共查询到20条相似文献,搜索用时 15 毫秒
1.
Yablochkova E. N. Bolotnikova O. I. Mikhailova N. P. Nemova N. N. Ginak A. I. 《Microbiology》2003,72(4):414-417
The activity and the cofactor specificity of xylose reductase and xylitol dehydrogenase were studied in extracts of yeasts from the genera Candida, Kluyveromyces, Pachysolen, Pichia,and Torulopsis grown under microaerobic conditions. It was found that xylitol dehydrogenase in all of the yeast species studied is specific for NAD+; xylose reductase in the xylitol-producing species C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, Kl. fragilis, Kl. marxianus, P. guillermondii, andT. molishiama is specific for NADPH; and xylose reductase in the ethanol-producing species P. stipitis, C. shehatae, and Pa. tannophilus is specific for both NADPH and NADH. 相似文献
2.
M. A. Alexander V. W. Yang T. W. Jeffries 《Applied microbiology and biotechnology》1988,29(2-3):282-288
Summary
Candida shehatae exhibits different fermentative capacities when grown under different aeration conditions. These studies investigated the titers of xylose reductase, xylitol dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase in crude extracts ofCandida shehatae grown in continuous culture with various specific aeration rates. Carbon source, aeration rate, dilution rate and temperature were examined as variables. Xylose reductase and xylitol dehydrogenase were induced by xylose and were largely absent in glucose-grown cells. Alcohol dehydrogenae levels were higher in glucose-grown cells than in xylose-grown cells. The levels of this enzyme also correlated with the fermentative character of metabolism, having a low value under fully aerobic conditions, a high value under anaerobic conditions, and intermediate levels under various semi-aerobic conditions. Temperature had no effect on any enzyme level over the range of 20–30°C.Maintained in cooperation with the University of Wisconsin-Madison 相似文献
3.
The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His6-tagged fusion protein. The recombinant XR had Km values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was
specific for d-xylese. 相似文献
4.
Yablochkova E. N. Bolotnikova O. I. Mikhailova N. P. Nemova N. N. Ginak A. I. 《Microbiology》2004,73(2):129-133
The activities of xylitol dehydrogenase and xylose reductase in the yeasts Candida shehatae, C. didensiae, C. intermediae, C. tropicalis, Kluyveromyces marxianus, Pichia stipitis, P. guillermondii, Pachysolen tannophilus, and Torulopsis molishiama were studied at different oxygen transfer rates (OTRs) to the fermentation medium (0, 5, and 140 mmol O2/(l h)). The activities of these enzymes were maximum in the yeasts P. stipitis and C. shehatae. The xylitol dehydrogenase of all the yeasts was NAD+-dependent, irrespective of the intensity of aeration. The xylose reductase of the yeasts C. didensiae, C. intermediae, C. tropicalis, Kl. marxianus, P. guillermondii, and T. molishiama was NADPH-dependent, whereas the xylose reductase of P. stipitis, C. shehatae, and Pa. tannophilus was specific for both NADPH and NADH. The effect of OTR on the activities of the different forms of xylitol dehydrogenase and xylose reductase in xylose-assimilating yeasts is discussed. 相似文献
5.
The relationship between the degree of aerobiosis, xylitol production and the initial two key enzymes of d-xylose metabolism were investigated in the yeasts Pichia stipitis, Candida shehatae and C. tenuis. Anoxic conditions severely curtailed growth and retarded ethanol productivity. This, together with the inverse relationship between xylitol accumulation and aeration level, suggested a degree of redox imbalance. The ratios of NADH- to NADPH-linked xylose reductase were similar in all three yeasts and essentially independent of the degree of aerobiosis, and thus did not correlate with their differing capacities for ethanol production, xylitol accumulation or growth under the different conditions of aerobiosis. Under anoxic conditions the enzyme activity of Pichia stipitis decreased significantly, which possibly contributed to its weaker anoxic fermentation of xylose compared to C. shehatae. 相似文献
6.
Summary Three different NAD(P)H-FMN reductases were extracted from Beneckea harveyi MB-20 cells and separated by DEAE-Sephadex A50 column chromatography. Further purification was achieved by affinity chromatography. In determinations of Km values for NADH, NADPH, and FMN, these three reductases exhibited different specificities and kinetic parameters. One reductase utilizes NADH, whereas a second one utilizes NADPH as the preferred substrate. The third, a newly described reductase species, exhibits about the same reaction rates with NADH and NADPH. The reaction mechanisms of the three enzyme forms have been deduced by steady state kinetic analysis. The highly pure (based on gel electrophoresis) NADPH-FMN reductase still exhibited a low (approximately 2%) activity for NADH, which activity was increased upon storage at 4° but suppressed completely by the replacement of the phosphate buffer with sodium citrate buffer. This high specificity of NADPH-FMN reductase for NADPH under these conditions is useful for the assay of NADPH, notably in systems coupled to bacterial luciferase. 相似文献
7.
Peter M. Bruinenberg Peter H. M. de Bot Johannes P. van Dijken W. Alexander Scheffers 《Applied microbiology and biotechnology》1984,19(4):256-260
Summary The kinetics and enzymology of d-xylose utilization were studied in aerobic and anaerobic batch cultures of the facultatively fermentative yeasts Candida utilis, Pachysolen tannophilus, and Pichia stipitis. These yeasts did not produce ethanol under aerobic conditions. When shifted to anaerobiosis cultures of C. utilis did not show fermentation of xylose; in Pa. tannophilus a very low rate of ethanol formation was apparent, whereas with Pi. stipitis rapid fermentation of xylose occurred. The different behaviour of these yeasts ist most probably explained by differences in the nature of the initial steps of xylose metabolism: in C. utilis xylose is metabolized via an NADPH-dependent xylose reductase and an NAD+-linked xylitol dehydrogenase. As a consequence, conversion of xylose to ethanol by C. utilis leads to an overproduction of NADH which blocks metabolic activity in the absence of oxygen. In Pa. tannophilus and Pi. stipitis, however, apart from an NADPH-linked xylose reductase also an NADH-linked xylose reductase was present. Apparently xylose metabolism via the NADH-dependent reductase circumvents the imbalance of the NAD+/NADH redox system, thus allowing fermentation of xylose to ethanol under anaerobic conditions. The finding that the rate of xylose fermentation in Pa. tannophilus and Pi. stipitis corresponds with the activity of the NADH-linked xylose reductase activity is in line with this hypothesis. Furthermore, a comparative study with various xylose-assimilating yeasts showed that significant alcoholic fermentation of xylose only occurred in those organisms which possessed NADH-linked aldose reductase. 相似文献
8.
Zhang B Zhang L Wang D Gao X Hong J 《Journal of industrial microbiology & biotechnology》2011,38(12):2001-2010
Kluyveromyces marxianus is thermotolerant yeast that is able to utilize a wider range of substrates and has greater thermal tolerance than most other
yeast species. K. marxianus can assimilate xylose, but its ability to produce ethanol from xylose in oxygen-limited environments is poor. In the present
study, the K. marxianus xylose reductase (KmXR) gene (Kmxyl1) was cloned and the recombinant enzyme was characterized to clarify the factors that limit xylose fermentation in K. marxianus NBRC1777. KmXR is a key enzyme in the xylose metabolism of K. marxianus, which was verified by disruption of the Kmxyl1 gene. The Km of the recombinant KmXR for NADPH is 65.67 μM and KmXR activity is 1.295 U/mg, which is lower than those of most reported
yeast XRs, and the enzyme has no activity with coenzyme NADH. This result demonstrates that the XR from K. marxianus is highly coenzyme specific; combined with the extremely low XDH activity of K. marxianus with NADP+, the limitation of xylose fermentation is due to a redox imbalance under anaerobic conditions and low KmXR activity. 相似文献
9.
Xylose reductase catalyzes the NAD(P)H-dependent reduction of xylose to xylitol and is essential for growth on xylose by yeasts. To understand the nature of coenzyme binding to the Pichia stipitis xylose reductase, we investigated the role of the strictly conserved Lys270 in the putative IPKS coenzyme binding motif by site-directed mutagenesis. The Lys270Met variant exhibited lower enzyme activity than the wild-type enzyme. The apparent affinity of the variant for NADPH was decreased 5–16-fold, depending on the substrate used, while the apparent affinity for NADH, measured using glyceraldehyde as the substrate, remained unchanged. This resulted in 4.3-fold higher affinity for NADH over NADPH using glyceraldehyde as the substrate. The variant also showed a 14-fold decrease in Km for xylose, but only small changes were observed in Km values for glyceraldehyde. The wild-type enzyme, but not the Lys270Met variant, was susceptible to modification by the Lys-specific pyridoxal 5′-phosphate. Results of our chemical modification and site-directed mutagenesis study indicated that Lys270 is involved in both NADPH and d-xylose binding in the P. stipitis xylose reductase. 相似文献
10.
George A. Khoury Hossein Fazelinia Jonathan W. Chin Robert J. Pantazes Patrick C. Cirino Costas D. Maranas 《Protein science : a publication of the Protein Society》2009,18(10):2125-2138
In this study we introduce a computationally‐driven enzyme redesign workflow for altering cofactor specificity from NADPH to NADH. By compiling and comparing data from previous studies involving cofactor switching mutations, we show that their effect cannot be explained as straightforward changes in volume, hydrophobicity, charge, or BLOSUM62 scores of the residues populating the cofactor binding site. Instead, we find that the use of a detailed cofactor binding energy approximation is needed to adequately capture the relative affinity towards different cofactors. The implicit solvation models Generalized Born with molecular volume integration and Generalized Born with simple switching were integrated in the iterative protein redesign and optimization (IPRO) framework to drive the redesign of Candida boidinii xylose reductase (CbXR) to function using the non‐native cofactor NADH. We identified 10 variants, out of the 8,000 possible combinations of mutations, that improve the computationally assessed binding affinity for NADH by introducing mutations in the CbXR binding pocket. Experimental testing revealed that seven out of ten possessed significant xylose reductase activity utilizing NADH, with the best experimental design (CbXR‐GGD) being 27‐fold more active on NADH. The NADPH‐dependent activity for eight out of ten predicted designs was either completely abolished or significantly diminished by at least 90%, yielding a greater than 104‐fold change in specificity to NADH (CbXR‐REG). The remaining two variants (CbXR‐RTT and CBXR‐EQR) had dual cofactor specificity for both nicotinamide cofactors. 相似文献
11.
MAHENDRA K. MOHANTY FRISSELL R. HUNTER JOSEPH B. MYERS 《The Journal of eukaryotic microbiology》1977,24(2):335-340
SYNOPSIS. Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (Km) determined were as follows: NADH diaphorase, 350 μM; NADPH diaphorase, 200 μM; NADH cytochrome c reductase, 13 μM; NADPH cytochrome c reductase, 9 μM; NADH oxidase, 100 μM; NADPH oxidase 150 μM; NADH lipoyl dehydrogenase, 0.35 μM. Enzyme activities after storage at –5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles. 相似文献
12.
Jinxin Zhang Min Yang Shen Tian Yazhen Zhang Xiushan Yang 《Applied Biochemistry and Microbiology》2010,46(4):415-420
The inability oft Saccharomyces cerevisiae to utilize xylose is attributed to its inability to convert xylose to xylulose. Low xylose reductase (XR) and xylitol dehydrogenase
(XDH) activities in S. cerevisiae are regarded as the reason of blocking the pathway from xylose to xylulose. We had found that Candida shehatae could also be another source for XR gene except Pichia stipitis in the previous study. In this study, we tried to investigate if the expressed XR from C. shehatae could work with the over-expressed endogenous XDH together to achieve the same goal of converting xylose to ethanol in S. cerevisiae. The XR gene (XYL1) from C. shehatae and endogenous XDH gene (XYL2) were both cloned and over-expressed in host S. cerevisiae cell. The specific enzyme activities of XR and XDH were measured and the result of fermentation revealed that the new combination
of two enzymes from different sources other than P. stipitis could also coordinate and work with each other and confer xylose utilization ability to S. cerevisiae. 相似文献
13.
Cornelis Verduyn Johannes Frank Jzn Johannes P. van Dijken W.Alexander Scheffers 《FEMS microbiology letters》1985,30(3):313-317
Abstract Cell-free extracts of xylose-grown Pachysolen tannophilus exhibited xylose reductase activity with both NADPH and NADH. The ratio of the NADPH- and NADH-dependent activities varied with growth conditions. Affinity chromatography of cell-free extracts resulted in a separation of two xylose reductases. One was active with both NADPH and NADH, the other was specific for NADPH. Apart from this coenzyme specificity, the two enzymes also differed in their affinities for xylose and NADPH. The role of the two enzymes in xylose metabolism is discussed in relation to attempts to use P. tannophilus for the alcoholic fermentation of wood sugars. 相似文献
14.
Jin Hou Goutham N. Vemuri Xiaoming Bao Lisbeth Olsson 《Applied microbiology and biotechnology》2009,82(5):909-919
During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A
deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation.
However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor
imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the
native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains
was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO2 to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic
NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol
to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall,
although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of
NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated. 相似文献
15.
Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene encoding xylose reductase
(Texr) was isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli and purified to homogeneity. Texr encodes a 320 amino acid protein with a molecular weight of 36 kDa, which exhibited high sequence identity with other xylose
reductase sequences and was shown to be a member of the aldoketoreductase (AKR) superfamily with a preference for reduced
nicotinamide adenine dinucleotide phosphate (NADPH) as coenzyme. Given the potential application of xylose reductase enzymes
that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation
of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed mutagenesis. The TeXRK271R+N273D double mutant displayed an altered coenzyme preference with a 16-fold improvement in NADH utilization relative to the wild
type and therefore has the potential to reduce redox imbalance of xylose fermentation in recombinant S. cerevisiae strains. Expression of Texr was shown to be inducible by the same carbon sources responsible for the induction of genes encoding enzymes relevant to
lignocellulose hydrolysis, suggesting a coordinated expression of intracellular and extracellular enzymes relevant to hydrolysis
and metabolism of pentose sugars in T. emersonii in adaptation to its natural habitat. This indicates a potential advantage in survival and response to a nutrient-poor environment. 相似文献
16.
The activity and the cofactor specificity of xylose reductase and xylitol dehydrogenase were studied in extracts of yeasts from the genera Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis grown under microaerobic conditions. It was found that xylitol dehydrogenase in all of the yeast species studied is specific for NAD+; xylose reductase in the xylitol-producing species C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, Kl. fragilis, Kl. marxianus, P. guillermondii, and T. molishiama is specific for NADPH; and xylose reductase in the ethanol-producing species P. stipitis, C. shehatae, and Pa. tannophilus is specific for both NADPH and NADH. 相似文献
17.
Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K
m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K
m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells. 相似文献
18.
Background
Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP+ or NAD+ dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR) was previously shown to promote NADH dependent reduction of aromatic α-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR. 相似文献19.
Summary Induced wildtype cells ofA. nidulans rapidly lost NADPH — linked nitrate reductase activity when subjected to carbon and or nitrogen starvation. A constitutive mutant at the regulatory gene for nitrate reductase,nirA
c1, rapidly lost nitrate reductase activity upon carbon starvation. This loss of activity is thought to be due to a decrease in the NADPH concentration in the cells. Cell free extracts from wild-type cells grown in the presence of nitrate, rapidly lost their nitrate reductase activity when incubated at 25° C. NADPH prevented this loss of activity. Wildtype cells grown in the presence of nitrate and urea have a higher initial NADPH: NADP+ ratio and cell free extracts from such cells lost their nitrate reductase activity slower than extracts of cells grown with nitrate alone.The Pentose Phosphate Pathway mutant,pppB-1, had a lower NADPH concentration compared with the wildtype grown under the same conditions and cell free extracts lost their nitrate reductase activity more rapidly than the wildtype. Cell free extracts ofnirA
c-1 and a non-inducible mutant for nitrate reductase,nirA
--14, upon incubation lost little of their nitrate reductase activity. 相似文献
20.
《BBA》1987,893(3):386-397
Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q1. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant. 相似文献