Appl Microbiol Biotechnol (2006) 70: 135–139 Asymmetric oxidation by Gluconobacter oxydans Received: 29 August 2005 / Revised: 12 December 2005 / Accepted: 16 December 2005 / Published online: 24 January 2006 Abstract Asymmetric oxidation is of great value and a been well performed and dramatically developed (Marinomajor interest in both research and application. This review focuses on asymmetric oxidation of organic com- Biotransformation may offer distinct advantages over pounds by Gluconobacter oxydans. The microbe can be classical chemical synthetic technology. They can be used for bioproduction of several kinds of important chiral carried out under mild and ecologically compatible con- compounds, such as vitamin C, 6-(2-hydroxyethyl)amino- ditions, being often chemo-, regio- and enantioselective 6-deoxy-α-l-sorbofuranose, (S)-2-methylbutanoic acid, (Garcia-Granados et al. ; Taylor et al. ). Ap- (R)-2-hydroxy-propionic acid and 5-keto-D-gluconic acid.
plication of whole microbial cells is very attractive when Characteristics of the bacteria and research progress on the oxidations are involved since cofactor and recycle enantioselective biotransformation process are introduced.
systems form part of the cells’ metabolism (Pearce etal. ).
Oxidative fermentations have been well established for a long time, especially in vinegar and in L-sorbose produc-tion. Recently, information on the enzyme systems in- Chirality is an intrinsic universal feature of various levels volved in these oxidative fermentations has accumulated, of matter. Molecular chirality plays a key role in science and new developments are possible based on these findings and technology. In particular, life depends on molecular chirality, and many biological functions are inherently Gluconobacter oxydans is an obligate aerobic Gram- negative bacterium. It has a respiratory metabolism char- Asymmetric catalysis, since its infancy in the 1960s, has acterized by incomplete oxidation of sugars, alcohols and dramatically changed the procedures of chemical synthesis.
acids in which partially oxidized organic compounds are It has also resulted in a great progression to a level that excreted as end products (Deppenmeier et al. The technically approximates or sometimes even exceeds that corresponding products (aldehyde, ketone and organic of natural biological processes (Noyori ). The 2001 acid) are excreted almost completely into the medium. In Nobel Prize in Chemistry was shared by three scientists most cases, the reactions are catalyzed by dehydrogenases who devised techniques for asymmetric synthesis.
connected to the respiratory chain. Thus, reducing equiv- During the last decade, the discovery and syntheses of alents derived from the oxidative processes are finally new chiral drug substances and chiral intermediates have transferred to oxygen. Electron transfer is coupled to thegeneration of the electrochemical proton gradient which isthe driving force for ATP synthesis.
Since the reactive centres of the enzymes are oriented towards the periplasmic space, transportation of substratesand products into and out of the cell is not necessary.
Thus, rapid accumulation of incompletely oxidized prod-ucts in the medium is facilitated (Deppenmeier et al.
State Key Laboratory of Bioreactor Engineering, Therefore, this organism generates nearly quantitative East China University of Science and Technology, yields of the oxidation products, making G. oxydans im- portant for industrial use (Tkac et al. ; Zigova et al.
). This paper presents recent development and re- Tel.: +86-21-64252981Fax: +86-21-64250068 search on asymmetric synthesis by G. oxydans.
(Herrmann et al. This enzyme is involved in thenon-phosphorylative ketogenic oxidation of D-glucose and The rapid incomplete oxidation of carbon substrates by oxidizes gluconate to 5-KGA. It also utilizes a number of Gluconobacter strains is applied for several biotechnolog- compounds as substrate, including D-arabitol, D-sorbitol, ical processes (Tkac et al. ; Zigova et al. The D-glucosaminic acid, D-mannitol, 2,3-butandiol, xylitol, great advantage is that the substrates are regio- and G. oxydans is equipped with two topologically separated This feature is employed in combined biotechnological– enzyme systems for redox reactions with glucose to form 5- chemical synthesis of sugar derivatives that could only KGA (Pronk et al. ). The first enzyme system oxidizes otherwise be obtained by complex protection group chem- glucose to glucono-δ-lactone by the membrane-bound istry. Furthermore, it is worth mentioning that the process glucose dehydrogenase. This intermediate is converted to of incomplete oxidation is accompanied by enormous heat gluconic acid by a lactonase. Further oxidation of gluconic development. Hence, the isolation of thermotolerant Glu- acid by the membrane-bound enzymes gluconate dehydro- conobacter bacteria (e.g. grown at 37–40°C) might be genase and 2-ketogluconate dehydrogenase results in the advantageous for industrial applications to reduce the costs formation of 2-KGA and 2,5-diketogluconic acid (Meyer of cooling during fermentation (Moonmangmee et al.
and Hauer (Fig. ). The membrane-bound gluconate oxidoreductase converts gluconic acid to 5-KGA. The sec-ond enzyme system is located in the cytoplasm, where thesoluble NADP+-dependent glucose dehydrogenase cat- Asymmetric synthesis of chiral intermediate: alyzes the formation of glucono-δ-lactone, which is then converted to gluconic acid by a lactonase (Fig. Thecytoplasmic gluconate:NADP-5 oxidoreductase converts 5-Keto-D-gluconic acid (5-KGA) is an interesting com- pound with a number of potential applications. It isparticularly important in producing L-(+)-tartaric acidthrough oxidation of 5-KGA with oxygen, catalyzed by Asymmetric biosynthesis of D-α-hydroxyl acid vanadate or noble metals (Miasnikov and Jacobsen Xylaric acid can also be produced from 5-KGA by oxi- Oxidation of primary alcohols has been proven an easy and dation with air under alkaline conditions (Fleche 5- efficient means for the production of structurally diverse KGA is also a precursor for the synthesis of a number of carboxylic acids (Jurgen et al. Ian-Lucas and Jens- savoury flavor compounds, e.g. 4-hydroxy-5-methyl-2,3- Michael ; Švitel and Šturdík ; Gandolfi et al.
dihydrofuranone-3 (De Rooij and Johannes ). Moreover, the method can be employed for the The current world production of L-(+)-tartaric acid is enantioselective oxidation of different racemic primary estimated to be 35,000 tonne/year, with the price of about alcohols for the production of optically pure organic acids €6.00/kg (Lichtenthaler ). Tartaric acid has many (Molinari et al. ). The enantioselectivity of qui-applications, for example, as an antioxidant in food in- noehaemoprotein dehydrogenases in Gluconobacer sp. has dustry, a chiral reagent in organic synthesis and an acidic been recently reviewed, and the mechanisms accounting reducing agent in the textile industry. Additionally, it could for their stereopreference have been proposed (Jongejan et be an interesting alternative to citric acid as an acidulant in G. oxydans is applied to the conversion of D-glucose to 5-KGA (Fig. which is of considerable interest for the chemical industry (Saito and Loewus ; Bhat et al.
To increase 5-KGA formation, Gluconobacter strains were genetically engineered, and a plasmid-encoded copy of the gene encoding the gluconate:NADP-5 oxidoreduc- tase was overexpressed in G. oxydans strain DSM 2434 Reaction sequence of ketogluconate formation catalyzed by Gluconobacter oxydans (Herrmann et al. ). GADH Gluconate Fig. 1 Biosynthesis of 5-keto-D-gluconic acid. Conditions: D-glucose dehydrogenase, GDH glucose dehydrogenase, GNO gluconate: G. oxydans is an important strain for production of 2-keto- L-gluconic acid (2-KGA), precursor of vitamin C. Keyenzyme in the metabolic pathway of the biotransformation Fig. 3 Biosynthesis of (R)-2-hydroxy-propionic acid. Conditions:(R,S )-1,2-propanediol 10 g/l in aqueous solution, 30°C, pH 6.0 process is L-sorbose dehydrogenase (SDH) (Hao et al.
Production of (R)-2-hydroxy-propionic acid through The biotransformation of D-sorbitol to L-sorbose by G.
microbial oxidation of racemic 1,2-propanediol by G.
oxydans is an important step in the Reichstein method for oxydans is firstly carried out by the authors (Su et al. production of L-ascorbic acid (Saito et al. This is one (Fig. The biotransformation was processed with high of the most economically important industrial processes, enantiomeric excess (e.e.; more than 99%) and near the- with world vitamin C production estimated at around oretical yield (48% of racemic 1,2-propanediol) when the substrate concentration was lower than 20 g/l. When the Industrial biotransformation systems involving D-sorbi- substrate concentration was increased, maintaining the pH tol to L-sorbose usually involve a series of stirred tanks at 6.0 helped improve the enantioselectivity. It is possibly operating as chemostats in sequence to achieve maximum because the lower pH influences the dehydrogenases on the yield per unit time, and the timing of transfer from one tank selectivity of different configurations. The yield of D-lactic to the subsequent one is crucial to conversion efficiency.
acid is not markedly improved by the pH adjustment, Analysis of the composition of the system is necessary to which needs more approaches such as semi-continuous optimize the process. Thus, a rapid assay of sorbitol/ addition of the substrate or immobilization of the cells (Su sorbose would be of great benefit. In analytical terms, this system is especially difficult not only because of theclose chemical similarity between the two key analytesbut also because of the ratio that large quantities of one analyte is associated with low quantities of the other. Few 6-(2-hydroxyethyl)amino-6-deoxy-α-l-sorbofuranose analytical methods can achieve such discrimination.
Macauley-Patrick et al. () offered the method of in- Miglitol, a new α-glucosidase inhibitor developed by frared spectroscopy, which conferred greater advantages Bayer Co., was approved by FDA in December 1996 to over other monitoring options. Multiple analytes can be treat type II diabetes. It smoothes postprandial plasma glu- measured simultaneously by the method, and the mea- cose levels and is generally well tolerated. Now, it has been surement can be achieved in a fraction of the time taken the first choice for the treatment of type II diabetes.
Miglitol was manufactured by the combined methods of biotransformation and chemical synthesis. 6-(2-Hydroxy-ethyl)amino-6-deoxy-α-L-sorbofuranose is a precursor and key intermediate of miglitol. The authors apply G. oxydansto asymmetric biosynthesis of the intermediate. The sub- (S)-2-methylbutanoic acid (2-MBA) and its esters, par- strate of N-(2-hydroxyethyl)glucamine originally obtained ticularly 2-methylbutanoic acid ethyl ester, are well-known from Sigma and then synthesized by the author is suc- natural aroma compounds of fruits. These volatiles, de- cessfully converted by G. oxydans into 6-(2-hydroxyethyl) scribed as impact compounds of several fruits such as amino-6-deoxy-α-L-sorbofuranose (Fig. The structures apple, pineapple, strawberry and many more, are of high of the key intermediate and the final product are verified by economic interest (Perez et al. ). The database of mass spectrometry (MS) and 13C nuclear magnetic reso- volatile compounds in food of the TNO Nutrition and Food Research reports 2-MBA to be an ingredient in 26 fruits, 4 Strain of G. oxydans with high dehydrogenase activity vegetables and 26 other products (Schumacher et al. was screened and preserved by the author. By optimizing the Therefore, it is often used in aroma compositions. The acid cultural condition, biomass of the strain piles up to 6–7 g/l itself is used as well as its esters.
(dry cell weight). The asymmetric synthesis of 6-(2- G. oxydans is applied to bioconversion of 2-methyl- hydroxyethyl)amino-6-deoxy-α-l-sorbofuranose is finished butanol (2-MBOH) to 2-methylbutanoic acid (Fig. in 8 h, with conversion of more than 90%.
Besides the almost enantiopure (S)-2-MBOH, a racemic of the racemic mixture of 2-chloropropanol occurs with much lower enantioselectivity. The highest e.e. is below 50%. Higher rates are generally observed with 2-chloro-propanol, which may indicate that less bulky substituents (e.g. chloro vs phenyl) have a positive effect on velocity anda negative effect on enantioselectivity (Romano et al.
Fig. 5 Bioconversion of 2-methylbutanol (2-MBOH). Conditions:2-MBOH 5 g/l in aqueous solution, 30°C, for 1 week Low e.e. observed with whole cells may be due to a mixture and mixtures of known enantiomeric ratios are number of factors, such as the presence of various dehy- used as starting materials. The conversion rates and enan- drogenases with different enantioselectivities, the action tiomeric distribution of chiral compounds before and after of a single enzyme with low enantioselectivity and race- the bioconversion reaction are given. The results clearly mization of the formed product. Racemization was ruled prove that the enantiomers of 2-MBOH are differentially out by carrying out experiments in which optically pure metabolized by the bacteria (Schumacher et al. ).
S-chloropropanoic acid was added to the cells and Differing enthalpy or entropy changes during the gen- observing that no significant change in the enantiomeric eration of the enzyme–enantiomer complex may serve as an composition took place (Romano et al. ). The explanation. On the other hand, the phenomenon observed enantioselectivity is generally very low: one reason for might be caused by two or more different enzymes. Further the high rates and low enantiospecificity may be the investigations are necessary to determine the reasons.
distance between the stereocentre and the reacting site.
G.oxydans has been successfully applied to many asymmetric oxidations, and quite a lot of interesting com- Enantioselective oxidation of chiral and prochiral pounds have been synthesized. However, there are still much to be investigated, such as dehydrogenase and re-spective gene sequence, reaction mechanism exploration, Many kinds of chiral and prochiral alcohol and diols could biotransformation optimization and modulation of the be oxidized by G. oxydans to produce carboxylic acid. The dehydrogenation of alcohol and diol with bacteria has beenthe object of recent studies (Tsuji et al. but no recentsystematic information is available about the stereochem- ical outcomes of these oxidations. Romano et al. carried out enantioselective biotransformation of several Ameyama M (1987) Oxidative fermentation. 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