Production of natural and rare pentoses using microorganisms and their enzymes
Ministry of Education, Science, Sports and Culture of Japan under
scholarship program for foreign students.
Biotransformation of carbohydrates is a classical example of the application of the regiospecificity of enzymes. Next to significance to the production of monosaccharides from the corresponding biopolymers, the microbial transformations of monosaccharides have become an important bioprocess. In the past few years, the medicinal application of L-carbohydrates and their derived nucleosides have greatly increased. Several modified nucleosides derived from L-sugars have shown to be potent antiviral agents and also usable in antigens therapy. Derivatives of rare sugar have also been used as an anti-hepatitis B virus and human immunodeficiency virus (HIV) agents as antitumor agent such as bleomycin are active against several murine tumors and thereby making it useful for cancer treatment. Moreover, rare sugars (unnatural sugars) are usually sweet like the natural sugars, but unlike them, rare sugars are either not metabolized by the body or metabolized to a lesser extent than natural sugars. Due to these features, rare sugars are desirable as low-calorie sweeteners and are well tolerated by diabetics. It was also found that L-monosaccharides have antineoplastic characteristics and are useful in combination with all major forms of cancer therapy including surgery, biological, chemical and radiation therapies and hyperthermia. In addition to increasing the mortality rate of neoplastic cells, they can reduce the metastatic potential of the tumor, and slow down the growth of malignant cells. Other advantage of rare sugars is the absence of an objectionable aftertaste, commonly experienced with artificial sweeteners such as saccharin or cyclamates. However, in spite of the demand for these rare sugars, their commercial availability, application or usefulness is negligible as they are expensive to prepare and unavailable in nature. Biochemical methods, usually microbial or enzymatic, are suitable for the production of unusual or rare monosaccharides.
Production of keto-pentoses using whole cell and cell -free oxidoreductases
L-Xylulose is a ketopentose, which is not abundant in nature, but it can be easily produced from xylitol by dehydrogenation. The bacteria Klebsiella pneumoniae, Alcaligenes sp. 701B and Erwina uredovora Dm 122 were reported to produce L-xylulose from xylitol. It seems that the corresponding dehydrogenase is mostly responsible for the whole-cell transformation of xylitol to L-xylulose. D-xylulose can be produced by whole cell or enzyme biocatalysis. Microbial and enzymatic production of D-xylulose from D-arabitol has been reported previously. Acetobacter sp. IFO 3281 was extremely active in the transformation of D-arabitol to D-xylulose at a relatively high substrate concentration (~50%) and showed no product consumption or by-product formation. Nearly stoichiometric conversion of D-arabitol to D-xylulose was achieved with A. aceti IFO 3281. D-xylulose can also be produced from D-xylose. The production of L-ribulose from ribitol has been studied using various acetic acid bacteria and the highest activity was found in G. frateurii IFO 3254, followed by A. aceti IFO 3281. An enzyme, NAD-dependent ribitol dehydrogenase (EC 22.214.171.124) identified from Gluconobacter suboxydans, responsible for L-ribulose production from the oxidation of ribitol. The oxidation of ribitol to L-ribulose, in all these cases, was followed by a long lag period to attain complete oxidation; resulting in the formation of by-products. In contrast, A. aceti IFO 3281 transformed ribitol to L-ribulose at high substrate concentration (~20%) without any by-product formation and did not show any tendency of product consumption.
Production of pentitols using whole cell and cell -free oxidoreductases
Pentitol catabolism is an interesting phenomenon in microbial metabolism, which can serve as an excellent model system for studying the acquisition of new metabolic capabilities by microbes. Extensive studies have been carried out on the production of polyols, such as glycerol, erythritol, xylitol, D-arabitol and D-mannitol, during the fermentation of soy sauce by halotolerent yeasts. It was revealed that Candida sp. was one of the most potent microorganisms for D-arabitol production. C. famata R28 produced D-arabitol from D-glucose without producing any by-product. In one report it was proposed that Candida spp. produced D-arabitol from D-ribulose with NAD-dependent D-arabitol dehydrogenase in which D-ribulose was derived by dephosphorylating D-ribulose-5-PO4 in the pentose pathway. Candida pelliculosa produced D-arabitol from D-glucose but concomitantly produced, D-ribose as a by-product. Certain osmophilic yeast (such as Pichia misa) grown in the presence of high glucose concentration (~30%) produced, in addition to ethanol and CO2, a variety of polyhydric alcohols: glycerol, erythritol, D-arabitol and mannitol. It was also reported that D-arabitol is the intermediate in the interconversion of aldose and ketose in Candida albicans, C. utilis and Penicillium chrysogenum. The synthesis of xylitol from natural products is based on the chemistry of pentosans occurring in many plants. Xylan, a constituent of pentosan, is a polysaccharide that can be hydrolyzed into D-xylose, which is also known as wood sugar. Xylitol can be synthesized by hydrogenation of xylose. Xylitol can also be produced by microbial transformation reactions, such as from D-xylose by yeast or from D-glucose by yeast and bacteria. Xylitol has also been produced from D-xylulose using Mycobacterium smegmatis and from D-xylose using the commercial immobilized D-xylose isomerase of Bacillus coagulans or immobilized cells of M. smegmatis. Sud-Chemie AG, Munich, Germany in their patents granted in 1976 had described a process for preparing xylitol by acid hydrolysis of xylan. Another US patent, applied from Finland and granted in 1977, describes a method of producing xylitol on commercial scale by acid hydrolysis of raw materials (e.g. wood, corncobs, straw, bran, cotton-seed hulls, etc.) containing pentosan (such as xylans). Ribitol is a naturally occurring polyol, which is commercially available and very cheap. In one previous report, it was found that D-ribose could be reduced to give an optically inactive ribitol. In one report, it was found that ribitol can be produced as end product from D-ribose dissimilation by the salt-tolerant yeast Candida polymorpha.
Production of aldo-pentoses using whole cell and cell -free oxidoreductases
D-Lyxose is unavailable and thus rare in nature, and can be obtained by chemical synthesis. The microbial production of D-lyxose from D-xylulose was reported using L-ribose isomerase of Acinetobacter sp. DL 28. The success of this D-lyxose synthesis was due to three factors: (a) all reactions were selective only for product formation; (b) the production of D-lyxose from D-glucose was a continuous process, which means that product separation or purification at each step was not needed; and (c) most importantly the final product (D-lyxose) was very easily separated from the reaction mixture with no effect on the desired product and without producing by-products. On the other hand, L-lyxose is very expensive due to its scarcity in nature and it can be obtained only by chemical synthesis. In a previous study, an enzyme biotrasformation of L-lyxose from ribitol was reported. L-ribose is not abundant in nature and therefore is an expensive and rare aldo-pentose that can only be obtained by a series of chemical reactions. It was found in one report that the purified L-ribose isomerase (L-RI) enzyme can catalyze the isomerization of D-mannose, D-lyxose and L-ribose, with the highest activity observed on L-ribose. L-ribose can also be produced from ribitol by whole cell oxidation of Acetobacter aceti IFO 3281 and isomerization using L-RI. Several methods have been reported for the preparation of D-ribose and many of them are patented. The production of D-ribose by fermentation has received much attention lately, possibly because of its use for the production of antiviral and anticancer drugs. It has been reported that D-ribose can be obtained from D-glucose by fermentation using Bacillus subtilis strain ATCC 21951 or Candida pelliculosa. One report was found for the synthesis of D-ribose from D-glucose. A mutant BS-9 Strain of Bacillus subtilis can produce D-ribose from D-glucose. A lead-catalyzed system was developed for the synthesis of D-ribose at established conditions that selectively precipitated ribose from this mixture of four pentoses in the presence of lead nitrate. Since L- and D-ribose form distinct aggregates, therefore it should be possible to isolate D-ribose. L-Arabinose can only be obtained by a series of chemical reactions. During the study on carbohydrate metabolism by Mycobacterium smegmatis strain SMDU, it was found that this strain can transform about 90% L-ribulose into L-arabinose by means of its L-arabinose isomerase.
The configurations of various diastereoisomeric sugars are related to a natural alditol, such as, D-arabinose, D-arabitol, and D-lyxose and this relationship offers a simple route for the synthesis of unnatural aldoses from natural ones. Most abundant six carbon sugars in nature are D-glucose, D-fructose, D-galactose and D-mannose. Several studies have been carried out with microorganisms and their enzymes to produce various rare L- and D- sugars from inexpensive carbohydrates. Many aldose-ketose isomerization of free sugars have also been reported. Of the total of eight aldopentoses and four pentitols, three pentoses (D-xylose, L-arabinose and D-ribose) and two pentitols (ribitol and D-arabitol) are common in nature. The remaining pentoses are unknown or rare and are not abundant in nature, being xenobiotic compounds. These rare carbohydrates are difficult to produce and can only be produced by chemical reactions. However, the chemical route is time-consuming, requires many steps and costly chemicals and produce unnecessary by-products being not feasible for mass production of these rare sugars. Therefore, the developments of simple methods are extremely important for increasing the production of these sugars. Moreover, to produce enough amounts of these rare aldopentoses, natural substrates have to be used which are abundant in nature and cheap. D-Glucose is the most abundant aldose occurring in nature. If, it is possible to produce these aldopentoses economically from D-glucose through whole microbial cell or enzyme biocatalysts it will also be possible to use them as valuable starting materials for the manufacture of various live saving drugs and other important high-value products.
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