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A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395, and its activity was studied in Escherichia coli. The 3.9-kb operon comprising three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EIIMtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD), and a mannitol operon repressor (MtlR). The transport of [3H]mannitol by the cloned mannitol operon in E. coli was 13.8 ± 1.4 nmol/min/mg protein. The insertional inactivation of EIIMtl abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram-positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon.  相似文献   

3.
To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and 13C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.  相似文献   

4.
The nutritionally versatile soil bacterium Acinetobacter baylyi ADP1 copes with salt stress by the accumulation of compatible solutes, a strategy that is widespread in nature. This bacterium synthesizes the sugar alcohol mannitol de novo in response to osmotic stress. In a previous study, we identified MtlD, a mannitol‐1‐phosphate dehydrogenase, which is essential for mannitol biosynthesis and which catalyses the first step in mannitol biosynthesis, the reduction of fructose‐6‐phosphate (F‐6‐P) to the intermediate mannitol‐1‐phosphate (Mtl‐1‐P). Until now, the identity of the second enzyme, the phosphatase that catalyses the dephosphorylation of Mtl‐1‐P to mannitol, was elusive. Here we show that MtlD has a unique sequence among known mannitol‐1‐phosphate dehydrogenases with a haloacid dehalogenase (HAD)‐like phosphatase domain at the N‐terminus. This domain is indeed shown to have a phosphatase activity. Phosphatase activity is strictly Mg2+ dependent. Nuclear magnetic resonance analysis revealed that purified MtlD catalyses not only reduction of F‐6‐P but also dephosphorylation of Mtl‐1‐P. MtlD of A. baylyi is the first bifunctional enzyme of mannitol biosynthesis that combines Mtl‐1‐P dehydrogenase and phosphatase activities in a single polypeptide chain. Bioinformatic analysis revealed that the bifunctional enzyme is widespread among Acinetobacter strains but only rarely present in other phylogenetic tribes.  相似文献   

5.
d-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO2 as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol L−1 and a production rate of 0.15 g mannitol L−1 day−1. This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO2 in cyanobacteria.  相似文献   

6.
Sorbitol is a sugar alcohol largely used in the food industry as a low-calorie sweetener. We have previously described a sorbitol-producing Lactobacillus casei (strain BL232) in which the gutF gene, encoding a sorbitol-6-phosphate dehydrogenase, was expressed from the lactose operon. Here, a complete deletion of the ldh1 gene, encoding the main l-lactate dehydrogenase, was performed in strain BL232. In a resting cell system with glucose, the new strain, named BL251, accumulated sorbitol in the medium that was rapidly metabolized after glucose exhaustion. Reutilization of produced sorbitol was prevented by deleting the gutB gene of the phosphoenolpyruvate: sorbitol phosphotransferase system (PTSGut) in BL251. These results showed that the PTSGut did not mediate sorbitol excretion from the cells, but it was responsible for uptake and reutilization of the synthesized sorbitol. A further improvement in sorbitol production was achieved by inactivation of the mtlD gene, encoding a mannitol-1-phosphate dehydrogenase. The new strain BL300 (lac::gutF Δldh1 ΔgutB mtlD) showed an increase in sorbitol production whereas no mannitol synthesis was detected, avoiding thus a polyol mixture. This strain was able to convert lactose, the main sugar from milk, into sorbitol, either using a resting cell system or in growing cells under pH control. A conversion rate of 9.4% of lactose into sorbitol was obtained using an optimized fed-batch system and whey permeate, a waste product of the dairy industry, as substrate.  相似文献   

7.
Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar-based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2-dependent CO2 reductase and furthermore belong to different classes of ‘Rnf’- and ‘Ech-acetogens’. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood–Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g−1 h−1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO-based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.  相似文献   

8.
With multiple applications in food, pharmaceutical, and chemical industries as antioxidant or nonmetabolizable sweetener; the bioproduction of d -mannitol is gaining global attention, especially with photosynthetic organisms as hosts. Considering the sustainability prospects, the current work encompasses metabolic engineering of a widely used cyanobacterial strain, Synechococcus elongatus PCC 7942, and two newly isolated fast-growing cyanobacterial strains; S. elongatus PCC 11801 and S. elongatus PCC 11802, for mannitol production. We engineered these strains with a two-step pathway by cloning genes for mannitol-1-phosphate dehydrogenase (mtlD) and mannitol-1-phosphatase (mlp), where the mtlD expression was under the control of different promoters from PCC 7942, namely, Prbc225, PcpcB300, PcpcBm1, PrbcLm17, and PrbcLm15. The strains were tested under the “switch conditions,” where the growth conditions were switched after the first 3 days, thereby resulting in differential promoter activity. Among the engineered strains of PCC 11801 and PCC 11802, the strains possessing Prbc225-mtlD module produced relatively high mannitol titers of 401 ± 18 mg/L and 537 ± 18 mg/L, respectively. The highest mannitol titer of 701 ± 15 mg/L (productivity 60 mg/L.d, yield 895 µM/OD730) was exhibited by the engineered strain of PCC 7942 expressing PcpcB300-mtlD module. It is by far the highest obtained mannitol yield from the engineered cyanobacteria.  相似文献   

9.
The bacterial gene mtlD, which encodes mannitol 1-phosphate dehydrogenase (E. C. 1. 1. 1. 17), was transformed into Arabidopsis thaliana and expressed under control of the CaMV 35S promoter. MtlD-transformants accumulated mannitol, a sugar alcohol that is not normally found in Arabidopsis. Amounts of soluble carbohydrates, sucrose, glucose, fructose, myo-inositol and mannitol were determined in different tissues of wild-type and transgenic plants. We estimated that less than 1& of the carbon assimilated was converted into mannitol by the transgenic plants. The establishment of individual transformed lines (after self-crossing three times) resulted in high and low mannitol-producing lines which were stably maintained. The presence of mannitol did not alter plant appearance or growth habit. When MtlD-expressing seeds and control seeds (T3 generation) were imbibed with solutions containing NaCl (range 0 to 400 mol m?3), transgenic seeds containing mannitol germinated in medium supplemented with up to 400 mol m?3 NaCl, while control seeds ceased germination at 100 mol m?3 NaCl. It is doubtful whether the ability to germinate in high salt was a result of an osmotic effect exerted by elevated levels of mannitol, considering that mannitol concentrations were in the mol m?3 range in seeds. A specific effect of polyols, for example on the integrity of subcellular membranes or enzymes, cannot be excluded.  相似文献   

10.
Cupriavidus necator H16 is one of the most researched carbon dioxide (CO2)-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO2 utilising precursors derived from the CBB cycle.  相似文献   

11.
To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.  相似文献   

12.
Sorghum bicolor L. Moench cv. SPV462 was transformed with the mtlD gene encoding for mannitol-1-phosphate dehydrogenase from E. coli with an aim to enhance tolerance to water deficit and NaCl stress. Transgene (pCAM mtlD) integration and expression were successfully confirmed by PCR, Southern, RT-PCR and Western analysis. Segregation analysis based on germination of T0 seed on hygromycin-supplemented medium revealed an expected Mendelian ratio 3:1 in lines 5, 72 and 75. Retention of leaf water content was remarkably higher in transgenic leaf segments when exposed to polyethylene glycol 8000 (−2.0 MPa), as compared to the untransformed controls. Another significant finding is that the transgenics maintained a 1.7 to 2.8 fold higher shoot and root growth, respectively, under NaCl stress (200 mM) when compared to untransformed controls. These results demonstrate that engineering mannitol biosynthetic pathway into sorghum can impart enhanced tolerance to water deficit and salinity.  相似文献   

13.
Several representatives of marine brown macroalgae (Phaeophyceae) including Fucus serratus L., Fucus spiralis L. and Fucus vesiculosus L. as well as Laminaria digitata (Huds.) Lamour., Laminaria hyperborea (Gunn.) Foslie and Laminaria saccharina (L.) Lamour. were investigated with particular regard to features of biosynthesis of the storage product mannitol. The respective catalytic system involved in the last step of mannitol formation, mannitol-1-phosphate dehydrogenase, appears to be a cytoplasmic enzyme as may be judged from the degree of correlation with the chloroplast key enzyme ribulose-1,5-bisphosphate carboxylase in different tissues of Laminaria digitata and Laminaria saccharina. Activity of mannitol-1-phosphate dehydrogenase in vitro is not affected by mannitol-l-phosphate or free mannitol, suggesting that mannitol biosynthesis in vivo) is mainly controlled by the environment and/or developmental stage. Certain inorganic ions such as NO3- (including K+) exert a strong influence on the activity of mannitol1-phosphate dehydrogenase thus suggesting that the intracellular pools of stored NO3- and mannitol are confined to spatially separated cellular compartments.  相似文献   

14.
A metabolic pathway, known as the mannitol cycle in fungi, has been identified as a new entity in the eulittoral mangrove red algaCaloglossa leprieurii (Montagne) J. Agardh. Three specific enzymes, mannitol-1-phosphate dehydrogenase (Mt1PDH; EC 1.1.1.17), mannitol-1-phosphatase (MtlPase; EC 3.1.3.22), mannitol dehydrogenase (MtDH; EC 1.1.1.67) and one nonspecific hexokinase (HK; EC 2.7.1.1) were determined and biochemically characterized in cell-free extracts. Mannitol-1-phosphate dehydrogenase showed activity maxima at pH 7.0 [fructose-6-phosphate (F6P) reduction] and pH 8.5 [oxidation of mannitol-1-phosphate (Mt1P)], and a very high specificity for both carbohydrate substrates. TheK m values were 1.4 mM for F6P, 0.09 mM for MOP, 0.020 mM for NADH and 0.023 mM for NAD+. For the dephosphorylation of MOP, MtlPase exhibited a pH optimum at 7.2, aK m value of 1.2 mM and a high requirement of Mg2+ for activation. Mannitol dehydrogenase had activity maxima at pH 7.0 (fructose reduction) and pH 9.8 (mannitol oxidation), and was less substrate-specific than Mt1PDH and MtlPase, i.e. it also catalyzed reactions in the oxidative direction with arabitol (64.9%), sorbitol (31%) and xylitol (24.8%). This enzyme showedK m values of 39 mM for fructose, 7.9 mM for mannitol, 0.14 mM for NADH and 0.075 mM for NAD+. For the non-specific HK, only theK m values for fructose (0.19 mM) and glucose (7.5 mM) were determined. The activities of the anabolic enzymes Mt1PDH and MtlPase were always at least two orders of magnitude higher than those of the degradative enzymes, indicating a net carbon flow towards a high intracellular mannitol pool. The function of mannitol metabolism inC. leprieurii as a biochemical adaptation to the environmental extremes in the mangrove habitat is discussed.Abbreviations F6P fructose-6-phosphate - HK hexokinase - Mt1P mannitol-1-phosphate - Mt1PDH mannitol-1-phosphate dehydrogenase - Mt1Pase mannitol-1-phosphatase - MtDH mannitol dehydrogenase  相似文献   

15.
The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V max (∼0.033 μmol Pi/min/mg protein) was low. Li+ was found to be an inhibitor (IC50 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na+, K+ NH4+) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg2+ dependent, which can be substituted partially by Mn2+. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.  相似文献   

16.
Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase 14C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent Km (M6P) was 15.8 millimolar, but the apparent Km (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg2+, Ca2+, Zn2+, and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPH-dependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.  相似文献   

17.
The speckled peacock bass Cichla temensis is a popular sport and food fish that generates substantial angling tourism and utilitarian harvest within its range. Its popularity and value make this species important for management and a potential aquaculture candidate for both fisheries enhancement and food fish production. However, little is known of optimal physiochemical conditions in natural habitats, which also are important for the development of hatchery protocols for handling, spawning and grow-out. Speckled peacock bass have been documented to have high sensitivity to extreme temperatures, but the metabolic underpinnings have not been evaluated. In this study, the effects of temperature (25, 30 and 35°C) on the standard metabolic rate (SMR) and lower dissolved oxygen tolerance (LDOT) of juvenile speckled peacock bass (mean ± standard error total length 153 ± 2 mm and wet weight 39.09 ± 1.37 g) were evaluated using intermittent respirometers after an acclimation period of 2 weeks. Speckled peacock bass had the highest SMR at 35°C (345.56 ± 19.89 mgO2 kg−1 h−1), followed by 30°C (208.16 ± 12.45 mgO2 kg−1 h−1) and 25°C (144.09 ± 10.43 mgO2 kg−1 h−1). Correspondingly, the Q10, or rate of increase in aerobic metabolic rate (MO2) relative to 10°C, for 30–35°C was also greater (2.76) than from 25 to 30°C (2.08). Similarly, speckled peacock bass were the most sensitive to hypoxia at the warmest temperature, with an LDOT at pO2 of 90 mmHg (4.13 mg l−1) at 35°C compared to pO2 values of 45 mmHg (2.22 mg l−1) and 30 mmHg (1.61 mg l−1) at 30 and 25°C, respectively. These results indicate that speckled peacock bass are sensitive to temperatures near 35°C, therefore we recommend managing and rearing this species at 25–30°C.  相似文献   

18.
Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L−1 l-lysine from mannitol at a yield of 0.26 mol mol−1 and a maximum productivity of 2.1 g L−1 h−1. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol−1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol−1. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.  相似文献   

19.
The enzyme 2-deoxy-d-ribose-5-phosphate aldolase (DERA) is a useful tool for synthesizing statin side-chain intermediates. In this work, we identified the DERA from Streptococcus suis (SsDERA) by structural and sequence alignment and highly expressed it in Escherichia coli BL21. The recombinant SsDERA had a specific activity of 18.2 U mg−1, KM of 0.8 mM, and Vmax of 32.9 μmol min−1 mg−1 toward 2-deoxy-d-ribose-5-phosphate under the optimal conditions: 40 °C and pH 7.0. The enzyme retained 23.3 % activity after incubation in 200 mM acetaldehyde for 2 h and 58.2 % activity in 100 mM chloroacetaldehyde for 2 h. The enzyme showed moderate activity and aldehyde tolerance compared with reported DERAs. The SsDERA-catalyzed reaction between 200 mM acetaldehyde and 100 mM chloroacetaldehyde generated (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose in 76 % yield in 8 h. This work provides a new DERA for the synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranose, which is a potential candidate for the industrial synthesis of statin intermediates.  相似文献   

20.
Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 °C and retains 63% of its activity at 120 °C but shows no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min at 80 °C and 6 min at 95 °C. Although TmMtDH has a higher V max with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with NAD+ than with NADP+. This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196) downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 °C.  相似文献   

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