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1.
Two cytosolic NADPH-dependent carbonyl reductases from Gluconobacter oxydans 621H, Gox0644 and Gox1615, were heterologously produced in Escherichia coli. The recombinant proteins were purified to homogeneity and characterized. Gox0644 and Gox1615 were dimers with native molecular masses of 66.1 and 74.5 kDa, respectively. The enzymes displayed broad substrate specificities and reduced α-ketocarbonyls at the keto moiety most proximal to the terminus of the alkyl chain to produce alpha-hydroxy carbonyls, as demonstrated by NMR. With respect to stereoselectivity, protein Gox0644 specifically reduced 2,3-pentanedione to 2R-hydroxy-pentane-3-one, whereas Gox1615 produced 2S-hydroxy-pentane-3-one. Both enzymes also reduced 1-phenyl-1,2-propanedione to 2-hydroxy-1-phenylpropane-1-one, which is a key intermediate in the production of numerous pharmaceuticals, such as antifungal azoles and antidepressants. Gox0644 displayed highest activities with 2,3-diones, α-ketoaldehydes, α-keto esters, and 2,5-diketogluconate. Gox1615 was less active with these substrates, but displayed a broader substrate spectrum reducing a variety of α-diketones and aldehydes.  相似文献   

2.
Gluconobacter oxydans enable to oxidize sugars and polyols incompletely to corresponding materials with potential industrial applications, containing around 75 putative dehydrogenases. One of these putative dehydrogenases, Gox2181, was cloned and expressed in Escherichia coli BL21 (DE3), and its X-ray crystal structure was determined to a resolution of 1.8 Å. Gox2181 formed a homo-tetramer in the crystal that was coincident with the apparent molecular mass determined in the solution. Gox2181 displayed α/β-folding patterns, the conserved catalytic tetrad of Asn119-Ser147-Tyr162-Lys166, and the NAD-binding pocket, which aligned well with the ‘classical’ type of short-chain dehydrogenase/reductase (SDR) enzymes. Gox2181 was denoted SDR51C based on the SDR nomenclature system. The purified recombinant Gox2181 was demonstrated to be NAD(H)-dependent and active towards a wide range of substrates, including sugar alcohols, secondary alcohols, ketones, and ketoses. Among the substrates tested, Gox2181 displayed preference for secondary hydroxyl or carbonyl groups, showing low Km values with d-arabitol and butanedione.  相似文献   

3.
The versatile carbonyl reductases from Gluconobacter oxydans in the enantioselective reduction of ketones to the corresponding alcohols were exploited by genome search approach. All purified enzymes showed activities toward the tested ketoesters with different activities. In the reduction of 4-phenyl-2-butanone with in situ NAD(P)H regeneration system, (S)-alcohol was obtained with an e.e. of up to 100% catalyzed by Gox0644. Under the same experimental condition, all enzymes catalyzed ethyl 4-chloroacetoacetate to give chiral products with an excellent e.e. of up to 99%, except Gox0644. Gox2036 had a strict requirement for NADH as the cofactor and showed excellent enantiospecificity in the synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate. For the reduction of ethyl 2-oxo-4-phenylbutyrate, excellent e.e. (>99%) and high conversion (93.1%) were obtained by Gox0525, whereas the other enzymes showed relatively lower e.e. and conversions. Among them, Gox2036 and Gox0525 showed potentials in the synthesis of chiral alcohols as useful biocatalysts.  相似文献   

4.
Two cytosolic nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductases, Gox1899 and Gox2253, from Gluconobacter oxydans 621H were overproduced and purified from Escherichia coli. The purified proteins exhibited subunit masses of 26.4 (Gox1899) and 36.7 kDa (Gox2253). Both proteins formed homo-octamers exhibiting native masses of 210 and 280 kDa, respectively. The substrate spectra, optimal reaction conditions, and kinetic constants were determined for Gox1899 and Gox2253. Both enzymes efficiently catalyzed the reduction of medium/long-chain aldehydes. However, Gox1899 had a wider substrate spectrum and was more catalytically efficient. The best activity with Gox1899 was found for aliphatic aldehydes of C6-C10. In contrast, Gox2253 had a limited substrate spectrum and reduced octanal, nonanal, and decanal. Both enzymes were unable to oxidize primary alcohols. Aldehyde removal may be of particular importance for Gluconobacter because the membrane-bound alcohol dehydrogenase rapidly oxidizes short to long-chain alcohols, and large quantities of aldehydes could enter the cell, making detoxification necessary.  相似文献   

5.
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.  相似文献   

6.
A gene encoding an NADH-dependent short-chain dehydrogenase/reductase (gox2036) from Gluconobacter oxydans 621H was cloned and heterogeneously expressed in Escherichia coli. The protein (Gox2036) was purified to homogeneity and biochemically characterized. Gox2036 was a homotetramer with a subunit size of approximately 28 kDa. Gox2036 had a strict requirement for NAD+/NADH as the cofactor. Gox2036 displayed preference for oxidation of secondary alcohols and 2,3-diols as well as for reduction of α-diketones, hydroxy ketones, α-ketoesters, and β-ketoesters. However, Gox2036 was poorly active on 1,2-diols and acetoin and showed no activity on primary alcohols, polyols, and aldehydes. The optimum pH values for the oxidation and reduction reactions were 9 and 6, respectively. Gox2036 was highly selective in the reduction of various β-ketones and β-ketoesters. Among the substrates tested, ethyl 4-chloro acetoacetate was reduced to ethyl (R)-4-chloro-3-hydroxybutanoate ester with an excellent conversion yield of 96.9 % and optical purity of >99 % e.e. using an efficient in situ NADH-recycling system involving glucose and a glucose dehydrogenase from Bacillus subtilis (BsGDH).  相似文献   

7.
Activities and a few properties of alkaline phosphatase and 5’-nucleotidase were compared in the developing human placenta. Both the enzymes were mostly membrane-bound and displayed similar developmental patterns with the highest activities at 24/26 weeks of the placenta. L-Phenylalanine, L-tryptophan and L-leucine were inhibitors of alkaline phosphatase, whereas they had no effect on the 5’-nucleotidase. Alkaline phosphatase from a late stage of gestation appeared to be almost heat-stable. An appreciable part of 5’-nucleotidase was also resistant to heat inactivation and this fraction varied with gestational age of the tissue. For both the enzymes, Vmax changed without alteringK m values with periods of gestation. Ca2+, Mg2+ and Mn2+ ions stimulated the alkaline phosphatase activity and Hg2+, Zn2+, Cu2+, Ni2+ were inhibitory. 5’-Nucleotidase was not activated by any of these cations. EDTA and Concanavalin A inhibited both the enzymes, although the extent of inhibition was different and also varied with gestation.  相似文献   

8.
The study was targeted to saccharify foodwastes with the cellulolytic and amylolytic enzymes obtained from culture supernatant ofTrichoderma harzianum FJ1 and analyze the kinetics of the saccharification in order to enlarge the utilization in industrial application.T. harzianum FJ1 highly produced various cellulolytic (filter paperase 0.9, carboxymethyl cellulase 22.0, β-glucosidase 1.2, Avicelase 0.4, xylanase 30.8, as U/mL-supernatant) and amylolytic (α-amylase 5.6, β-amylase 3.1, glucoamylase 2.6, as U/mL-supernatant) enzymes. The 23–98 g/L of reducing sugars were obtained under various experimental conditions by changing FPase to between 0.2–0.6 U/mL and foodwastes between 5–20% (w/v), with fixed conditions at 50°C, pH 5.0, and 100 rpm for 24 h. As the enzymatic hydrolysis of foodwastes were performed in a heterogeneous solid-liquid reaction system, it was significantly influenced by enzyme and substrate concentrations used, where the pH and temperature were fixed at their experimental optima of 5.0 and 50°C, respectively. An empirical model was employed to simplify the kinetics of the saccharification reaction. The reducing sugars concentration (X, g/L) in the saccharification reaction was expressed by a power curve (X=K·t n) for the reaction time (t), where the coefficient,K andn, were related to functions of the enzymes concentrations (E) and foodwastes concentrations (S), as follow:K=10.894 Ln(E·S 2)-56.768,n=0.0608·(E/S)−0.2130. The kinetic developed to analyze the effective saccharification of foodwastes composed of complex organic compounds could adequately explain the cases under various saccharification conditions. The kinetics results would be available for reducing sugars production processes, with the reducing sugars obtained at a lower cost can be used as carbon and energy sources in various fermentation industries.  相似文献   

9.
Irrigation of farm field with water mixed with pulp and paper mill effluent from Century pulp and paper mill in Uttrakhand state of India for over last 25 years in succession increased streptomycetes population (120 × 105) compared to the fresh water irrigated fields (48 × 103 in WIF). Denaturing gradient gel electrophoresis, amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, BIOLOG™ substrate usage, production of extracellular enzymes (xylanase and cellulase) and plant growth promoting attributes were applied to monitor changes in genetic and metabolic diversity of streptomycetes. Significant variation was observed for production of extracellular enzymes, Indolic compounds, siderophore and P-solubilisation among isolates. Metabolic substrate usage of Streptomyces isolates was evaluated using the BIOLOG™ GP2 plates and unique carbon substrate usage profiles were observed. Based on 16S rRNA gene sequencing, the isolates were identified as Streptomyces variabilis, Streptomyces spp. S. glaucescens, S. viridochromogenes, S. cinnabarinus, S. aburaviensis, S. viridis, S. xylophagus, S. macrosporeus, S. thermocarboxydus, and S. albogriseolus. The diversity index parameters like Shannon index, reciprocal of Simpson’s index (1/D), and Pielou index of evenness based on ARDRA revealed that streptomycetes community in effluent irrigated field (EIF) was more diverse. DGGE profiles of Streptomyces specific 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar in both soils.  相似文献   

10.
DNA photolyases (EC 4.1.99.3) are enzymes that catalyze photoreversion of cis,syn-thymine photodimer (T[c,s]T), which is one of major photolesion products in DNA, by utilizing UV light. In this work, we have designed and synthesized Zn2+–1,4,7,10-tetraazacyclododecane complexes bearing a lumiflavin and l-tryptophan (ZnL3) or l-phenylalanine (ZnL4) as artificial DNA photolyases. We have found that (ZnL3)red, whose flavin unit was reduced in situ by Na2S2O4, accelerates the photoreversion of T[c,s]T utilizing near-UV light in aqueous solution at pH 7.6 and 11. Interestingly, more efficient photoreversion of T[c,s]T was achieved by UV irradiation of an oxidized form of ZnL3 [(ZnL3)ox] in the presence of an excess amount of Et3N at pH 11. UV–vis and fluorescence measurements and action spectra showed that an oxidized form of flavin of (ZnL3)ox was photoreduced by Et3N into its reduced form (ZnL3)red, which promoted the photoreduction of T[c,s]T. Comparison of the photochemical properties of ZnL3 with those of ZnL4 suggested that a tryptophan unit in ZnL3 contributed to the stabilities of the flavin through intramolecular photoinduced electron transfer.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.  相似文献   

11.
Aplanospores ofHaematococcus pluvialis MUR 145 contained 0.7% carotenoids (dry wt. basis) consisting of β,β-carotene (5% of total carotenoid), echinenone (4%), canthaxanthin (4%), (3S,3′S)-astaxanthin diester (34%), (3S,3′S)-astaxanthin monoester (46%), (3S,3′S)-astaxanthin (1%) and (3R,3′R,6′R)-lutein (6%). The astaxanthin esters were examined by TLC and HPLC and VIS,1H NMR and mass spectra recorded. Their chirality was determined by the camphanate method (Vecchi & Müller, 1979) after anaerobic hydrolysis. The tough cell wall of the aplanospores required enzymatic treatment prior to pigment extraction. The potential use of this microalga as a feed ingredient in aquaculture is discussed briefly.  相似文献   

12.
The monoterpene lilac aldehyde (=2‐(5‐ethenyl‐5‐methyloxolan‐2‐yl)propanal) is a widespread flower scent. Lilac aldehyde is emitted in high amounts from nocturnal plant species, and it is highly attractive to nocturnal moth pollinators, such as Hadena bicruris, the pollinating seed predator of Silene latifolia. Lilac aldehyde possesses three stereogenic centers and can occur in eight stereoisomers which induce different antennal responses in H. bicruris. The distribution pattern of stereoisomers differs among plant species, and if H. bicruris has different receptors for detecting different isomers, it may use these differences to discriminate flowers of S. latifolia hosts from flowers of non‐host plants. To investigate the question whether the moths have in their antennae one olfactory receptor or several different receptors for the detection of the single lilac aldehyde isomers, (2S,2′S,5′S)‐lilac aldehyde was diastereoselectively synthesized. (2S,2′S,5′S)‐Lilac aldehyde and its isomeric mixture were tested electrophysiologically on antennae of H. bicruris. The results displayed antennal responses, which are characteristic for a single receptor that detects the different lilac aldehyde isomers.  相似文献   

13.
A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac5cdOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and 1H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac5cdOM]‐(Aib)4‐OEt ( 1) and Cbz‐(Aib)4‐[(S,S)‐Ac5cdOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 310‐helical structure was present in 1 and that a right‐handed (P) 310‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

14.
A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44°C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 ± 2.15 U mg−1) and 2′,3′,4′,5′,6′-pentafluoroacetophenone (8.57 ± 0.49 U mg−1) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2′,3′,4′,5′,6′-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).  相似文献   

15.
Di-nor-benzofuran neolignan aldehydes, Δ7-3,4-methylenedioxy-3′-methoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal A) 1, Δ7-3,4,5,3′-tetramethoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal B) 2, and macrophyllin-type bicyclo[3.2.1]octanoid neolignans (7R, 8R, 3′S, 4′S, 5′R)-Δ8′-4′-hydroxy-5′-methoxy-3,4-methylenedioxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol A) 3, (7R, 8R, 3′S, 4′S, 5′R)-Δ8′-4′-hydroxy-3,4,5′-trimethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol B) 4, (7R, 8R, 3′S, 4′S, 5′R)-Δ8′-4′-hydroxy-3,4,5,5′-tetramethoxy-2′,3′,4′,5′-tetrahydro-2′-oxo-7.3′,8.5′-neolignan (ocophyllol C) 5, as well as 2′-epi-guianin 6 and (+)-licarin B 7, were isolated and characterized from leaves of Ocotea macrophylla (Lauraceae). The structures and configuration of these compounds were determined by extensive spectroscopic analyses. Inhibition of platelet activating factor (PAF)-induced aggregation of rabbit platelets were tested with neolignans 1–7. Although compound 6 was the most potent PAF-antagonist, compounds 3–5 showed some activity.  相似文献   

16.
Summary A comparison of EST-5 grain esterase phenotypes from wheat-alien amphiploid, addition and substitution genotypes, resolved by flat-bed isoelectric focusing identified homoeologous Est-5 loci on chromosome 3H of Hordeum vulgare, 3Hch of H. chilense, 3Sb of Aegilops bicornis, 3S1 of Ae. sharonensis and Ae. longissima and 6R of Secale cereale and 6Rm of S. montanum. The Est-5 genes in alien species provide evidence for chromosome homoeology with wheat.  相似文献   

17.
Two novel Gram-positive actinobacteria, designated H97-3T and H83-5, were isolated from marine sediment samples and their taxonomic positions were investigated by a polyphasic approach. Both strains formed vegetative hyphae in the early phase of growth but the hyphae eventually fragmented into coccoid cells. The peptidoglycan type was found to be A4α. The predominant menaquinone was MK-9(H4), and the major fatty acids were anteiso-C15:0, anteiso-C17:0 and C16:0. The DNA G+C content was 74.0–74.9 mol %. 16S rRNA gene sequencing analysis revealed that strains H97-3T and H83-5 represented novel members of the family Cellulomonadaceae. Their nearest phylogenetic neighbours were the members of the genus Oerskovia, with a similarity of 98.3–98.4 %. However, strains H97-3T and H83-5 were distinguishable from the members of the genus Oerskovia and the other genera of the family Cellulomonadaceae in terms of chemotaxonomic characteristics and phylogenetic relationship. The result of the DNA–DNA hybridization indicated that strains H97-3T and H83-5 belonged to the same species. Therefore, strains H97-3T and H83-5 represent a novel genus and species of the family Cellulomonadaceae, for which the name Sediminihabitans luteus gen. nov., sp. nov. is proposed. The type strain of S. lutes is H97-3T (=NBRC 108568T = DSM 25478T).  相似文献   

18.
Enantiospecific microbial reduction of bicyclic ketones was described. Racemic Wieland–Miescher (1) and Hajos–Parrish (2) ketones were used as substrates. In a 4-h biotransformation of Hajos–Parrish ketone (2) using the strain of Didymosphaeria igniaria an optically pure ketone (R)-2 was obtained, whereas the (S)-2 ketone underwent reduction to (4aS,5S)-4 alcohol with 100% of enantiomeric excess and with over 60% of diastereoisomeric excess. Jones oxidation of the alcohol obtained in the biotransformation gave an optically pure ketone (S)-2. Enzymatic system of Coryneum betulinum reduced the (R)-2 ketone to (4aR,5S)-4 alcohol with a high enantiomerical purity in a 6-day reaction. Wieland-Miescher (1) ketone was transformed by these microorganisms in an analogous way, but the reaction times were longer.  相似文献   

19.
Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a [3Fe–4S]+ cluster accounting for 3–4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe–4S]+ cluster is observed that accounts for 34–45% of total iron. Addition of S-adenosylmethionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-l-methionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 ± 0.6 nmol min−1 mg−1, whereas no repair was observed for the 5S diastereomer.  相似文献   

20.
Two new sterols 1 and 2 and five known ones 3 – 7 were isolated for the first time from the fruiting bodies of Cortinarius glaucopus. Their structures were established by 1‐ and 2D‐NMR spectra and HR‐FABS‐MS. The relative configuration of 1 was firmly determined by comparison of the observed 1H–1H couplings and NOESY correlations, with those predicted for the computed geometries of the conformers. Calculations were performed by means of DFT with the B3LYP functional at 6‐31 + G(d,p) level of theory, in CHCl3 as the solvent. The structures of the new ergosterol derivatives, called glaucoposterol A ( 1 ) and B ( 2 ), were thus established as (3S,5R,7R,10R,13R,17R,20S,22R,23R,24R)‐5,6‐epoxy‐3,7,23‐trihydroxystrophast‐8‐en‐14‐one and (22E,3S,5S,9S,10R,13R,17R,20R,24R)‐3,5‐dihydroxyergosta‐6,8(14),22‐trien‐15‐one, respectively. Moreover, the configuration of known strophasterol C ( 3 ) was determined as (3S,5R,6S,7R,10R,13R,17R,20S,22S,24R). Glaucoposterol A ( 1 ) and strophasterol C ( 3 ) represent the second finding in nature of steroids with the rare strophastane skeleton.  相似文献   

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