Biochemical characterization of l-arabitol 2-dehydrogenase from Pantoea ananatis

Two oxidoreductases, XDH and LAD, were found in the same operon that was involved in sugar metabolism in Pantoea ananatis. LAD, whose endogenous substrate was unknown, was recombinantly prepared and biochemically analyzed. Consequently, LAD was identified as l-arabitol 2-dehydrogenase and its substrate specificity was complementary to that of XDH.     

Acetic acid removal by Saccharomyces cerevisiae during fermentation in oenological conditions. Metabolic consequences

The commercial Saccharomyces cerevisiae strains used in champagne winemaking were tested for their ability to metabolise acetic acid during alcoholic fermentation. Fermentation tests were performed in conditions close to oenological ones using a Chardonnay grape juice supplemented with acetic acid. The amount of acetic acid metabolised by wine yeast increased with increasing initial acetic acid concentration and this elimination occurred during the second part of the exponential growth phase. When the initial acetic acid concentration exceeds 1 g/l, and whatever the yeast strain used, the concentration of acetic acid in the resulting wine cannot be reduced to an acceptable level according to the current legislation. Acetic acid removal modified yeast metabolism, since more acetaldehyde, less glycerol and less succinic acid were produced. Considering the reduction of the NADPH/NADP⁺ ratio following acetic acid consumption, we propose, as a new hypothesis, that acetic acid could modify yeast metabolism by reducing the activity of the NADP⁺ dependent aldehyde dehydrogenase Ald6p.     

Efficient production of L-lactic acid from xylose by a recombinant Candida utilis strain

Efficient L-lactic acid production from xylose was achieved using a pyruvate decarboxylase-deficient Candida utilis strain expressing an L-lactate dehydrogenase, an NADH-preferring mutated xylose reductase (XR), a xylitol dehydrogenase and a xylulokinase. The recombinant strain showed 53% increased L-lactic acid production compared with the reference strain expressing native XR (NADPH-preferring).     

Favorable effect of very low initial KLa value on xylitol production from xylose by a self-isolated strain of Pichia guilliermondii

Xylitol production from xylose by a self-isolated furfural and 5-hydroxymethyl furfural assimilating Pichia guilliermondii was studied under oxygen limitation. An extremely low initial volumetric oxygen transfer coefficient (0.075 h^− 1) was found most favorable to the xylitol production with yield of 0.61 g g^− 1. Related enzymes activities were also investigated and discussed.     

Catalytic removal of acetaldehyde in saliva by a Gluconobacter strain

Acetaldehyde (AA) accumulates in the oral cavity after alcohol intake and is responsible for an increased risk of alcohol-related upper aerodigestive tract (UDAT) cancer among aldehyde dehydrogenase 2-inactive heterozygotes in particular. Thus, the removal of AA from the saliva to a level below its mutagenic concentration (50 μM) after drinking is a potentially straightforward method for reducing the risk of alcohol-related UDAT cancer. Although microbial cells with AA-decomposing activity could potentially serve as a useful agent for the catalytic removal of AA from the saliva without the supplemental addition of cofactors, these cells generally exhibit strong AA-producing activity from ethanol, which is present in excess (50mM) over AA (100 μM) in the saliva after drinking. In this study, we observed that Gluconobacter kondonii (GK) cells efficiently decomposed salivary AA (100-390 μM) without the supplemental addition of cofactors irrespective of the type of alcoholic beverages consumed, even in the presence of an excess of ethanol (63 mM). Hydrogen peroxide, which is carcinogenic in animal experiments, was not produced because of the AA removal. The GK cells incubated at 45 °C and pH 3.5 for 15 h were killed, but they retained 80% of their original AA-decomposing activity. The treated cells were used as nonviable microcapsules that harbor a membrane-bound AA-decomposing activity.     

Cloning and sequencing of a gene encoding of aldehyde oxidase in Pseudomonas sp. AIU 362

We have cloned a gene encoding an aldehyde oxidase (ALOD) oxidized glyoxal but not glyoxylic acid from Pseudomonas sp. AIU 362. The ALOD gene contained an open reading frame consisting of 888 nucleotides corresponding to 295 amino acid residues. The deduced amino acid sequence exhibited a high similarity to those of 3-hydroxyisobutyrate dehydrogenases (3-HIBDHs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant ALOD expressed in E.?coli BL21 was 20,000-fold higher than that of ALOD in Pseudomonas sp. AIU 362. The recombinant ALOD exhibited ALOD activity and 3-HIBDH activity. The 3-HIBDH from Pseudomonas putida KT2440 also exhibited ALOD activity. Thus, the ALOD from Pseudomonas sp. AIU 362 and 3-HIBDH from P.?putida KT2440 were classified into the same enzyme group.     

Stable disruption of ethanol production by deletion of the genes encoding alcohol dehydrogenase isozymes in Saccharomyces cerevisiae

We analyzed the effects of the deletions of genes encoding alcohol dehydrogenase (ADH) isozymes of Saccharomyces cerevisiae. The decrease in ethanol production by ADH1 deletion alone could be partially compensated by the upregulation of other isozyme genes, while the deletion of all known ADH isozyme genes stably disrupted ethanol production.     

Characterization of NAD-dependent alcohol dehydrogenase enzymes of strawberry's achenes (Fragaria x ananassa cv. Elsanta) and comparison with respective enzymes from Methylobacterium extorquens

Achenes were isolated from strawberries fruits, while the methylotroph Methylobacterium extorquens (strain with CABI registration number IMI 369321), which has been isolated from strawberry (Fragaria x ananassa cv. Elsanta) callus cultures, was grown on a mixture of methanol (0.25% v/v) and 1,2-propanediol (0.75% v/v). The Alcohol Dehydrogenase (ADH) enzymatic activities of the achenes were assessed and the optimum pH for ADH activity was found to be pH 10.0. Enzyme assays were carried out in order to define the best substrate specificity at pH = 10.0. The best substrates were found to be ethanol (Km = 5.950 mM) and methanol (Km = 12.610 mM). Only enzymes from the bacterium showed capability of using aldehydes - especially formaldehyde - as substrates. A wide variety of metals as well as EDTA and NaN₃ were shown to decrease the enzymatic activity. Furthermore, SDS-PAGE Electrophoresis experiments showed Molecular Weight of 47.0 kDa for the Alcohol Dehydrogenase from achenes of strawberries (Fragaria x ananassa). Additional experiments were conducted in order to define certain thermodynamic properties of the enzymes, by using the dehydrogenation activities of these three enzyme sources which were calculated by measuring the absorption of NADH at 340 nm, in the Arrhenius equation.     

Effects of 1-MCP and ethylene on expression of three CAD genes and lignification in stems of harvested Tsai Tai (Brassicachinensis)

Tsai Tai (Brassicachinensis) is produced mainly in China but also consumed overseas. The stems of Tsai Tai are subject to toughening due to lignification. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in lignin biosynthesis. Three full-length CAD cDNAs were isolated, encoding proteins of 288, 323, and 323 amino acids, respectively. Sequence analysis showed that they share a highly conserved putative NAD/NADP(H)-binding site at the amino terminus. All the three BcCAD genes responded to 1-MCP and ethylene within 2 h. Ethylene up-regulated expression of BcCAD1-1 and BcCAD2, while 1-MCP down-regulated them. Ethylene increased yellow leaves and stem lignin, and 1-MCP decreased them. These results suggest ethylene is involved in lignin growth in Tsai Tai, and induced expression of BcCAD1-1 and BcCAD2 could contribute to lignification. They also suggest removal of ethylene and application of 1-MCP could extend shelf-life and knockout of certain CAD genes could produce lines that store well.     

Carbon monoxide utilization of an extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4

CO utilization by a CO(2)-requiring extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4 was found when CO(2) was removed from the culture environment before cultivation, suggesting that this bacterium can convert CO into CO(2). However, the gene encoding putative CO dehydrogenase large subunit in N9T-4 was not induced by CO.     

Molecular structure and gene analysis of Ce3+ -induced methanol dehydrogenase of Bradyrhizobium sp. MAFF211645

The molecular structure and nucleotide sequence of Ce(3+)-induced methanol dehydrogenase (MDH) of Bradyrhizobium sp. MAFF211645 were investigated. The addition of 30 μM Ce(3+) to 1/10 nutrient broth containing 0.5% methanol remarkably increased MDH activity. Furthermore, La(3+) increased MDH activity, but other heavier rare earth and metal elements did not have the same effect. MDH increased by Ce(3+) was purified by sequential column chromatography, and the purified MDH migrated as a single band with an apparent molecular weight of 68 kDa on SDS-PAGE. The apparent molecular weight of native MDH was estimated to be 108,000 by gel chromatography. The MDH was comprised of two identical subunits. N-terminal 23-amino acid sequence, 1-NDELHKMAQNPKDWVMPAGDYAN-23, of the purified MDH exhibited 91.3% identity to that of the MDH large subunit-like protein encoded by mxaF' of Bradyrhizobium japonicum USDA110. Nucleotide sequencing of the MDH gene of strain MAFF211645 yielded a deduced amino acid sequence comprising 601 amino acid residues, an N-terminal signal peptide, and a mature MDH comprising 578 amino acid residues with a predicted molecular mass of 62,918 Da. Further analysis of the deduced amino acid sequence of mature MDH revealed that the functional amino acids in its active site, such as two adjacent Cys residues, and bacterial quinoprotein signatures 1 and 2 were conserved. These results indicate that Ce(3+)-induced MDH encoded by mxaF' may be involved in methanol metabolism in Bradyrhizobium sp. MAFF211645.     

Substrate specificity of alcohol dehydrogenase from the yeast Hansenyls polymorpha CBS 4732 and Candida utilis CBS 621

The substrate specificity of alcohol dehydrogenase (ADH) from Hansenula polymorpha and Candida utilis has been compared with that of the classical ADH from baker's yeast. Cell-free extracts of H. polymorpha and C. utilis exhibited a much higher ratio of butanol to ethanol oxidation than baker's yeast ADH. This was also observed with the purified enzymes. The ratio of activities with ethanol and butanol was pH-dependent. With the baker's yeast enzyme the activity strongly decreased with increasing chain length, whereas the enzymes form H. polymorpha and C. utilis showed a high reactivity with long-chain alcohols. In addition, the affinity constant for ethanol was more than tenfold lower than that of the baker's yeast enzyme. The purified preparation yielded several protein bands on polyacrylamide slab gels, each of which showed activity with both ethanol and butanol.     

Molecular structure of La3+-induced methanol dehydrogenase-like protein in Methylobacterium radiotolerans

La(3+) and not Ca(2+) increases methanol dehydrogenase (MDH) activity in Methylobacterium radiotolerans NBRC15690. La(3+)- and Ca(2+)-MDH-like proteins were found to be homodimeric (α(2)) and heterotetrameric (α(2)β(2)), respectively. N-terminal amino acid sequences of these proteins revealed that La(3+)- and Ca(2+)-MDH-like proteins were encoded by xoxF and mxaFI, respectively.     

嗜热厌氧乙醇杆菌组氨酸融合双活性乙醇脱氢酶的克隆、表达和酶学性质

乙醛脱氢酶和乙醇脱氢酶是嗜热厌氧乙醇菌Thermoanaerobacter ethanolicus乙醇代谢途径中的关键酶。根据高同源性的NADP(H)依赖型Ⅱ型乙醇脱氢酶(S-ADH)的序列设计引物,从T.ethanolicusJW200基因组中PCR扩增出编码S-ADH的基因,插入带组氨酸标签的pET-20(b),测序获得基因大小为1 059 bp,并在大肠杆菌JM109(DE3)中表达。表达产物经Ni亲和柱提纯达电泳纯。带组氨酸标签的重组NADP(H)依赖型乙醇脱氢酶具乙醇脱氢酶和乙醛脱氢酶双活性,能将乙酰辅酶A转化成乙醇.带组氨酸标签的重组酶酶学性质为乙醛脱氢酶活性最适值为pH 8.4,最适温度70℃;乙醇脱氢酶活性正、逆反应分别最适pH 8.0,最适T 55℃;最适pH8.9,最适为T 60℃。以乙酰辅酶A(Aceyl-CoA)为底物,该酶的Km为3.32 mmol/L,Vmax为12.5μmol/(min.mg)。T.ethanolicusJW200中双活性S-ADH的首次发现,建立了该菌乙醇代谢途径中从乙酰辅酶A到的乙醇的另一条通路。     

Developmental changes in pulp organic acid concentration and activities of acid-metabolising enzymes during the fruit development of two loquat (Eriobotrya japonica Lindl.) cultivars differing in fruit acidity

Changes in organic acid concentration and related enzyme activities in loquat (Eriobotrya japonica Lindl.) pulp were studied, using low-acid ‘Changhong 3’ and high-acid ‘Jiefangzhong’ cultivars. Both titratable acidity (TA) and malic acid concentration increased during the early stages of fruit development and decreased at the later stages. The difference in TA between the two cultivars could be explained by the difference in malic acid concentration, which could result from a difference in NAD-malate dehydrogenase (NAD-MDH) and NADP-malic enzyme (NADP-ME) activities. Although the difference in malic acid concentration between the two cultivars could not result from a difference in phosphoenolpyruvate carboxylase (PEPC) activity, malic acid concentration in both ‘Changhong 3’ throughout fruit development and ‘Jiefangzhong’ at the early stages increased linearly and curvilinearly with increasing PEPC activity, respectively. Therefore, NAD-MDH, NADP-ME and PEPC activities may play significant roles in malic acid biosynthesis and degradation.