氧化葡萄糖杆菌合成右旋糖酐糊精酶的pH两阶段控制策略

为了进一步提高氧化葡萄糖杆菌右旋糖酐糊精酶的产量,在3 L发酵罐水平上考察了pH(3.5?6.0)对菌体生长和产酶的影响。基于不同pH发酵过程中菌体生长及产物合成的变化,确定了pH两阶段控制策略,即0?6 h时控制pH 5.0,6 h后将pH调至4.0。通过采用这一优化策略,右旋糖酐糊精酶酶活有了较大的提高,可达4.03 U/mL,比不控制pH模式下提高了38.5%,是摇瓶水平的12.5倍,同时发酵时间从47 h缩短为15 h。     

Adsorptive bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003

An integrated bioprocess for the production of glycolic acid from ethylene glycol with Gluconobacter oxydans DSM 2003 and in situ product removal were investigated. A slight substrate inhibition was observed as substrate concentration was above 20 g/l and the product inhibition was much stronger. Bioconversion of glycolic acid is an end-product-inhibited reaction. In order to increase the productivity of glycolic acid and reduce the end-product inhibition of bioconversion, an adsorptive bioconversion for glycolic acid production from ethylene glycol catalyzed by resting cells of G. oxydans DSM 2003, was developed by using anion exchange resin D315 as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2 g/l, compared to 74.5 g/l obtained from a conventional fed-batch mode.     

Dextran dextrinase and dextran of Gluconobacter oxydans

Certain strains of Gluconobacter oxydans have been known since the 1940s to produce the enzyme dextran dextrinase (DDase; EC2.4.1.2)—a transglucosidase converting maltodextrins into (oligo)dextran. The enzyme catalyses the transfer of an α1,4 linked glucosyl unit from a donor to an acceptor molecule, forming an α1,6 linkage: consecutive glucosyl transfers result in the formation of high molecular weight dextran from maltodextrins. In the early 1990s, the group of K. Yamamoto in Japan revived research on DDase, focussing on the purification and characterisation of the intracellular DDase produced by G. oxydans ATCC 11894. More recently, this was taken further by Y. Suzuki and coworkers, who investigated the properties and kinetics of the extracellular DDase formed by the same strain. Our group further elaborated on fermentation processes to optimise DDase production and dextran formation, DDase characterisation and its use as a biocatalyst, and the physiological link between intracellular and extracellular DDase. Here, we present a condensed overview of the current scientific status and the application potential of G. oxydans DDase and its products, (oligo)dextrans. The production of DDase as well as of dextran is first described via optimised fermentation processes. Specific assays for measuring DDase activity are also outlined. The general characteristics, substrate specificity, and mode of action of DDase as a transglucosidase are described in detail. Two forms of DDase are produced by G. oxydans depending on nutritional fermentation conditions: an intracellular and an extracellular form. The relationship between the two enzyme forms is also discussed. Furthermore, applications of DDase, e.g. production of (oligo)dextran, transglucosylated products and speciality oligosaccharides, are summarized.     

应用细胞透性化技术快速提取氧化葡萄糖杆菌胞内右旋糖酐糊精酶

将细胞透性化技术应用到右旋糖酐酶的提取检测过程中,根据单因素实验和正交试验设计确定了从氧化葡萄糖杆菌(Gluconobacter oxydans)细胞中提取胞内右旋糖酐糊精酶(DDase)的最佳方法:甘氨酸(Gly)质量分数15%,Triton X-100体积分数1%,菌悬液OD600为0.5~6.0,pH为6.81,冰浴处理时间1 h,透性化试剂的提取效率可达到酶活最大值的90%以上。与超声波细胞破碎法相比,该方法条件温和,酶的释放率较高并易于大量试样的平行实验操作。     

Functions of membrane-bound alcohol dehydrogenase and aldehyde dehydrogenase in the bio-oxidation of alcohols in Gluconobacter oxydans DSM 2003

In this study a new insight was provided to understand the functions of membrane-bound alcohol dehydrogenase (mADH) and aldehyde dehydrogenase (mALDH) in the bio-oxidation of primary alcohols, diols and poly alcohols using the resting cells of Gluconobacter oxydans DSM 2003 and its mutant strains as catalyst. The results demonstrated that though both mADH and mALDH participated in most of the oxidation of alcohols to their corresponding acid, the exact roles of these enzymes in each reaction might be different. For example, mADH played a key role in the oxidation of diols to its corresponding organic acid in G. oxydans, but it was dispensable when the primary alcohols were used as substrates. In contrast to mADH, mALDH appears to play a relatively minor role in organic acid-producing reactions because of the possible presence of other isoenzymes. Aldehydes were, however, found to be accumulated in the mALDH-deficient strain during the oxidation of alcohols.     

Asymmetric oxidation by Gluconobacter oxydans

Asymmetric oxidation is of great value and a major interest in both research and application. This review focuses on asymmetric oxidation of organic compounds by Gluconobacter oxydans. The microbe can be used for bioproduction of several kinds of important chiral compounds, such as vitamin C, 6-(2-hydroxyethyl)amino-6-deoxy-α-l-sorbofuranose, (S)-2-methylbutanoic acid, (R)-2-hydroxy-propionic acid and 5-keto-d-gluconic acid. Characteristics of the bacteria and research progress on the enantioselective biotransformation process are introduced.     

Characterization of Gluconobacter oxydans immobilized in calcium alginate

Summary Gluconobacter oxydans cells were immobilized in calcium alginate and the preparation was used for the oxidation of glycerol to dihydroxyacetone. The characterization was done according to the guidelines given by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The pH optimum of the preparation was found to be 5.0 and the temperature optimum was 40°C. However, the operational stability was better at 30°C. The glycerol concentration required to obtain half the maximal reaction rate was about 5 mM for both immobilized and free cells. At low concentrations of glycerol and high concentrations of dihydroxyacetone a slight inhibition was noted. No loss of activity of the immobilized preparation was observed after storage for 68 days at +4°C. Investigation of the operational stability revealed a half-life of 5 days. Studies of the influence of particle size and cell densities as well as that of oxygen concentration revealed that the oxygen supply was the rate limiting step.     

Biotransformation of patulin by Gluconobacter oxydans

A bacterium isolated from patulin-contaminated apples was capable of degrading patulin to a less-toxic compound, ascladiol. The bacterium was identified as Gluconobacter oxydans by 16S rRNA gene sequencing, whereas ascladiol was identified by liquid chromatography-tandem mass spectrometry and proton and carbon nuclear magnetic resonance. Degradation of up to 96% of patulin was observed in apple juices containing up to 800 microg/ml of patulin and incubated with G. oxydans.     

Biotransformation of Patulin by Gluconobacter oxydans

A bacterium isolated from patulin-contaminated apples was capable of degrading patulin to a less-toxic compound, ascladiol. The bacterium was identified as Gluconobacter oxydans by 16S rRNA gene sequencing, whereas ascladiol was identified by liquid chromatography-tandem mass spectrometry and proton and carbon nuclear magnetic resonance. Degradation of up to 96% of patulin was observed in apple juices containing up to 800 μg/ml of patulin and incubated with G. oxydans.     

A study of dextran production from maltodextrin by cell suspensions of Gluconobacter oxydans NCIB 4943

This study investigated dextran synthesis from a commercial maltodextrin substrate using cell suspensions of G. oxydans NCIB 4943 as catalysts. Experiments were arranged according to a central composite statistical design. The effects of substrate concentration (10-100 g l-1), cell concentration (0.32-32.0 g wet weight l-1), time of reaction (8-48 h) and pH (3.5-5.5), each at three levels, on dextran yield and dextran molecular weight (MW), were investigated. Response surface methodology was used to assess factor interactions, and empirical models describing the two responses were fitted. Most of the variance in dextran yield could be explained by the fitted model (R2 = 0.96). Dextran yield ranged from 1.21 to 41.69%. The presence of significant negative quadratic effects of cell concentration and time indicated that dextran yield reached a plateau and thus, optimum levels of cell concentration and time could be identified to maximize dextran yield. Dextran MW ranged from 6.6 to 38 kDa and was characterized by the significant interactions of reaction time with substrate concentration and cell concentration. The model, however, could account for only 60% of the variance in dextran MW. Possible reasons for this are discussed.     

Determining different impact factors on the xylonic acid production using Gluconobacter oxydans DSM 2343

Xylonic acid is a promising compound for the substitution of gluconic acid. Gluconobacter oxydans DSM 2343 has proven to be a highly potent biocatalyst for the conversion of xylose to xylonic acid.In the present study, different nitrogen sources for the growth of G. oxydans and subsequent xylonic acid production were investigated for the first time with minimal medium. Application of 0.32 g/L glutamate supplemented with 0.15 g/L ammonium sulfate as a cheap nitrogen source enabled a xylonic acid productivity of 2.92 g/(Lh) which is similar to findings involving a complex medium (3.20 g/(Lh)). The study further investigated the impact of the xylose source on the growth and production of G. oxydans. Dose-response curves confirmed that G. oxydans is mainly insensitive towards the main inhibitory compounds, acetate and hydroxymethylfurfural, up to a concentration of 5 g/L and 2.5 g/L, respectively. However, batch investigations indicated that substitution of 25 % of the pure xylose with hemicellulosic xylose resulted in a xylonic acid yield of 90 % compared to the control approach without hemicellulosic xylose. The feeding of hemicellulosic xylose in a pulsed fed-batch mode even enabled the use of 50 g/L demonstrating that the proper selection of a feeding strategy for the hemicellulosic xylose greatly improves the production of xylonic acid.     

基于重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌

吡咯喹啉醌(Pyrroloquinoline quinone,PQQ)是一种重要的氧化还原酶辅基,具有多种生理生化功能,在食品、医药卫生及农业等领域具有广泛的应用。文中采用重组氧化葡萄糖酸杆菌生物合成吡咯喹啉醌。首先构建丙酮酸脱羧酶基因GOX1081敲除的重组菌G. oxydans T1,减少副产物乙酸的形成。然后利用筛选的内源性组成型启动子P0169融合表达pqqABCDE基因簇及tldD基因,构建重组菌G. oxydans T2。最后对发酵培养基添加物和发酵条件进行优化。结果显示重组菌G. oxydans T1、G. oxydans T2生物量较野生菌分别提高43.02%和38.76%,而PQQ的产量分别是野生菌的4.82倍和20.5倍。进一步优化G. oxydans T2碳源及培养条件,最终PQQ产量达(51.3241±0.8997)mg/L,是野生菌的345.62倍。通过基因工程手段,可以有效提高氧化葡萄糖酸杆菌的生物量和合成PQQ的产量,为改善PQQ生物合成效率奠定基础。     

Aldehyde production by alcohol oxidation with Gluconobacter oxydans

The microbial oxidation of various primary alcohols to the corresponding aldehydes has been investigated. A focused screening performed amongst some acetic acid bacteria showed that a newly isolated strain of Gluconobacter oxydans oxodizes various short-chain aliphatic alcohols to the corresponding aldehydes with negligible acid production. 3-Methyl-1-butanol (isoamyl alcohol) proved to be the better substrate with high yields (more than 90%) without by-product formation. This biotransformation also occurs with continuous or semicontinuous addition of substrate since the volatile product is removed from the medium under vigorous aeration conditions. Product recovery is attained either by the use of cold traps or by reversible complex formation.     

Characterization of enzymes involved in the central metabolism of Gluconobacter oxydans

Gluconobacter oxydans is an industrially important bacterium that lacks a complete Embden–Meyerhof pathway (glycolysis). The organism instead uses the pentose phosphate pathway to oxidize sugars and their phosphorylated intermediates. However, the lack of glycolysis limits the amount of NADH as electron donor for electron transport phosphorylation. It has been suggested that the pentose phosphate pathway contributes to NADH production. Six enzymes predicted to play central roles in intracellular glucose and gluconate flux were heterologously overproduced in Escherichia coli and characterized to investigate the intracellular flow of glucose and gluconates into the pentose phosphate pathway and to explore the contribution of the pentose phosphate pathway to NADH generation. The key pentose phosphate enzymes glucose 6-phosphate dehydrogenase (Gox0145) and 6-phosphogluconate dehydrogenase (Gox1705) had dual cofactor specificities but were physiologically NADP- and NAD-dependent, respectively. Putative glucose dehydrogenase (Gox2015) was NADP-dependent and exhibited a preference for mannose over glucose, whereas a 2-ketogluconate reductase (Gox0417) displayed dual cofactor specificity for NAD(P)H. Furthermore, a putative gluconokinase and a putative glucokinase were identified. The gluconokinase displayed high activities with gluconate and is thought to shuttle intracellular gluconate into the pentose phosphate pathway. A model for the trafficking of glucose and gluconates into the pentose phosphate pathway and its role in NADH generation is presented. The role of NADPH in chemiosmotic energy conservation is also discussed.     

Oxidative d-xylose metabolism of Gluconobacter oxydans

Summary Gluconobacter oxydans subsp. suboxydans ATCC 621 oxidizes d-xylose to xylonic acid very efficiently, although it cannot grow on xylose as sole carbon source. The oxidation of xylose was found to be catalyzed by a membrane-bound xylose dehydrogenase. The xylono--lactone formed in the oxidation reaction is subsequently hydrolyzed to xylonic acid by a -lactonase. The complete oxidation pathway of d-xylose in G. oxydans is evidently located in the periplasmic space.     

Asymmetric reduction of diketones by two Gluconobacter oxydans oxidoreductases

Two genes encoding recombinant cytosolic oxidoreductases from Gluconobacter oxydans, gox0313 and gox0646, were heterologously expressed in Escherichia coli and the resulting proteins were purified and characterized. GOX0313 was identified as a medium-chain alcohol dehydrogenase, whereas GOX0646 was classified as a ketocarbonyl reductase. GOX0313 had a broad substrate spectrum and oxidized various primary alcohols. However, GOX0313 had a preference for substrate reduction, reducing many aldehydes and α-diketones. In contrast, GOX0646 had a narrow substrate spectrum and reduced α-diketones, preferring short-chain ketocarbonyls. Both enzymes regio- and stereospecifically reduced α-diketones to the corresponding (S)-hydroxy ketone, as shown by NMR. These products are difficult to produce chemically, requiring complicated protecting group chemistry. Furthermore, hydroxy ketones find industrial application in the production of pheromones, fragrances, flavors, and pharmaceuticals. Hence, these enzymes are interesting biocatalysts for the production of enantiomerically pure building blocks that are difficult to prepare chemically.     

Inhibitory effects of glycerol on Gluconobacter oxydans

Summary The inhibitory effects of glycerol on Gluconobacter oxydans were measured separately. The kinetics of oxygen uptake rate representing the DHA production, the CO₂ evolution rate representing the assimilation of the product, and the specific growth rate were mathematically modelled. Glycerol does not inhibit DHA formation and CO₂-evolution.now: Institut für Biotechnologie, TU Graz, Petersgasse 12, 8010 Graz, Austria     

In vitro three-stage continuous fermentation of gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from maltodextrin as the source, were evaluated for their fermentability by the human colonic microflora. The selectivity of growth of desirable bacteria in the human colon was studied in a three-stage continuous model of the human large intestine. Populations of bacteria, and their fluctuations as a response to the fermentation, were enumerated using fluorescent in situ hybridization (FISH). The gluco-oligosaccharides resulted in increases in numbers of bifidobacteria and the Lactobacillus/Enterococcus group in all 3 vessels of the system, representing the proximal, transverse and distal colonic areas. The prebiotic indices of the gluco-oligosaccharides were 2.29, 4.23 and 2.74 in V1, V2 and V3 respectively.     

Purification of a stereospecific 2-ketoreductase from Gluconobacter oxydans

The 2-ketoreductase from Gluconobacter oxydans (SC 13851) catalyzes the reduction of 2-pentanone to (S)-(+)-2-pentanol. The 2-ketoreductase was purified 295-fold to homogeneity from G. oxydans cell extracts. The purified 2-ketoreductase had a molecular mass of 29 kDa with a specific activity of 17.7 U/mg. (S)-(+)-2-pentanol was prepared on a pilot scale (3.2 kg of 2-pentanone input) using Triton X-100-treated G. oxydans cells. After 46 h, 1.06 kg (32.3 M%) of (S)-(+)-2-pentanol of >99% enantiomeric excess (ee) was produced. Journal of Industrial Microbiology & Biotechnology (2000) 25, 171–175. Received 01 May 2000/ Accepted in revised form 28 June 2000