Purification and Characterization of Membrane-Bound 3-Dehydroshikimate Dehydratase from Gluconobacter oxydans IFO 3244, A New Enzyme Catalyzing Extracellular Protocatechuate Formation

3-Dehydroshikimate dehydratase (DSD) is the first known enzyme catalyzing aromatization from 3-dehydroshikimate (DSA) to protocatechuate (PCA). Differently from cytosolic DSD (sDSD), a membrane-bound 3-dehydroshikimate dehydratase (mDSD) was found for the first time in the membrane fraction of Gluconobacter oxydans IFO 3244, and DSA was confirmed to be the direct precursor of PCA. In contrast to weak and instable sDSD, the abundance of mDSD in the membrane fraction suggested the metabolic significance of mDSD as the initial step in aromatization. mDSD was solubilized only by a detergent and was readily purified to high homogeneity. Its molecular weight was estimated to be 76,000. Purified mDSD showed a sole peak at 280 nm in the absorption spectrum and no critical cofactor requirements. The Km of DSA was measured at 0.5 mM, and the optimum pH was observed at pH 6–8. mDSD appeared to react only with DSA, and was inert to other compounds, such as 3-dehydroquinate, quinate, and shikimate.     

Conversion of Quinate to 3-Dehydroshikimate by Ca-Alginate-Immobilized Membrane of Gluconobacter oxydans IFO 3244 and Subsequent Asymmetric Reduction of 3-Dehydroshikimate to Shikimate by Immobilized Cytoplasmic NADP-Shikimate Dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

Regulatory Properties of Sucrose Synthetase in Sweet Potato Roots

The molecular weight of the yeast tannase [E.C. 3.1.1.20, tannin acyl-hydrolase] of Candida sp. was determined to be 250,000 by gel filtration on Sephadex G–200. The enzyme was dissociable into two identical subunits with molecular weight of 120,000 on SDS-polyacrylamide gel electrophoresis. The amino acid analysis revealed that the enzyme consisted of 786 amino acid residues per protein molecule. The polypeptide moiety of the enzyme was 38 % by the Lowry-Folin reaction and 35% by the amino acid analysis. The enzyme contained 62% neutral sugars, which were identified as mannose and galactose on cellulose thin-layer chromatogram and 2.2 % hexosamines.     

Isolation of Functional RNA from Periderm Tissue of Potato Tubers and Sweet Potato Storage Roots

A reliable and efficient protocol is given for the isolation of mRNA from the periderm of potato tubers and sweet potato storage roots. The method relies on a urea-based lysis buffer and lithium chloride to concentrate total RNA away from most of the cytoplasmic components and to prevent oxidation of phenolic complexes. To enhance the physical separation of the RNA from other macromolecular components, the RNA fraction was incubated in the presence of the cationic surfactant Catrimox-14. Poly(A)⁺ mRNA was separated from total RNA and other contaminants by using Promega's MagneSphere technology. The mRNA was suitable for cDNA library construction and RNA fingerprinting.     

Functional analysis of the essential bifunctional tobacco enzyme 3-dehydroquinate dehydratase/shikimate dehydrogenase in transgenic tobacco plants

In plants, the shikimate pathway occurs in the plastid and leads to the biosynthesis of aromatic amino acids. The bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHD/SHD) catalyses the conversion of dehydroquinate into shikimate. Expression of NtDHD/SHD was suppressed by RNAi in transgenic tobacco plants. Transgenic lines with <40% of wild-type activity displayed severe growth retardation and reduced content of aromatic amino acids and downstream products such as cholorogenic acid and lignin. Dehydroquinate, the substrate of the enzyme, accumulated. However, unexpectedly, so did the product, shikimate. To exclude that this finding is due to developmental differences between wild-type and transgenic plants, the RNAi approach was additionally carried out using a chemically inducible promoter. This approach revealed that the accumulation of shikimate was a direct effect of the reduced activity of NtDHD/SHD with a gradual accumulation of both dehydroquinate and shikimate following induction of gene silencing. As an explanation for these findings the existence of a parallel extra-plastidic shikimate pathway into which dehydroquinate is diverted is proposed. Consistent with this notion was the identification of a second DHD/SHD gene in tobacco (NtDHD/SHD-2) that lacked a plastidic targeting sequence. Expression of an NtDHD/SHD-2-GFP fusion revealed that the NtDHD/SHD-2 protein is exclusively cytosolic and is capable of shikimate biosynthesis. However, given the fact that this cytosolic shikimate synthesis cannot complement loss of the plastidial pathway it appears likely that the role of the cytosolic DHD/SHD in vivo is different from that of the plastidial enzyme. These data are discussed in the context of current models of plant intermediary metabolism.     

The Composition of Saccharogenic Amylase from Rhizopus javanicus and the Isolation of Glycopeptides

Saccharogenic amylase from Rhizopus javanicus sp. 3–46 was known to be a glycoprotein which contained 27 residues of mannose and 4 residues of N-acetylglucosamine per mole of the saccharogenic amylase. Attempts have been made to obtain glycopeptides from the saccharogenic amylase. Three glycopeptides, GP-I-a, GP-I-b and GP-II, were separated from a Pronase digest of heat-denatured saccharogenic amylase by gel filtration on Sephadex G-50 and chromatography on DEAE-Sephadex A-25. GP-I-a contained asparagine, glycine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 6: 2. GP-I-b contained asparagine, threonine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 9:2. GP-II consisted of threonine, serine, proline, alanine and mannose in a molar ratio of 6: 2: 2: 2: 12.     

Isolation and Characterization of Thermotolerant Gluconobacter Strains Catalyzing Oxidative Fermentation at Higher Temperatures

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10°C to 37°C and had average optimum growth temperature between 30-33°C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37°C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37°C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264^T was unable to do such fermentation at 37°C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30°C. Even oxidative fermentation of D-fructose done at 37°C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37°C was superior to that observed at 30°C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.     

The conversion of 3-deoxyarabinoheptulosonate 7-phosphate to 3-dehydroquinate by sorghum seedling preparations

Partially purified preparations from etiolated sorghum seedlings catalyzed the conversion of DAHP to DHQ. The reaction catalysed by DHQ synthetase was stimulated by 0.1 μM to 0.1 mM NAD in the presence O-0.5 mM Co²⁺. NADH at 1 μM stimulated the reaction as much as 50% but became inhibitory at 100μM. Co²⁺ at 0.5mM stimulated enzyme activity 3-fold; Mg²⁺, Mn²⁺, Cu²⁺, and Zn²⁺ were not stimulatory. EDTA at 5 mM inhibited the reaction 95% but its effects were reversed by equal concentrations of Co²⁺. Phe, Tyr, Trp, t-cinnamate, several hydroxylated cinnamates, DHS, quinate, and shikimate at 0.3 mM failed to affect enzyme activity but slight inhibition occurred with DHQ and protocatechuic acid at 0.3 mM, inhibition being 14 % and 22 %, respectively. DHQ synthetase activity also was detected in spinach leaves and potato tuber tissue. Synthetase activity appeared to increase in response to injury of potato tuber and sweet potato root tissues.     

二级水灌溉对土壤及马铃薯块茎中大肠菌群数量的影响

采用多管发酵法检测二级水灌溉对马铃薯种植土壤中大肠菌群数量,结果表明:充分灌溉后的土壤中大肠菌群的数量均高于分根交替灌的土壤;地下滴灌的土壤中大肠菌群数量高于沟灌土壤的大肠菌群数量;二级水经加氯消毒后,土壤中大肠菌群数量明显下降。同样采用多管发酵法检测马铃薯块茎,结果发现:采用加氯消毒后的二级水灌溉的小区E和F中生长的马铃薯,其块茎组织中大肠菌群数量低于其他小区,采用地下滴灌灌溉的小区I和J所种植出的马铃薯块茎中大肠菌群的数量高于采用沟灌灌溉的小区K和L。结果表明:马铃薯块茎中大肠菌群的数量与其生境中大肠菌群的数量有密切关系。     

Reassessing the type I dehydroquinate dehydratase catalytic triad: Kinetic and structural studies of Glu86 mutants

Dehydroquinate dehydratase (DHQD) catalyzes the third reaction in the biosynthetic shikimate pathway. Type I DHQDs are members of the greater aldolase superfamily, a group of enzymes that contain an active site lysine that forms a Schiff base intermediate. Three residues (Glu86, His143, and Lys170 in the Salmonella enterica DHQD) have previously been proposed to form a triad vital for catalysis. While the roles of Lys170 and His143 are well defined—Lys170 forms the Schiff base with the substrate and His143 shuttles protons in multiple steps in the reaction—the role of Glu86 remains poorly characterized. To probe Glu86′s role, Glu86 mutants were generated and subjected to biochemical and structural study. The studies presented here demonstrate that mutant enzymes retain catalytic proficiency, calling into question the previously attributed role of Glu86 in catalysis and suggesting that His143 and Lys170 function as a catalytic dyad. Structures of the Glu86Ala (E86A) mutant in complex with covalently bound reaction intermediate reveal a conformational change of the His143 side chain. This indicates a predominant steric role for Glu86, to maintain the His143 side chain in position consistent with catalysis. The structures also explain why the E86A mutant is optimally active at more acidic conditions than the wild‐type enzyme. In addition, a complex with the reaction product reveals a novel, likely nonproductive, binding mode that suggests a mechanism of competitive product inhibition and a potential strategy for the design of therapeutics.     

The Occurrence of Quinic Acid in Sweet Potato Roots

The aroma extract dilution analysis of an extract prepared from pork stock and subsequent experiments led to the identification of 15 aroma-active compounds in the flavor dilution factor range of 64–2048. Omission experiments to select the most aroma-active compounds from the 15 odor compounds suggested acetol, octanoic acid, δ-decalactone, and decanoic acid as the main active compounds contributing to the aroma of pork stock. Aroma recombination, addition, and omission experiments of these four aroma compounds in taste-reconstituted pork stock showed that each compound had an individual aroma profile. A comparison of the overall aroma between this recombined mixture and pork stock showed strong similarity, suggesting that the key aroma compounds had been successfully identified.     

Isolation of 2-Amino-3-methyl-imidazo- [4,5-f]quinoline as Mutagen from the Heated Product of a Mixture of Creatine and Proline

Membrane-bound glucono-δ-lactonase (MGL) was purified to homogeneity from the membrane fraction of Gluconobacter oxydans IFO 3244. After solubilization with 1 M CaCl₂, MGL was purified in the presence of Ca²⁺ and detergent. A single band corresponding to 60 kDa appeared in SDS–PAGE. The molecular weight of MGL was judged to be 120k. Differently from cytoplasmic lactonases, MGL showed optimum pH in an acidic range of 5–5.5. It was highly sensitive to metal-chelating agents such as EDTA, and the lost MGL activity was restored to the original level by the addition of divalent cations such as Ca²⁺ or Mg²⁺. The purified MGL was strictly dependent on Ca²⁺ and underwent rapid denaturing precipitation on Ca²⁺ depletion even in the presence of detergent. This communication can be the first one dealing with the solubilization, purification and properties of MGL.     

Induction of Furano-terpenoids in Sweet Potato Roots by the Larval Components of the Sweet Potato Weevils

When sweet potato root tissues were infested by the larvae of sweet potato weevil, Cylas formicarius and West Indian sweet potato weevil, Euscepes postfasciatus, furano-terpenoids and coumarins were produced in brown necrotic layer formed during the infestation.

The larval homogenates of both weevils also induced in the tissue the production of furano-terpenoids and coumarins, as well as the formation of necrotic layer. The larval homogenate of sweet potato weevil induced also ethylene formation, the marker of injury in the tissue. Investigations on the furano-terponoid inducing factor demonstrated that the factor was 20 mm KCl-soluble, non-dialyzable, acetone-precipitable, (NH₄)₂SO₄-precipitable, heat-unstable, passing through Sephadex G–25 column without sieving and partially inactivated by pronase, indicating that the factor was a high molecular weight compound, perhaps of a proteinacious property. It is likely that the factor causes injury or death to sweet potato root tissue, leading to the formation of ethylene and necrotic layer, and then to production of furano-terpenoids and coumarins.     

Membrane-bound glycerol dehydrogenase catalyzes oxidation of D-pentonates to 4-keto-D-pentonates,D-fructose to 5-keto-D-fructose,and D-psicose to 5-keto-D-psicose

A novel oxidation of D-pentonates to 4-keto-D-pentonates was analyzed with Gluconobacter thailandicus NBRC 3258. D-Pentonate 4-dehydrogenase activity in the membrane fraction was readily inactivated by EDTA and it was reactivated by the addition of PQQ and Ca²⁺. D-Pentonate 4-dehydrogenase was purified to two different subunits, 80 and 14 kDa. The absorption spectrum of the purified enzyme showed no typical absorbance over the visible regions. The enzyme oxidized D-pentonates to 4-keto-D-pentonates at the optimum pH of 4.0. In addition, the enzyme oxidized D-fructose to 5-keto-D-fructose, D-psicose to 5-keto-D-psicose, including the other polyols such as, glycerol, D-ribitol, D-arabitol, and D-sorbitol. Thus, D-pentonate 4-dehydrogenase was found to be identical with glycerol dehydrogenase (GLDH), a major polyol dehydrogenase in Gluconobacter species. The reaction versatility of quinoprotein GLDH was notified in this study.     

Taxonomic distribution of isoenzymes of dehydroquinate hydrolyase in the angiosperms

The distribution of a dehydroquinate hydrolase (E.C. 4.2.1.10) isoenzyme activated by shikimic acid was studied in angiosperms through a simple diagnos     

芳香族化合物微生物代谢工程研究进展

代谢工程从20世纪90年代初期发展至今已有近30年历史,对微生物菌种改良和选育工作起到了极大的推动作用.芳香族化合物是一类可以通过微生物发酵生产的化学品,广泛应用于医药、食品、饲料和材料等领域.利用代谢工程手段对莽草酸和芳香族氨基酸合成途径进行理性改造,微生物细胞可以定向地大量积累人们需要的各种芳香族化合物.笔者对近3...     

Effect of Storage in Darkness and in Light on the Content of Membrane Lipids of Potato Tubers

The lipid composition of tubers from the potato varieties Bintje and Desirée was investigated during storage. Storage in total dark ness after harvest gave only small changes in the amounts of triglycerides. monogalactosyl diglycerides and digalactosyl digtycerides. Storage in light resulted in changes in these lipids and in their fatty acid composition. The absolute amount and the relative content of linolenic acid in the galactolipids increased. A simultaneous and equivalent decrease in the percentage of linoleic acid took place without any marked percentage changes in the other major fatty acids. The light induced changes of the lipids, which occur simultaneously with greening of the tuber, are discussed and related to the development of thylakoid membrane systems in the plastids.