinant strains of G. oxydans that had up to 11-fold greater XDH activity than the wild-type strain. When used in the production of xylitol from D-arabitol under controlled aeration and pH conditions, the strain harboring the xdh expression plasmids produced 57 g/l xylitol from 225 g/l D-arabitol, whereas the control strain produced 27 g/l xylitol. These results demonstrated that increasing XDH activity in G. oxydans improved xylitol productivity.
We isolated a β-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of β-N-acetylglucosaminidase in a wheat bran solid culture. The β-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21% and 0.15% yield, respectively.