Coupling protein scaffold and biosilicification: A sustainable and recyclable approach for d-mannitol production via one-step purification and immobilization of multienzymes.

Enzymatic synthesis of biochemicals in vitro is vital in synthetic biology for its efficiency, minimal by-products, and easy product separation. However, challenges like enzyme preparation, stability, and reusability persist. Here, we introduced a protein scaffold and biosilicification coupled system, providing a singular process for the purification and immobilization of multiple enzymes. Using d-mannitol as a model, we initially constructed a self-assembling EE/KK protein scaffold for the co-immobilization of glucose dehydrogenase and mannitol dehydrogenase. Under an enzyme-to-scaffold ratio of 1:8, a d-mannitol yield of 0.692 mol/mol was achieved within 4 h, 2.16-fold higher than the free enzymes. The immobilized enzymes retained 70.9 % of the initial joint activity while the free ones diminished nearly to inactivity after 8 h. Furthermore, we incorporated the biosilicification peptide CotB into the EE/KK scaffold, inducing silica deposition, which enabled the one-step purification and immobilization process assisted by Spy/Snoop protein-peptide pairs. The coupled system demonstrated a comparable d-mannitol yield to that of EE/KK scaffold and 1.34-fold higher remaining activities after 36 h. Following 6 cycles of reaction, the immobilized system retained the capability to synthesize 56.4 % of the initial d-mannitol titer. The self-assembly co-immobilization platform offers an effective approach for enzymatic synthesis of d-mannitol and other biochemicals.

[Efficient biosynthesis of D-mannitol by coordinated expression of a two-enzyme cascade].

D-mannitol is widely used in the pharmaceutical and medical industries as an important precursor of antitumor drugs and immune stimulants. However, the cost of the current enzymatic process for D-mannitol synthesis is high, thus not suitable for commercialization. To address this issue, an efficient mannitol dehydrogenase LpGDH used for the conversion and a glucose dehydrogenase BaGDH used for NADH regeneration were screened, respectively. These two enzymes were co-expressed in Escherichia coli BL21(DE3) to construct a two-enzyme cascade catalytic reaction for the efficient synthesis of d-mannitol, with a conversion rate of 59.7% from D-fructose achieved. The regeneration of cofactor NADH was enhanced by increasing the copy number of Bagdh, and a recombinant strain E. coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh was constructed to address the imbalance between cofactor amount and key enzyme expression level in the two-enzyme cascade catalytic reaction. An optimized whole cell transformation process was conducted under 30 ℃, initial pH 6.5, cell mass (OD600) 30, 100 g/L D-fructose substrate and an equivalent molar concentration of glucose. The highest yield of D-mannitol was 81.9 g/L with a molar conversion rate of 81.9% in 5 L fermenter under the optimal conversion conditions. This study provides a green and efficient biotransformation method for future large-scale production of D-mannitol, which is also of great importance for the production of other sugar alcohols.

Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex.

Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.

Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase.

Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses.

Time-averaged distributions of solute and solvent motions: exploring proton wires of GFP and PfM2DH.

Proton translocation pathways of selected variants of the green fluorescent protein (GFP) and Pseudomonas fluorescens mannitol 2-dehydrogenase (PfM2DH) were investigated via an explicit solvent molecular dynamics-based analysis protocol that allows for direct quantitative relationship between a crystal structure and its time-averaged solute-solvent structure obtained from simulation. Our study of GFP is in good agreement with previous research suggesting that the proton released from the chromophore upon photoexcitation can diffuse through an extended internal hydrogen bonding network that allows for the proton to exit to bulk or be recaptured by the anionic chromophore. Conversely for PfM2DH, we identified the most probable ionization states of key residues along the proton escape channel from the catalytic site to bulk solvent, wherein the solute and high-density solvent crystal structures of binary and ternary complexes were properly reproduced. Furthermore, we proposed a plausible mechanism for this proton translocation process that is consistent with the state-dependent structural shifts observed in our analysis. The time-averaged structures generated from our analyses facilitate validation of MD simulation results and provide a comprehensive profile of the dynamic all-occupancy solvation network within and around a flexible solute, from which detailed hydrogen-bonding networks can be inferred. In this way, potential drawbacks arising from the elucidation of these networks by examination of static crystal structures or via alternate rigid-protein solvation analysis procedures can be overcome. Complementary studies aimed at the effective use of our methodology for alternate implementations (e.g., ligand design) are currently underway.

Characterization of mannitol-2-dehydrogenase in Saccharina japonica: evidence for a new polyol-specific long-chain dehydrogenases/reductase.

Mannitol plays a crucial role in brown algae, acting as carbon storage, organic osmolytes and antioxidant. Transcriptomic analysis of Saccharina japonica revealed that the relative genes involved in the mannitol cycle are existent. Full-length sequence of mannitol-2-dehydrogenase (M2DH) gene was obtained, with one open reading frame of 2,007 bp which encodes 668 amino acids. Cis-regulatory elements for response to methyl jasmonic acid, light and drought existed in the 5'-upstream region. Phylogenetic analysis indicated that SjM2DH has an ancient prokaryotic origin, and is probably acquired by horizontal gene transfer event. Multiple alignment and spatial structure prediction displayed a series of conserved functional residues, motifs and domains, which favored that SjM2DH belongs to the polyol-specific long-chain dehydrogenases/reductase (PSLDR) family. Expressional profiles of SjM2DH in the juvenile sporophytes showed that it was influenced by saline, oxidative and desiccative factors. SjM2DH was over-expressed in Escherichia coli, and the cell-free extracts with recombinant SjM2DH displayed high activity on D-fructose reduction reaction. The analysis on SjM2DH gene structure and biochemical parameters reached a consensus that activity of SjM2DH is NADH-dependent and metal ion-independent. The characterization of SjM2DH showed that M2DH is a new member of PSLDR family and play an important role in mannitol metabolism in S. japonica.

Analytical method development for directed enzyme evolution research: a high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers.

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality L-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a ß-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C(18) guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4×96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM ß-cyclodextrin was used as electrolyte. 0.35%of D-ribose in L-ribose can be detected which can be translated into 99.3% ee of L-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement.

Dynamic mechanism of proton transfer in mannitol 2-dehydrogenase from Pseudomonas fluorescens: mobile GLU292 controls proton relay through a water channel that connects the active site with bulk solvent.

The active site of mannitol 2-dehydrogenase from Pseudomonas fluorescens (PfM2DH) is connected with bulk solvent through a narrow protein channel that shows structural resemblance to proton channels utilized by redox-driven proton pumps. A key element of the PfM2DH channel is the "mobile" Glu(292), which was seen crystallographically to adopt distinct positions up and down the channel. It was suggested that the "down → up" conformational change of Glu(292) could play a proton relay function in enzymatic catalysis, through direct proton shuttling by the Glu or because the channel is opened for water molecules forming a chain along which the protons flow. We report evidence from site-directed mutagenesis (Glu(292) → Ala) substantiated by data from molecular dynamics simulations that support a role for Glu(292) as a gate in a water chain (von Grotthuss-type) mechanism of proton translocation. Occupancy of the up and down position of Glu(292) is influenced by the bonding and charge state of the catalytic acid base Lys(295), suggesting that channel opening/closing motions of the Glu are synchronized to the reaction progress. Removal of gatekeeper control in the E292A mutant resulted in a selective, up to 120-fold slowing down of microscopic steps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD(+)-mannitol complex is promoted by a corresponding position change of Glu(292), which at physiological pH is associated with obligatory deprotonation of Lys(295) to solvent. These results underscore the important role of conformational dynamics in the proton transfer steps of alcohol dehydrogenase catalysis.

From alcohol dehydrogenase to a "one-way" carbonyl reductase by active-site redesign: a mechanistic study of mannitol 2-dehydrogenase from pseudomonas fluorescens.

Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD(+)-mannitol (K(int)) was altered dramatically (10(4)- to 10(5)-fold) from being balanced in the wild-type enzyme (K(int) ≈ 3) to favoring enzyme-NAD(+)-mannitol in the single site mutants, N191D and N300D. The change in K(int) reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn --> Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD(+)-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn-->Asp substitutions behaved as a slow "fructose reductase" at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively "one-way" reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis.

Crystal structure of the NADP-dependent mannitol dehydrogenase from Cladosporium herbarum: Implications for oligomerisation and catalysis.

The ascomycete Cladosporium herbarum is a prominent fungal inducer of Type I allergy. The only major allergen identified so far is Cla h 8, a NADP-dependent mannitol dehydrogenase (MtDH). MtDH, a cytoplasmic protein of 28.5kDa, belongs to the Short chain Dehydrogenases/Reductases (SDR), acting as a NADP-dependent oxidoreductase. In this study, we found that C. herbarum MtDH can exist as monomers, dimers and tetramers in solution and, correspondingly, forms tetramers and higher oligomers in two crystal structures. Additionally, we identified a unique adaptive binding site for the metal ions Na(+) and Zn(2+) that were distinguished by an anomalous dispersion experiment. A Translation-Libration-Screw analysis confirmed the stabilising effect of Zn(2+) for the tetrameric assembly. Moreover, the zinc containing structure explains the mode of MtDH multimerisation by metal bridging of the tetramers. The formation of oligomers and higher multimers of MtDH provides a missing link to its allergenic properties. Based on the well defined active site region and a comparative analysis with related structures, we can also clarify the atypical enzymatic properties of MtDH by two alternative binding modes of the substrate to the active site.

Data-independent liquid chromatography/mass spectrometry (LC/MS(E)) detection and quantification of the secreted Apium graveolens pathogen defense protein mannitol dehydrogenase.

Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, mannitol dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted mannitol. In the current study, LC/MS(E) was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MS(E) can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis.

The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites.

The side chains of Asn191 and Asn300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagine residues by alanine. The rate constants for the overall hydride transfer to and from C-2 of mannitol, which were estimated as approximately 5 x 102 s-1 and approximately 1.5 x 103 s-1 in the wild-type enzyme respectively, were selectively slowed, between 540- and 2700-fold, in single-site mannitol 2-dehydrogenase mutants. These effects were additive in the corresponding doubly mutated enzyme, suggesting independent functioning of the two asparagine residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by >or=1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme-NAD+-mannitol complex. The oxyanion hole of mannitol 2-dehydrogenase is suggested to drive a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is 'activated' for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 (fructose reduction). In the subsequent hydride transfer step, the two asparagine residues provide approximately 40 kJ/mol of electrostatic stabilization.

Polyol-specific long-chain dehydrogenases/reductases of mannitol metabolism in Aspergillus fumigatus: biochemical characterization and pH studies of mannitol 2-dehydrogenase and mannitol-1-phosphate 5-dehydrogenase.

Functional genomics data suggests that the metabolism of mannitol in the human pathogen Aspergillus fumigatus involves the action of two polyol-specific long-chain dehydrogenases/reductases, mannitol-1-phosphate 5-dehydrogenase (M1PDH) and mannitol 2-dehydrogenase (M2DH). The gene encoding the putative M2DH was expressed in Escherichia coli, and the purified recombinant protein was characterized biochemically. The predicted enzymatic function of a NAD(+)-dependent M2DH was confirmed. The enzyme is a monomer of 58kDa in solution and does not require metals for activity. pH profiles for M2DH and the previously isolated M1PDH were recorded in the pH range 6.0-10.0 for the oxidative and reductive direction of the reactions under conditions where substrate was limiting (k(cat)/K) or saturating (k(cat)). The pH-dependence of logk(cat) was usually different from that of log(k(cat)/K), suggesting that more than one step of the enzymatic mechanism was affected by changes in pH. The greater complexity of the pH profiles of log(k(cat)/K) for the fungal enzymes as compared to the analogous pH profiles for M2DH from Pseudomonas fluorescens may reflect sequence changes in vicinity of the conserved catalytic lysine.

Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase.

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 degrees C and retains 63% of its activity at 120 degrees 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 degrees C and 6 min at 95 degrees 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 degrees C.

Isomalt production by cloning, purifying and expressing of the MDH gene from Pseudomonas fluorescens DSM 50106 in different strains of E. coli.

A NADH-dependent mannitol dehydrogenase gene (mtlD) was cloned from Pseudomonas fluorescens, subcloned into an expression vector (pDEST110) and entered into different strains of E. coli to compare their protein expression and the enzyme specific activity. Purifications were accomplished by Ni(2+)-NTA affinity chromatography. Using this approach, the efficiency of purification process significantly increased (up to 90%) so that the purified enzyme gave a sharp single band (55 kDa) in SDS-PAGE. The results showed that among the strains, BL21 (DE3) plysS exhibited the maximum expression level of MDH(mannitol dehydrogenase) (11 mg L(-1)). Results from activity assay with fructose as substrate also showed that in this strain the specific activity of 63 U mg(-1) protein monitored for the enzyme, the record not reported before. Resazurin staining also indicated that the enzyme reduced fructose, whereas oxidized other substrates including mannitol, sorbitol and arabitol under optimal assay condition. From HPLC analysis it was showed for the first time that the enzyme could convert substrate isomaltulose to the specific products, GPM and GPS. Interestingly, because of the high specificity of the enzyme for substrate, the method can be used as an alternative approach to substitute nonspecific conventional method of isomalt production.

Crystallization, preliminary X-ray diffraction and structure analysis of Thermotoga maritima mannitol dehydrogenase.

Diffraction data have been collected from a crystal of Thermotoga maritima mannitol dehydrogenase at the Canadian Light Source. The crystal diffracted to 3.3 A resolution and belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 83.43, b = 120.61, c = 145.76 A. The structure is likely to be solved by molecular replacement.

Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis.

The commercial production of mannitol involves high-pressure hydrogenation of fructose using a nickel catalyst, a costly process. Mannitol can be produced through fermentation by microorganisms. Currently, a few Lactobacillus strains are used to develop an efficient process for mannitol bioproduction; most of the strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer Lactobacillus strains to increase fructose-to-mannitol conversion with decreased production of other products. We cloned the gene mtlK encoding mannitol-2-dehydrogenase (EC 1.1.1.67) that catalyzes the conversion of fructose into mannitol from Lactobacillus brevis using genomic polymerase chain reaction. The mtlK clone contains 1328 bp of DNA sequence including a 1002-bp open reading frame that consisted of 333 amino acids with a predicted molecular mass of about 36 kDa. The functional mannitol-2-dehydrogenase was produced by overexpressing mtlK via pRSETa vector in Escherichia coli BL21pLysS on isopropyl-beta-D-thiogalactopyranoside induction. The fusion protein is able to catalyze the reduction of fructose to mannitol at pH 5.35. Similar rates of catalytic reduction were observed using either the NADH or NADPH as cofactor under in vitro assay conditions. Genetically engineered Lactobacillus plantarum TF103 carrying the mtlK gene of L. brevis indicated increased mannitol production from glucose. The evaluation of mixed sugar fermentation and mannitol production by this strain is in progress.

Purification and characterization of a novel mannitol dehydrogenase from Lactobacillus intermedius.

Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol. Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol. The MDH from this bacterium was purified from the cell extract to homogeneity by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel Hydroxyapatite HTP column chromatography. The purified enzyme (specific activity, 331 U/mg protein) was a heterotetrameric protein with a native molecular weight (MW) of about 170 000 and subunit MWs of 43 000 and 34 500. The isoelectric point of the enzyme was at pH 4.7. Both subunits had the same N-terminal amino acid sequence. The optimum temperature for the reductive action of the purified MDH was at 35 degrees C with 44% activity at 50 degrees C and only 15% activity at 60 degrees C. The enzyme was optimally active at pH 5.5 with 50% activity at pH 6.5 and only 35% activity at pH 5.0 for reduction of fructose. The optimum pH for the oxidation of mannitol to fructose was 7.0. The purified enzyme was quite stable at pH 4.5-8.0 and temperature up to 35 degrees C. The K(m) and V(max) values of the enzyme for the reduction of fructose to mannitol were 20 mM and 396 micromol/min/mg protein, respectively. It did not have any reductive activity on glucose, xylose, and arabinose. The activity of the enzyme on fructose was 4.27 times greater with NADPH than NADH as cofactor. This is the first highly NADPH-dependent MDH (EC 1.1.1.138) from a LAB. Comparative properties of the enzyme with other microbial MDHs are presented.

Purification and characterization of a novel mannitol dehydrogenase from a newly isolated strain of Candida magnoliae.

Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37 degrees C and 10.0 and 40 degrees C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k(cat) = 823 s(-1), K(m) = 28.0 mM, and k(cat)/K(m) = 29.4 mM(-1) s(-1)) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.

Role of glucose in the bioconversion of fructose into mannitol by Candida magnoliae.

The most efficient substrate for mannitol production by Candida magnoliae HH-01 is fructose; glucose and sucrose can also be converted into mannitol but with lower conversion yields. Mannitol dehydrogenase was purified and characterized; it had the highest activity with fructose as the substrate and used only NADPH. In fed-batch fermentation with glucose, the production of mannitol from fructose ceased when the glucose was exhausted but it was reinitiated with the addition of glucose, implying that glucose plays an important role in NADPH regeneration.