Abstract:
Nucleic acids encoding  Thermotoga maritima  mannitol dehydrogenase and the  Thermotoga maritima  mannitol dehydrogenase polypeptide are disclosed. Further provided are an electrochemical bioreactor system and a bioreactor electrode that can be used to convert glucose or fructose to mannitol.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims benefit to U.S. Provisional Application Serial No. 60/837,039, filed Aug. 11, 2006, which is incorporated herein by reference in its entirety. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       [0002]     This work was supported by a grant from the U.S. Department of Agriculture (USDA). The U.S. government has certain rights to this invention. 
     
    
     BACKGROUND OF THE INVENTION  
       [0003]     (1) Field of the Invention  
         [0004]     The present invention relates generally to a thermostable mannitol dehydrogenase from  Thermotoga maritima.  Specifically, the present invention relates to the use of this enzyme on a bioreactor electrode in a bioreactor system to produce mannitol from glucose or fructose.  
         [0005]     (2) Description of the Related Art  
         [0006]     Currently, 50,000 tons/year of mannitol are produced by hydrogenation of a 50% fructose/50% glucose syrup at high pressures and temperatures using a Raney nickel catalyst. The fructose/glucose syrup is converted to a 30% mannitol-70% sorbitol mixture from which mannitol is purified by low-temperature crystallization. Developing a new, simplified, enzyme-catalyzed process for mannitol production could lower product costs. By starting from 100% glucose, it would increase the chemical yield (one mole mannitol produced per mole of glucose), and lower the downstream processing costs by eliminating the crystallization step. It would also allow mannitol to be called a natural product.  
         [0007]     Mannitol is used as a low-caloric and low-cariogenic sweetener (in particular in diabetic foodstuffs), as a pharmaceutical formulating agent (e.g., used as a diuretic in the manufacture of intravenous fluids and tablets, in dental hygiene products, and as a low reactivity drug filler), as a specialty chemical, and in plastic manufacturing. SpecChem Online estimates the global market for mannitol to be about $28 million (www.specchemonline.com, Sugaring the pill, Oct. 21, 2004).  
         [0008]     While the related art teaches mannitol production, there still exists a need for an improved method of producing mannitol.  
       OBJECTS  
       [0009]     It is an object of the present invention to provide a thermostable mannitol dehydrogenase which is immobilized on a bioreactor electrode and used in an electrochemical bioreactor system to produce mannitol. It is also an object to provide a process which is reliable and economically favorable for producing mannitol. These and other objects will become increasingly apparent by reference to the following description and the drawings.  
       SUMMARY OF THE INVENTION  
       [0010]     The present invention provides a bioreactor electrode comprising: an electrode substrate having an exposed surface; a first thermostable enzyme having a mannitol dehydrogenase activity immobilized on the surface of the electrode substrate; and a second thermostable enzyme having a xylose isomerase activity immobilized on the surface of the electrode substrate, wherein the first enzyme and the second enzyme convert glucose to mannitol when used in an electrochemical bioreactor system. In further embodiments, the first thermostable enzyme is  Thermotoga maritima  mannitol dehydrogenase. In still further embodiments, the second thermostable enzyme is  Thermotoga neapolitana  xylose isomerase. Further still, preferably the first thermostable enzyme is  Thermotoga maritima  mannitol dehydrogenase and the second thermostable enzyme is  Thermotoga neapolitana  xylose isomerase. Further, preferably NADH or NADPH is immobilized on the electrode.  
         [0011]     The present invention provides an electrochemical bioreactor system comprising: a glucose solution; an electrochemical reactor electrode in the glucose solution comprising an electrode substrate having an exposed surface; a first thermostable enzyme having a mannitol dehydrogenase activity immobilized on the surface of the electrode substrate; a second thermostable enzyme having a xylose isomerase activity immobilized on the surface of the electrode substrate, wherein the first enzyme and the second enzyme convert glucose to mannitol when used in an electrochemical reactor system; a cofactor comprising NADH or NADPH; and a power source electrically connected to the electrochemical reactor electrode, wherein when the power source provides an electrical current the cofactor is recycled so that the glucose in the glucose solution is converted to mannitol. Further still, preferably the NADH or NADPH is immobilized on the electrode substrate. Still further, the first thermostable enzyme is  Thermotoga maritima  mannitol dehydrogenase and the second thermostable enzyme is  Thermotoga neapolitana  xylose isomerase.  
         [0012]     Further, the present invention provides a process for producing mannitol which comprises: a glucose solution; an electrochemical reactor electrode in the glucose solution comprising an electrode substrate having an exposed surface; a first thermostable enzyme having a mannitol dehydrogenase activity immobilized on the surface of the electrode substrate; a second thermostable enzyme having a xylose isomerase activity immobilized on the surface of the electrode substrate, wherein the first enzyme and the second enzyme convert glucose to mannitol when used in an electrochemical reactor system; a cofactor comprising NADH or NADPH; and a power source electrically connected to the electrochemical reactor electrode, wherein when the power source provides an electrical current the cofactor is recycled so that the glucose in the glucose solution is converted to mannitol; and introducing the electrical current from the power source to produce the mannitol from the glucose. Further, the first thermostable enzyme is preferably  Thermotoga maritima  mannitol dehydrogenase. Still further, preferably the second thermostable enzyme is  Thermotoga neapolitana  xylose isomerase. Further still, preferably the first thermostable enzyme comprises a polypeptide having the amino acid sequence of SEQ ID NO 2. Further, the NADH or NADPH is immobilized on the substrate. Still further, preferably the first thermostable enzyme is  Thermotoga maritima  mannitol dehydrogenase and the second thermostable enzyme is  Thermotoga neapolitana  xylose isomerase. Further, preferably the second thermostable enzyme is  Thermotoga neapolitana  xylose isomerase containing mutations V186T, L283P, and F187S.  
         [0013]     In further embodiments, a bioreactor electrode comprising: an electrode substrate having an exposed surface; a thermostable enzyme having a mannitol dehydrogenase activity immobilized on the surface of the electrode substrate; and wherein the enzyme converts fructose to mannitol when used in an electrochemical bioreactor system. Further, the thermostable enzyme is preferably  Thermotoga maritima  mannitol dehydrogenase. Still further, the enzyme comprises the amino acid sequence of SEQ ID NO 2. Further still, the invention relates to a process for producing mannitol which comprises using the electrode to convert fructose to mannitol. Finally, the invention relates to a system for producing mannitol which comprises using the system to produce the mannitol from a glucose isomerase mixture. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0014]      FIG. 1  is a drawing showing SEQ ID NO 1.  
         [0015]      FIG. 2  is a drawing showing SEQ ID NO 2.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0016]     All patents, patent applications, government publications, government regulations, and literature references cited in this specification are hereby incorporated herein by reference in their entirety. In case of conflict, the present description, including definitions, will control.  
         [0017]      Thermotoga neapolitana  xylose isomerase is described in U.S. Pat. No. 7,198,933 to Zeikus et al. hereby incorporated herein by reference in its entirety.  Thermotoga neapolitana  xylose isomerase containing mutations V186T, L283P, and F187S is described in the &#39;933 patent. The strains  Thermotoga maritima  DSM 3109, the strains  Thermotoga elfii  DSM 9442 and ATCC 51869, and the strains  Thermotoga neapolitana  DSM 4359 and ATCC 49049 are described in U.S. Patent No. 5,935,837 to Rasmussen hereby incorporated herein by reference in its entirety. Rasmussen teaches  Thermotoga maritima  xylose isomerase, useful for the electrochemical bioreactor system of the present invention. Xylose isomerase also known as glucose isomerase is well known to those skilled in the art.  
         [0018]     The present invention provides a gene encoding thermostable mannitol dehydrogenase from  Thermotoga maritima  and use of the enzyme in a bioreactor system to produce mannitol from glucose. The present invention replaces the current synthetic mannitol production process by the use of an enzyme catalyzed process. For this purpose, a thermostable mannitol dehydrogenase has been cloned and characterized which is used to produce mannitol from fructose or, from glucose in a bioelectrochemical reactor. Used alone, this enzyme is able to produce mannitol from a fructose syrup. Used in combination with a thermostable xylose isomerase (glucose isomerase), this enzyme would be able to produce mannitol directly from a glucose syrup.  
       EXAMPLE  
       [0019]     The  T. maritima  mannitol dehydrogenase gene was obtained by DNA amplification using  T. maritima  (MSB8) genomic DNA as the template and oligonucleotides 5′-CG CATATG AAAGTACTTTTGATAG-3′ (where CATATG creates an NdeI site) (SEQ ID NO. 3) and 5′-CT CTCGAG AGAAAAAATTCCCTTCATC-3′ (where CTCGAG creates a Xhol site) (SEQ ID NO. 4) as the primers. The PCR product has cloned into the Ndel and Xhol sites of pET24(a)+(Novagen) and transformed into  Escherichia coli  BL21(DE3) for protein expression. In this construct, the recombinant  T. maritima  mannitol dehydrogenase was expressed as a fusion protein with a C-terminal (His) 6  tag. The recombinant  T. maritima  mannitol dehydrogenase was routinely over expressed in  E. coli  by growing cultures in SB medium and inducing with IPTG (0.6 mM) when OD 600  reaches 1.4. Expression was induced for sixteen (16) hours. After resuspension in 50 mM Tris-HCl pH 8.5 containing 10 mM β-mercaptoethanol (buffer A), the bacteria were lysed using a French pressure cell, the crude extract was centrifuged for 40 min. at 25,000×g, the supernatant was heat treated at 85° C. for 20 min. to denature most  E. coli  proteins, the heat-treated extract was centrifuged for 20 min. at 20,000×g, and the supernatant was finally purified on a Ni-NTA affinity column.  
         [0020]     The recombinant  T. maritima  mannitol dehydrogenase expression and purification systems are currently acceptable for routine bench-top scale preparations, biochemical characterization, and testing in prototype bioelectrochemical reactors. Activity levels on fructose as the substrate and with NADH as the cofactor can be increased by mutagenesis to make this enzyme even more performing for industrial mannitol production. In particular, the affinity for fructose relative to mannitol can be increased. Since the three-dimensional structure of mannitol dehydrogenase is unknown, random mutagenesis can be used followed by screening for activity at room temperature to select for  T. maritima  mannitol dehydrogenase derivatives with increased activity levels.  
         [0021]     It is possible to convert 100% fructose into 100% mannitol using an immobilized enzyme system, as it is done today for fructose syrup (42%) production in an immobilized glucose isomerase reactor. Fructose is more expensive than glucose, though, and it is produced directly from glucose. Since a large selection of thermostable glucose isomerases is available, one can also produce the mannitol dehydrogenase bioreactor. Such a system with the robust thermostable mannitol dehydrogenase can be used with the pyrimidine nucleotide cofactor which can be easily recycled. By using electrochemical recycling, glucose can be converted stoichiometrically into mannitol in a single electrochemical reactor system at 60° C. containing both immobilized thermostable mannitol dehydrogenase (MtDH) and glucose isomerase.  
         [0022]     An NAD-dependent thermostable mannitol dehydrogenase was cloned.  T. maritima  mannitol dehydrogenase is increasingly active up to 90° C. The enzyme shows four times higher affinity for NADH than for NADPH. The optimum pH for fructose reduction is 6.0 and the optimum pH for mannitol oxidation is 8.3. When co-immobilized on an electrochemical reactor&#39;s electrode, this enzyme and a thermostable xylose isomerase are able to produce mannitol directly from glucose when the cofactor is recycled using electrons provided by an electrical current.  
         [0023]     While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.