Cofactor Regeneration in Biocatalysis in Organic Media

Methods used for the regeneration of cofactors in organic media are reviewed. Substratedriven regeneration methods include the use of a second substrate of the same enzyme and the use of a second enzyme and its substrate. The use of mediators in oxidoreductions is described and examples of photochemical and electrochemical regeneration methods are presented. General problems and possibilities of cofactor regeneration in organic media are discussed.     

NADPH Regeneration by Glucose Dehydrogenase from Gluconobacter scleroides for l-Leucovorin Synthesis

A new process for (6S)-tetrahydrofolate production from dihydrofolate was designed that used dihydrofolate reductase and an NADPH regeneration system. Glucose dehydrogenase from Gluconobacter scleroides KY3613 was used for recycling of the cofactor. The reaction mixture contained 200 mM dihydrofolate, 220mM glucose, 2mM NADP, 14.4U/ml dihydrofolate reductase, and 14.4U/ml Glucose dehydrogenase, and the reaction was complete after incubation at pH 8.0, and 40°C for 2.5 hr. With (6S)-tetrahydrofolate as the starting material, l-leucovorin was synthesized via a methenyl derivative. The purity of the l-leucovorin was 100%, and its diastereomeric purity was >99.5% d.e. as the (6S)-form.     

Kinetic and Structural Characterization for Cofactor Preference of Succinic Semialdehyde Dehydrogenase from Streptococcus pyogenes

The γ-Aminobutyric acid (GABA) that is found in prokaryotic and eukaryotic organisms has been used in various ways as a signaling molecule or a significant component generating metabolic energy under conditions of nutrient limitation or stress, through GABA catabolism. Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde to succinic acid in the final step of GABA catabolism. Here, we report the catalytic properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) regarding its cofactor preference. Kinetic analysis showed that SpSSADH prefers NADP⁺ over NAD⁺ as a hydride acceptor. Moreover, the structures of SpSSADH were determined in an apo-form and in a binary complex with NADP⁺ at 1.6 Å and 2.1 Å resolutions, respectively. Both structures of SpSSADH showed dimeric conformation, containing a single cysteine residue in the catalytic loop of each subunit. Further structural analysis and sequence comparison of SpSSADH with other SSADHs revealed that Ser158 and Tyr188 in SpSSADH participate in the stabilization of the 2’-phosphate group of adenine-side ribose in NADP⁺. Our results provide structural insights into the cofactor preference of SpSSADH as the gram-positive bacterial SSADH.     

Isolation and Biochemical Characterization of a Glucose Dehydrogenase from a Hay Infusion Metagenome

Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.     

Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product.     

Codon-Optimized NADH Oxidase Gene Expression and Gene Fusion with Glycerol Dehydrogenase for Bienzyme System with Cofactor Regeneration

NADH oxidases (NOXs) play an important role in maintaining balance of NAD⁺/NADH by catalyzing cofactors regeneration. The expression of nox gene from Lactobacillus brevis in Escherichia coli BL21 (BL21 (DE3)) was studied. Two strategies, the high AT-content in the region adjacent to the initiation codon and codon usage of the whole gene sequence consistent with the host, obtained the NOX activity of 59.9 U/mg and 73.3 U/mg (crude enzyme), with enhanced expression level of 2.0 and 2.5-folds, respectively. Purified NOX activity was 213.8 U/mg. Gene fusion of glycerol dehydrogenase (GDH) and NOX formed bifuctional multi-enzymes for bioconversion of glycerol coupled with coenzyme regeneration. Kinetic parameters of the GDH-NOX for each substrate, glycerol and NADH, were calculated as V _{max(Glycerol)} 20 μM/min, K _m(Glycerol) 19.4 mM, V _{max (NADH)} 12.5 μM/min and K _{m (NADH)} 51.3 μM, respectively, which indicated the potential application of GDH-NOX for quick glycerol analysis and dioxyacetone biosynthesis.     

Stability of Glucose 6-Phosphate Dehydrogenase Complexed with Its Substrate or Cofactor in Aqueous and Micellar Environment

Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), or cofactor, NADP⁺, has been studied within the range 20–40°C in three media: (a) 0.04 M NaOH–glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, k _in(s^–1). In the case of G6PDH–NADP⁺complexes, the values of k _inwere independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of k _infor the complex G6PDH–GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of k _incorresponded to the degree of hydration W ₀= 16.7; at W ₀> 16.7 and W ₀< 16.7, k _inincreased. Within the range 20–40°C, the values of k _inmeasured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of k _inwere characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6°C to 40°C. In all media studied, NADP⁺complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH–NADP⁺melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.     

解木聚糖类芽孢杆菌木葡聚糖酶的分离纯化及酶学性质研究

解木聚糖类芽孢杆菌(Paenibacillus xylanilyticus)发酵液经硫酸铵分级沉淀、HiPrep26/10 Desalting柱脱盐、HiPrepDEAE FF16/10阴离子交换柱、HiPrep 16/60 Sephacryl S-100凝胶柱、HiPrep 16/10 Source 30S阳离子交换柱等,最终纯化出单一组分的木葡聚糖酶,经过SDS-PAGE电泳分析,此木葡聚糖酶相对分子量约为39 kD。该菌所产木葡聚糖酶的最适反应温度是50℃,在60℃以下较稳定;最适反应pH是7.0,在pH5.0-10.0范围内酶活力较为稳定。酶的动力学研究显示Km为65 g/L,Vmax为6.49μmol/min,kcat=10.86 s-1。底物特异性研究表明对木葡聚糖具有较高比活力。酶蛋白经质谱分析,比对结果显示与来源于Paenibacillus pabuli的木葡聚糖酶有较高同源性。本研究为首次报道解木聚糖类芽孢杆菌(P.xylanilyticus)产木葡聚糖酶。     

甲基营养菌MP688萄糖脱氢酶基因分离鉴定及性质研究

目的：鉴定甲基营养菌MP688中的葡萄糖脱氢酶基因。方法：对甲基营养菌MP688基因组序列进行比对和分析,找到与已知细菌葡萄糖脱氢酶同源性最高的基因序列mpq_2164,且该基因所编码蛋白经分析具有跨膜结构域。设计51物扩增mpq_2164和缺失跨膜区域序列的s-mpq_2164,将PCR产物克隆到表达载雄pET-15b上,在大肠杆菌BL21中完成异源重组表达,然后通过组氨酸标签镍柱亲和层析纯化,采用DCIP法测定葡萄糖脱氢酶的活力。结果：分离了甲基营养菌MP688中的葡糖糖脱氢酶基因,并实现了s-mpq_2164的高效异源重组表达;MPQ2164的氯基酸序列与已知的葡萄糖脱氢酶相似性很低,但酶活测定结果表明S-MPQ-2164具有很高的葡糖糖脱氢酶活性。结论：MPQ_2164是-个依赖于吡咯喹啉醌的葡萄糖脱氢酶,去掉跨膜结构域有利于该蛋白的异源嘉{大,     

Cofactor Regeneration by a Soluble Pyridine Nucleotide Transhydrogenase for Biological Production of Hydromorphone

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP⁺-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.     

Phylogeny and Characterization of Three nifH-Homologous Genes from Paenibacillus azotofixans

In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.     

Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes

Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (adh) genes from metagenomes based on the Leifsonia species adh gene (lsadh), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of lsadh, and PCR was used to amplify nearly full-length adh genes from metagenomic DNAs. All adh preparations were fused with lsadh at the terminal region and used to construct Escherichia coli plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as adh positive (∼60%). Finally, 40 adh genes, Hladh-001 to Hladh-040 (for homologous Leifsonia adh), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The Hladh genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols.     

Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB₁-C-A-B₂, from which two proteins, OdhAB₁C and OdhAB₂C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB₁ and OdhB₂ in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.     

Purification and Characterization of Glucose 6-phosphate Dehydrogenase from Sheep Erythrocytes and Inhibitory Effects of some Antibiotics on Enzyme Activity

Glucose 6-phosphate dehydrogenase (d -glucose 6-phosphate: NADP + oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2′, 5′-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4°C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I 50 values were determined from Activity %-[Drug] graphs and K i values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Simultaneous Production of Amino Acid Dehydrogenases and Glucose Dehydrogenase by Bacillus Megaterium and Their Utilization for L-Valine Synthesis with Nadh Regeneration

An improved method for the simultaneous production of valine dehydrogenase and glucose dehydrogenase by Bacillus megaterium (ATCC 39118) is described. The highest yields in volumetric activities (8200 U.S^-1' of glucose dehydrogenase and 7200 U.S¹ of valine dehydrogenase) were obtained using a fed batch cultivation technique with glucose, yeast extract and corn steep liquor in the feed medium. The main characteristics (stability, optimal pH, Michaelis constants, substrate and product inhibitions) of valine dehydrogenase and glucose dehydrogenase from crude extracts were determined. B. megaterium crude extract was suitable for synthesis of L-valine from $aL-keto isovalerate with glucose dehydrogenase as the NADH-regenerating enzyme and the conditions of the conversion have been optimized. $aL-Keto acid was supplied in fed batch mode in order to avoid substrate inhibition and was not involved in side reactions. With the optimized system, a concentration of 95 mM L-valine was obtained in 45 hours with a molar conversion yield close to 100%.     

Enzymatic Removal of Diacetyl from Beer : III. Enzyme Protection and Regeneration of Cofactor

Use of diacetyl reductase, a reduced nicotinamide adenine dinucleotide (NADH)-requiring enzyme, to eliminate diacetyl off-flavor in beer was studied. The crude enzyme was extracted from Aerobacter aerogenes and partially purified by ammonium sulfate precipitation or Sephadex chromatography. In the semipure state, the enzyme was inactivated by lyophilization; in a crude state, the lyophilized extract remained stable for at least 4 months at - 20 C. A 50% reduction in specific activity within 5 min was observed when crude diacetyl reductase was suspended (5 mg of protein/ml) in phosphate buffer at pH 5.5 or below; a similar inactivation rate was observed when the crude enzyme was dissolved in a 5% aqueous ethyl alcohol solution. Effective crude enzyme activity in beer at a natural pH of 4.1 required protection of the enzyme in 10% gelatin. Incorporation of yeast cells with the gel-protected enzyme provided regeneration of NADH. Combinations of yeast, enzyme, and gelatin were tested to obtain data analyzed by regression analysis to determine the optimal concentration of each component of the system required to reduce the level of diacetyl in spiked (0.5 ppm) beer to less than 0.12 ppm within 48 hr at 5 C. The protected enzyme system was also effective in removing diacetyl from orange juice (pH 3.8) and some distilled liquors.     

一种葡聚糖芯片的再生方法、表征及其应用*

表面等离子体共振（surface plasmon resonance, SPR）生物传感器,作为一种适时快捷,无需标记的生物分子相互作用研究工具,已广泛应用于生物化学分析与研究。羧甲基化葡聚糖修饰的CM5传感芯片是Biacore 系列仪器应用最为普遍的核心部件,目前CM5芯片主要从法玛西亚公司购买,价格昂贵,且一旦共价交联的受体分子失活,就不能重复利用。阐述了一种简便、低成本、用于SPR生物传感器的葡聚糖修饰金膜芯片的再生方法及其表征和应用。用此方法再生的芯片能被循环伏安法和原子力显微镜很好地表征,并成功地用于抗前列腺特异性抗原(prostate-specific antigen,PSA)固定和PSA检测, 同时测定了PSA与其抗体之间的动力学和亲和常数。     

Purification and Characterization of a Soluble and a Particulate Glutamate Dehydrogenase from Rat Brain

Glutamate dehydrogenase (GDH) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low-speed pellet (480 g for 10 min). Approximately 60% of the high-speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similar Km values for the two GDH fractions for the substrates alpha-ketoglutarate, ammonia, and glutamate; however, there were small but significant differences in Km values for NADH and NADPH. Although the allosteric stimulation by ADP and L-leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mM ADP, with apparent Ki values of 18.5 and 9.0 microM GTP for the soluble and particulate fractions, respectively. The Hill plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45 degrees C and pH 7.4 in 50 mM phosphate buffer.     

Characterization and analysis of nifH genes from Paenibacillus sabinae T27

Paenibacillus sabinae T27 (CCBAU 10202=DSM 17841) is a gram-positive, spore-forming diazotroph with high nitrogenase activities. Three nifH clusters were cloned from P. sabinae T27. Phylogenetic analysis revealed that NifH1, NifH2 and NifH3 cluster with Cyanobacterium. Each of the coding regions of nifH1, nifH2 and nifH3 from P. sabinae T27 under the control of the nifH promoter of Klebsiella pneumoniae could partially restore nitrogenase activity of K. pneumoniae nifH^? mutant strain 1795, which has no nitrogenase activity. This suggests that the three nifH genes from P. sabinae T27 have some function in nitrogen fixation. RT-PCR showed that all three nifH genes were expressed under nitrogen-fixing growth conditions. Using promoter vectors which have promoterless lacZ gene, three putative promoter regions of nifH genes were identified.