葡萄糖酸氧化杆菌木糖醇脱氢酶基因的克隆与表达

【目的】获得葡萄糖酸氧化杆菌(Gluconobacter oxydans CGMCC 1.637)的木糖醇脱氢酶基因,研究其酶学性质及碳源特别是D-阿拉伯醇和木糖醇对该酶活性的影响。【方法】通过已报道序列的木糖醇脱氢酶的保守区设计引物,用聚合酶链式反应(polymerase chain reaction,PCR)扩增获得目的基因片段。根据获得的片段序列设计引物克隆目的基因的5’和3’片段,将所获得的片段拼接,获得完整的木糖醇脱氢酶基因。通过构建工程菌获得重组蛋白,并利用氧化还原反应测定重组酶的活性。用含不同碳源的培养基培养G.oxydans CGMCC 1.637,并测定其破胞上清液木糖醇脱氢酶氧化木糖醇的活性;用不同碳源培养的G.oxydans CGMCC 1.637转化木酮糖,用高效液相色谱法测定木糖醇的产量。【结果】获得一个新的798bp的木糖醇脱氢酶基因,所编码的木糖醇脱氢酶含265个氨基酸,属于短链脱氢酶家族。酶学性质研究发现,该木糖醇脱氢酶催化木糖醇氧化的最适合条件为35℃、pH 10.0,最高活性为23.27 U/mg,催化木酮糖还原为木糖醇的最适条件为30℃、pH 6.0。最高活性为255.55 U/mg;该木糖醇脱氢酶的对木糖醇的Km和Vmax分别为78.97 mmol/L和40.17 U/mg。碳源诱导实验表明,d-山梨醇对G.oxydans CGMCC 1.637木糖醇脱氢酶的活性有明显的促进作用,而葡萄糖、果糖、木糖、木糖醇、D-阿拉伯醇对木糖醇脱氢酶活性有明显的抑制作用。而在转化实验中,用d-甘露糖培养的G.oxydans CGMCC 1.637的转化能力明显高于其他碳源培养的G.oxydans CGMCC 1.637的转化能力,其中,用阿拉伯醇培养的G.oxydans CGMCC 1.637的转化能力最低,仅为对照的35%。【结论】克隆自G.oxydans CGMCC 1.637的木糖醇脱氢酶基因是一个新的基因,用阿拉伯醇培养的G.oxydans CGMCC 1.637破胞液木糖醇脱氢酶活性低;且阿拉伯醇对G.oxydans CGMCC 1.637木酮糖的还原能力具有抑制作用。     

Evidence that the gene YLR070c of Saccharomyces cerevisiae encodes a xylitol dehydrogenase.

The open reading frame YLR070c of Saccharomyces cerevisiae has high sequence similarity to S. cerevisiae sorbitol dehydrogenase and to xylitol dehydrogenase of Pichia stipitis. Overexpression of this open reading frame in S. cerevisiae resulted in xylitol dehydrogenase activity. The enzyme is specific for NADH. The following Michaelis constants were estimated: D-xylulose, 1.1 mM; NADH, 240 microM (at pH 7.0); xylitol, 25 mM; NAD, 100 microM (at pH 9.0). Xylitol dehydrogenase activity with the same kinetic properties can also be induced by xylose in wild type S. cerevisiae cells.     

Lactate dehydrogenase from gastrocnemius muscle of turtle

Five bands of lactate dehydrogenase (LDH) isoenzymes were seen by polyacrylamide gel electrophoresis in gastrocnemius muscle of the turtle (Kachuga smithi). The major band was of M2H2 type and was partially purified by gel filtration and affinity chromatography. The specific activity of the enzyme was 2.6 units/mg protein. The half-life of the enzyme at 4 degrees C, was about 7 days. The optimum temperature for enzyme activity was 30 degrees C and the enzyme was irreversibly inactivated at 40 degrees C. The optimum pH for the forward reaction (pyruvate to lactate) was 5.5, while for reverse reaction it was between 8.0 to 9.5. The apparent Km values for pyruvate, NADH, lactate and NAD+ were 0.20, 0.013, 25 and 0.333 mM, respectively. Oxalate was found to be the inhibitor of LDH with Ki of about 4.2 mM.     

Pentitol metabolism of Rhodobacter sphaeroides Si4: purification and characterization of a ribitol dehydrogenase.

The phototrophic bacterium Rhodobacter sphaeroides strain Si4 induced ribitol dehydrogenase (EC 1.1.1.56) when grown on ribitol- or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sulphate precipitation, affinity chromatography on Procion red, and chromatography on Q-Sepharose. For the native enzyme an isoelectric point of pH 6.1 and an apparent M(r) of 50,000 was determined. SDS-PAGE yielded a single peptide band of M(r) 25,000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-glucitol and D-arabitol were oxidized. The pH optimum of substrate oxidation was 10, and that of substrate reduction was 6.5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0.33 nM at pH 7.0 and 25 degrees C. The Km-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6.3, 12.5, 77 and 0.077 mM, respectively. Because of the favourable Km for ribitol, a method for quantitative ribitol determination was elaborated.     

Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154-->Cys forms of yeast xylitol dehydrogenase

Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10-0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19+/-0.03 s(-1) and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019+/-0.003% and 0.74+/-0.03% of wild-type catalytic efficiency (kcat/K(sorbitol)=7800+/-700 M(-1) x s(-1)) and kcat (=161+/-4 s(-1)) for NAD+-dependent oxidation of sorbitol at 25 degrees C respectively. The pH profile of kcat/K(sorbitol) for E154C decreased below an apparent pK of 9.1+/-0.3, reflecting a shift in pK by about +1.7-1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (approximately +0.2 log units), suggesting that the observed pK in the binary enzyme-NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7+/-0.2 (E154C, 1.7+/-0.1) and 1.9+/-0.3 (E154C, 2.4+/-0.2) on kcat/K(sorbitol) respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687+/-12 s(-1) in the pre-steady state, which features a turnover of 0.9+/-0.1 enzyme equivalents as NADH was produced with a rate constant of 409+/-3 s(-1). The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

Isolation and properties of cytoplasmic alpha-glycerol 3-phosphate dehydrogenase from the pectoral muscle of the fruit bat,Eidolon helvum

Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.     

Purification and characterization of pyridoxal 4-dehydrogenase from Aureobacterium luteolum

A pyridoxal dehydrogenase was purified to homogeneity from Aureobacterium luteolum, which can use pyridoxine as a carbon and nitrogen source, and characterized. The enzyme was a dimeric protein with a subunit molecular weight of 38,000. It had several properties distinct from those of the partially purified enzyme from Pseudomonas MA-1. The optimum pH (8.0-8.5) was 0.8-1.3 lower than that of the Pseudomonas enzyme. The Aureobacterium enzyme showed much higher and lower affinities for NAD+ (Km, 0.140 +/- 0.008 mM) and pyridoxal (0.473 +/- 0.109 mM), respectively, than those of the Pseudomonas enzyme. The Aureobacterium enzyme could use NADP+ as a substrate: the reactivity was 6.5% of NAD+. The enzyme was much more tolerant to metal-chelating agents. Irreversibility of the enzymatic reaction was shared by the two enzymes. No aldehyde dehydrogenase showed similarity to the amino-terminal amino acid sequence of the enzyme.     

Investigation and evaluation of a method for determination of ethanol with the SIRE Biosensor P100, using alcohol dehydrogenase as recognition element

A new method for rapid determination of ethanol was developed, using alcohol dehydrogenase as recognition element for the SIRE (sensors based on injection of the recognition element) Biosensor, which is an amperometric biosensor. The method was simple, fast, accurate, specific and cost-effective. The recognition element solution used was stable at least for 24 h in room temperature, and at least one month when lyophilised. The optimal potential versus the silver wire electrode, the optimal pH of the buffer and the optimal temperature of the water bath was determined to be +950 mV, 8.1 and 308 K, respectively. The optimal concentrations of alcohol dehydrogenase, BSA and NAD(+) were determined to be 200 U/ml, 20 mg/ml and 15 mM, respectively. The total analysis time was between 50 s and 4 min per analysis, depending on the concentration range. The linear range was 0-12.5 mM. The detection limit was less than 0.1 mM. The repeatability (%R.S.D.) was 3-5% (n=10). The reproducibility was 5-8% (n=5). Methanol gave no signal at all, but higher alcohols, such as propanol, pentanol and hexanol, gave significant signals, decreasing with increasing length of the carbon chain. The price for one measurement was calculated to be 0.052 euro. The results from measurements with the biosensor were compared to those from an established analysis kit for ethanol. The results correlated well (R(2)=0.9874). The concentration of ethanol in different alcoholic beverages was investigated and correlated well with the concentrations given by the manufacturers.     

米曲霉木糖醇脱氢酶的分子模建与对接

[目的]研究米曲霉木糖醇脱氢酶基因的结构与功能.[方法]克隆测序来源于米曲霉的木糖醇脱氢酶(XDH)基因,利用Swiss-MODEL和Modeller对XDH进行三级结构模建,通过PROCHECK和Prosa2003对得到的4个目标模型进行评价,从中得到一个最佳模型.在同源建模的基础上,通过分子对接软件MolsoftICM-Pro,对辅因子进行对接,预测了XDH与NAD+、Zn2+作用的相关残基.寻找底物木糖醇与XDH结合的可能活性口袋,用Molsoft模拟XDH与木糖醇的对接,预测了酶与底物作用的关键氨基酸残基.[结果]结构分析显示,米曲霉XDH含有醇脱氢酶家族锌指纹结构和典型醇脱氢酶Rossmann折叠的辅酶结合域,属于Medium-chain脱氢酶(MDR)家族.通过对接研究,预测了XDH与NAD+之间形成氢键的氨基酸有Asp206、Arg211、Ser255、Ser301和Arg303,这些氨基酸位于结合域,与Zn2+形成氢键的氨基酸有His72和Glu73,位于催化域,与天然底物木糖醇形成氢键的氨基酸有Ile46、Ile349、Lys350和Thr351,位于催化域.[结论]所得信息对XDH分子定向改造、拓展米曲霉工业应用范围有重要意义.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.     

Purification and properties of tauropine dehydrogenase from the shell adductor muscle of the ormer, Haliotis lamellosa

Tauropine dehydrogenase (tauropine:NAD oxidoreductase) was purified from the shell adductor muscle of the ormer, Haliotis lamellosa. The enzyme was found to utilize stoichiometrically NADH as co-enzyme and pyruvate and taurine as substrates producing tauropine [rhodoic acid; N-(D-1-carboxyethyl)-taurine]. The enzyme was purified to a specific activity of 463 units/mg protein using a combination of ammonium sulphate fractionation, ion-exchange and affinity chromatography. The relative molecular mass was 38,000 +/- 1000 when assessed by gel filtration on Ultrogel AcA 54 and 42,000 +/- 150 by electrophoresis on 5-10% polyacrylamide gels in the presence of 1% sodium dodecyl sulphate; the data suggest a monomeric structure. Tauropine and pyruvate were found to be the preferred substrates. Among the amino acids tested for activity with the enzyme, only alanine is used as an alternative substrate, but with a rate less than 6% of the enzyme activity with taurine. Of the oxo acids tested, 2-oxobutyrate and 2-oxovalerate were also found to be substrates. Apparent Km values for the substrates NADH, pyruvate and taurine are 0.022 +/- 0.003 mM, 0.64 +/- 0.07 mM and 64.7 +/- 5.4 mM, respectively, at pH 7.0 and for the products, NAD+ and tauropine, are 0.29 +/- 0.01 mM and 9.04 +/- 1.27 mM, respectively, at pH 8.3. Apparent Km values for both pyruvate and taurine decrease with increasing co-substrate (taurine or pyruvate) concentration. NAD+ and tauropine were found to be product inhibitors of the forward reaction. NAD+ was a competitive inhibitor of NADH, whereas tauropine gave a mixed type of inhibition with respect to pyruvate and taurine. Succinate was found to inhibit non-competitively with respect to taurine and pyruvate with an apparent Ki value in the physiological range of this anaerobic end product. The inhibition by L-lactate, not an end product in the ormer, was competitive with respect to pyruvate. The physiological role or tauropine dehydrogenase during anaerobiosis is discussed.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

Development of a screen-printed amperometric biosensor for the determination of L-lactate dehydrogenase level.

We attempted to develop a screen-printed biosensor for the amperometric determination of L-lactate dehydrogenase (LDH) level on the basis of NAD(+)/NADH-dependent dehydrogenase reaction. The printing ink for the working electrode consisted of L-lactate, NAD(+), composite polymer of hydroxyethyl cellulose with ethylene glycol, 3,4-dihydroxybenzaldehyde (3,4-DHB) as an electron transferring mediator, and graphite as the conducting material. The 3,4-DHB was electropolymerized on the carboneous working electrode by potential cycling between -200 and +300 mV vs. Ag/AgCl reference electrode. Through the electrocatalytic reaction with immobilized 3,4-DHB, the NADH generated by the LDH reaction could be efficiently oxidized at lower potential than the unmodified carbon electrode. The analytical performance of the electrode was characterized in terms of linear sensing range and detection limit for LDH. The response from the developed biosensor was linear up to 500 U/l of LDH, and the detection limit of 50 U/l was observed at the signal-to-noise ratio of 3.     

D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure.

1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents.     

Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli. I: Purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium

Escherichia coli containing the Bacillus subtilis glucose dehydrogenase gene on a plasmid (prL7) was used to produce the enzyme in high quantities. Gluc-DH-S was purified from the cell extract by (NH4)2SO4-precipitation, ion-exchange chromatography and Triazine-dye chromatography to a specific activity of 375 U/mg. The enzyme was apparently homogenous on SDS-PAGE with a subunit molecular mass of 31.5 kDa. Investigation of Gluc-DH-S was performed for comparison with the corresponding properties of Gluc-DH-M. The limiting Michaelis constant at pH 8.0 for NAD+ is Ka = 0.11 mM and for D-glucose Kb = 8.7 mM. The dissociation constant for NAD+ is Kia = 17.1 mM. Similar to Gluc-DH-M, Gluc-DH-S is inactivated by dissociation under weak alkaline conditions at pH 9.0. Complete reactivation is attained by readjustment to pH 6.5. Ultraviolet absorption, fluorescence and CD-spectra of native Gluc-DH-S, as well as fluorescence- and CD-backbone-spectra of the dissociated enzyme were nearly identical to the corresponding spectra of Gluc-DH-M. The aromatic CD-spectrum of dissociated Gluc-DH-S was different, representing a residual ellipticity of tryptophyl moieties in the 290-310 nm region. Density gradient centrifugation proved that this behaviour is due to the formation of inactive dimers in equilibrium with monomers after dissociation. In comparison to Gluc-DH-M, the kinetics of inactivation as well as the time-dependent change of fluorescence intensity at pH 9.0 of Gluc-DH-S showed a higher velocity and a changed course of the dissociation process.     

Purification and characterisation of NAD+-dependent xylitol dehydrogenase from Fusarium oxysporum

An NAD⁺-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M _r 48 000, and pI 3.6. It was optimally active at 45 °C and pH 9–10. It was fully stable at pH 6–7 for 24 h and 30 °C. K _m values for d-xylitol and NAD⁺ were 94 mM and 0.14 mM, respectively. Mn²⁺ at 10 mM increased XDH activity 2-fold and Cu²⁺ at 10 mM inhibited activity completely.     

Construction and co-expression of plasmid encoding xylitol dehydrogenase and a cofactor regeneration enzyme for the production of xylitol from D-arabitol

The biotransformation of D-arabitol into xylitol was investigated with focus on the conversion of D-xylulose into xylitol. This critical conversion was accomplished using Escherichia coli to co-express a xylitol dehydrogenase gene from Gluconobacter oxydans and a cofactor regeneration enzyme gene which was a glucose dehydrogenase gene from Bacillus subtilis for system 1 and an alcohol dehydrogenase gene from G. oxydans for system 2. Both systems efficiently converted D-xylulose into xylitol without the addition of expensive NADH. Approximately 26.91 g/L xylitol was obtained from around 30 g/L D-xylulose within system 1 (E. coli Rosetta/Duet-xdh-gdh), with a 92% conversion yield, somewhat higher than that of system 2 (E. coli Rosetta/Duet-xdh-adh, 24.9 g/L, 85.2%). The xylitol yields for both systems were more than 3-fold higher compared to that of the G. oxydans NH-10 cells (7.32 g/L). The total turnover number (TTN), defined as the number of moles of xylitol formed per mole of NAD(+), was 32,100 for system 1 and 17,600 for system 2. Compared with that of G. oxydans NH-10, the TTN increased by 21-fold for system 1 and 11-fold for system 2, hence, the co-expression systems greatly enhanced the NADH supply for the conversion, benefiting the practical synthesis of xylitol.     

Cloning,Purification, and Characterization of Diaminobutyrate Acetyltransferase from the Halotolerant Methanotroph <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

L-2,4-Diaminobutyrate (DAB) acetyltransferase (DABAcT) catalyzes one of the key reactions of biosynthesis of the bacterial osmoprotectant ectoine--acetylation of L-2,4-DAB yielding Ngamma-acetyl-2,4-DAB. Gene ectA encoding DABAcT was cloned from DNA of the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z and expressed in Escherichia coli with an additional six His residues at the C-terminus. Homogeneous enzyme preparation with specific activity 200 U/mg was obtained by affinity metal-chelating chromatography. DABAcT was found to be a homodimer with molecular mass 40 kD. The enzyme is most active at pH 9.5 and 20 degrees C, and its activity increased threefold in the presence of 0.1-0.2 M NaCl or 0.2 M KCl. The Km values of recombinant DABAcT measured at the optimal pH and temperature in the presence of 0.2 M KCl were 460 and 36.6 microM for L-2,4-DAB and acetyl-CoA, respectively. The enzyme is specific for L-2,4-DAB and acetyl-CoA and is also active against propionyl-CoA (20%). Zn2+ and Cd2+ at 1 mM concentration completely inhibit the recombinant enzyme; 10 mM ATP inhibits 26% of the enzyme activity, whereas EDTA, o-phenanthroline, ADP, NAD(P), and NAD(P)H do not significantly effect the enzyme activity. The possible participation of DABAcT in regulation of ectoine biosynthesis in M. alcaliphilum 20Z is discussed.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.