Polyol Conversion Specificity of Bacillus Pallidus

Biosci. Biotechnol. Biochem., 72 (1), 231–235, 2008 Note Polyol Conversion Specificity of Bacillus pallidus

y Wayoon POONPERM, Goro TAKATA, and Ken IZUMORI

Rare Sugar Research Center, Kagawa University, 2393 Ikenobe, Miki, Kagawa 7610795, Japan

Received July 27, 2007; Accepted October 2, 2007; Online Publication, January 7, 2008 [doi:10.1271/bbb.70475]

The conversion specificity of Bacillus pallidus Y25 for and xylitol have been found to be the main substrates polyols, including elusive rare sugar alcohols, was of B. pallidus, followed by D-iditol, L-arabitol, ribitol, investigated. B. pallidus cells showed transformation D-arabitol, and galactitol.4,5) In this report, we describe potential for several rare polyols, including allitol, L- the structural dependency of sugar substrates in the mannitol, D/L-talitol, and D-iditol, and converted them cellular reaction of this bacterium due to -OH config- to their corresponding ketoses. This indicates that the urations at C2 and C3 positions of polyols, and its bacterium had two polyol dehydrogenases specific for potential application in the production of various rare polyols that have D-erythro and D-threo configurations. sugars. By combination with intrinsic isomerases, polyols were All sugars not commercially available were produced converted directly to various aldoses, including L-xylose, in our laboratory. Erythritol, D/L-threitol, ribitol, D/L- L-talose, D-altrose, and L-glucose. arabitol, xylitol, D-glucitol, and galactitol were pur- chased from Wako (Osaka, Japan) or Sigma-Aldrich (St Key words: Bacillus pallidus; polyol; ribitol dehydro- Louis, MO). D/L-Iditol (from D/L-sorbose), L-glucitol genase; xylitol dehydrogenase (from D-sorbose), D/L-talitol (from D/L-tagatose), allitol (from D-psicose), and L-mannitol (from L-fructose) are Rare sugars, monosaccharides and their derivatives elusive rare polyols, and they were prepared in our that are rarely found in nature, are one of the most laboratory. B. pallidus was grown in yeast extract worthy targets for various applications in the food medium (0.5% yeast extract, 0.5% polypepton, and industry, as well as in the pharmaceutical and nutrition 0.5% NaCl, pH 8.5) containing 1% D-mannitol as a industries, for various products such as non-calorie growth enhancer at 55 C for 36 h. Cells were harvested sweeteners, bulking agents, precursor substances, and by centrifugation at 14;000 g for 5 min. The collected nucleoside analogues. Although rare sugars originate cells were then washed twice with double-distilled mainly by chemical synthesis, the reactions require water, and resuspended in 50 mM sodium phosphate multiple steps, are costly, and sometimes lead to the buffer (pH 7.0), and the conversion reaction was production of unnecessary by-products that are not examined at 55 C with shaking (100 rpm) in a reaction conducive to the mass production of rare sugars. mixture containing the cell suspension (final cell Various biotransformation processes have been re- density, OD600 ¼ 30) and 1% substrate. The reaction ported for carbohydrates, mediated by oxidation, reduc- was terminated after 48 h, with occasional sampling, and tion, isomerization, and epimerization, using micro- cells were removed from the reaction mixture by organisms screened from the soil.1–5) In addition, centrifugation at 14;000 g for 10 min. The quantity bioconversion strategies for all rare sugars have been of ketoses was estimated by cystein-carbazole or schematized.6) Biotransformation is generally recog- Somogyi-Nelson method.7,8) Sugar components of the nized as environmentally friendly compared to other reaction mixture were analyzed by HPLC (Shimadzu, chemical processes, and it is economically competitive Kyoto) using a GL-C611 separation column (Hitachi, in terms of cost and productivity. Our aim is to develop Tokyo). The optical activity of the product was and introduce simple and effective biochemical methods determined using a polarimeter (Nihon Bunko, Tokyo). for the mass production of rare sugars. The substrate specificity of B. pallidus was inves- Thermophilic bacteria have been screened widely for tigated using all the polyols. In addition to seven thermostable enzymes, which have a lot of biotechno- previously known substrates (allitol, xylitol, D-iditol, logical potential. Recently, we isolated Bacillus pallidus L-arabitol, ribitol, D-arabitol, and galactitol), the reaction Y25, a novel, moderately thermophilic bacterium capa- development was also observed by colorimetric method ble of producing rare ketoses from polyols.4) This when further six polyols were used as substrates bacterium showed broad substrate specificity, oxidizing (L-mannitol, L-talitol, L-glucitol, erythritol, D-talitol, various polyols to their corresponding ketoses. Allitol and D-threitol) (Fig. 1). From the results of HPLC, we

y To whom correspondence should be addressed. Fax: +81-87-891-3021; E-mail: [email protected] 232 W. POONPERM et al. a bcd

allitol D-psicose D-manmitol D-fructose D-glucitol D-fructose D-talitol D-psicose

L-mannitol L-fructose allitol L-psicose L-talitol L-psicose L-glucitol L-fructose

L-talitol L-tagatose D-glucitol L-sorbose L-iditol L-sorbose galactitol L-tagatose

L-glucitol D-sorbose D-talitol D-tagatose galactitol D-tagatose D-iditol D-sorbose

ribitol D-ribulose D-arabitol D-xylulose xylitol D-xylulose D-arabitol D-ribulose

L-arabitol L-xylulose ribitol L-ribulose L-arabitol L-ribulose xylitol L-xylulose

erythritolD-erythrulose erythritol L-erythrulose L-threitol L-erythrulose D-threitol D-erythrulose

Fig. 1. Proposed Classification of the Reaction of Polyols and Ketoses According to the -OH Configurations at C2 and C3 Positions of the Polyols. The proposed reactions of ribitol 2-dehydrogenase (a), ribitol 4-dehydrogenase (b), xylitol 2-dehydrogenase (c), and xylitol 4-dehydrogenase (d) are indicated. The reactions of B. pallidus mediated by two polyol dehydrogenases are boxed around the sugar structures. The vertical line indicates the carbon backbone, single lines branches are hydroxyl groups, and double line branches are keto groups of the sugars. Polyol Conversion Specificity of B. pallidus 233

a b c L-fructose D-psicose L-tagatose L-galactose D-altrose L-mannose L-mannitol L-talitol L-glucose D-allose galactitol L-talose

d e f ribitol L-glucitol L-arabitol L-xylulose D-gulose L-fructose D-ribulose D-sorbose xylitol D-arabitol L-ribulose L-xylose

g h i D-iditol D-talitol galactitol D-sorbose L-galactose L-tagatose D-psicose

j k l D-arabitol L-xylulose L-xylulose L-arabitol xylitol L-ribulose L-xylose m L-xylose L-arabitol L-ribulose xylitol L-arabitol L-xylulose D-arabinose D-ribulose xylitol L-ribulose

Fig. 2. HPLC of the Reaction Mixture of B. pallidus. The initial substrates, allitol (a), L-mannitol (b), L-talitol (c), L-glucitol (d), ribitol (e), L-arabitol (f), D-talitol (g), galactitol (h), D-iditol (i), D-arabitol (j), xylitol (k), L-xylulose (l), and L-ribulose (m), were mixed with the cells and incubated at 55 C for 48 h with shaking.

confirmed the transformation of allitol to D-psicose were the L- and D-formula respectively (D-threo config- (Fig. 2a), L-mannitol to L-fructose (Fig. 2b), L-talitol to uration). As for other reactions, the products were L-tagatose (Fig. 2c), L-glucitol to D-sorbose (Fig. 2d), not detectable by HPLC (data not shown). Reverse ribitol to D-ribulose (Fig. 2e), and L-arabitol to L- reactions from ketose to polyols were also achieved xylulose (Fig. 2f), in which the -OH configurations of using L-xylulose (Fig. 2l), L-ribulose (Fig. 2m), D-ribu- C2 and C3 positions of these polyols were D-formula lose, D-sorbose, L-tagatose, L-fructose, and D-psicose as (D-erythro configuration), and the transformation of substrates. Moreover, by HPLC, we observed various D-talitol to D-psicose (Fig. 2g), L-glucitol to L-fructose unexpected product peaks in the reaction mixture, with (Fig. 2d), galactitol to L-tagatose (Fig. 2h), D-iditol to a prolonged reaction period, i.e., L-xylose, L-xylulose, D-sorbose (Fig. 2i), D-arabitol to D-ribulose (Fig. 2j), L-ribulose, L-arabitol, and xylitol (Fig. 2e, f, l, and m); and xylitol to L-xylulose (Fig. 2k), in which the -OH D-ribulose, D-arabinose, and D-arabitol (Fig. 2j and k); configurations of C2 and C3 positions of these polyols L-tagatose, L-galactose, L-talose, galactitol, and L-talitol 234 W. POONPERM et al. a L-Xylose L-Lyxose L-Arabinose L-Ribose DAI L-Xylulose L-Arabitol L-Ribulose DH

Xylitol Ribitol

DH D-Xylulose D-Arabitol D-Ribulose DAI D-Xylose D-Lyxose D-Arabinose D-Ribose

b L-Galactose L-Talose L-Altrose L-Allose DAI LRhI

L-Tagatose L-Talitol L-Psicose DH

Galactitol Allitol

DH D-Tagatose D-Talitol D-Psicose DAI LRhI D-Galactose D-Talose D-Altrose D-Allose

D-Idose D-Gulose L-Glucose L-Mannose LRhI DAI LRhI D-Sorbose L-Glucitol L-Fructose DH DH DH DH D-Iditol L-Mannitol

Fig. 3. Summary of the Conversion Reactions of B. pallidus. The reactions of pentitols and pentoses (a) and hexitols and hexoses (b) are indicated. Black arrows indicate the route and gray arrows indicate the missing route of sugar conversion by B. pallidus. The black and boxed sugars are the starting materials for the production of various rare sugars. The black sugars indicate the products and the gray sugars are the missing products from the reactions with B. pallidus. DAI, LRhI and DH are the enzymes of DAI, D-arabinose isomerase; LRhI, L-rhamnose isomerase; and DH, polyol dehydrogenase of B. pallidus.

(Fig. 2c and h); L-mannose, L-glucose, L-mannitol, and We suggest based on these results that the enzymes L-fructose (Fig. 2b); D-psicose, D-allose, D-altrose, and corresponding to polyol dehydrogenase (ketose reduc- allitol (Fig. 2a); and D-gulose, D-sorbose, L-fructose, and tase) are to be classified into four groups based on the L-glucitol (Fig. 2d). The aldose products of the reaction, difference in -OH configurations at C2 and C3 positions L-xylose, D-arabinose, D-altrose, L-galactose, and L- of all polyols. Ribitol 2-dehydrogenase (EC 1.1.1.16), glucose, had to be obtained by the catalytic reaction of also called L-arabinitol 4-dehydrogenase (EC 1.1.1.12) D-arabinose isomerase with L-xylulose, D-ribulose, D- or erythrulose reductase (EC 1.1.1.162), acts on polyols psicose, L-tagatose, and L-fructose respectively; and the with a D-erythro configuration, such as allitol, L- products, L-talose, L-mannose, D-allose, and D-gulose mannitol, L-talitol, L-glucitol, ribitol, L-arabitol, and had to be obtained by the reaction of L-rhamnose erythritol, to produce the corresponding ketoses isomerase with L-tagatose, L-fructose, D-psicose, and D- (Fig. 1a). Ribitol 4-dehydrogenase, also called D-arabi- sorbose respectively (Fig. 3). Other possible aldose nitol 4-dehydrogenase (EC 1.1.1.11), D-mannitol 2- products, such as L-lyxose catalyzed by L-rhamnose dehydrogenase (EC 1.1.1.67), D-arabinitol dehydrogen- isomerase; L-arabinose, D-galactose, and L-altrose cata- ase (EC 1.1.1.287), or L-sorbose reductase (EC lyzed by L-arabinose isomerase; L-allose, D-talose, and 1.1.1.289), catalyses polyols with a L-erythro config- D-idose catalyzed by D-arabinose isomerase; D-ribose uration (-OH configurations at C2 and C3 positions were catalyzed by D-ribose isomerase; and L-ribose catalyzed L-formula), such as D-mannitol, allitol, D-glucitol, D- by L-ribose isomerase were not observed in this study talitol, D-arabitol, ribitol, and erythritol to their corre- (Fig. 3). sponding ketoses (Fig. 1b). Xylitol 2-dehydrogenase, Polyol Conversion Specificity of B. pallidus 235 also called D-xylulose reductase (EC 1.1.1.9), L-arabi- the mass production of rare sugars from polyols are low nitol 2-dehydrogenase (EC 1.1.1.13), L-iditol 2-dehy- cost, and the fact that the complete reaction can be drogenase (EC 1.1.1.14), or galactitol 2-dehydrogenase carried out without addition of a coenzyme. By the (EC 1.1.1.16), catalyses polyols with a L-threo config- synergistic effect of a resting cell reaction of isomerase uration(-OH configurations at C2 and C3 positions were and polyol dehydrogenase, we can improve the produc- D- and L-formula respectively), such as D-glucitol, L- tion yield by increasing the initial concentration of talitol, L-iditol, galactitol, xylitol, L-arabitol, and L- polyol, because the conversion reactions of polyol and threitol, to their corresponding ketoses (Fig. 1c). Xylitol isomerization are developed at the same time and the 4-dehydrogenase, also called L-xylulose reductase (EC concentration of intermediate ketose is maintained in the 1.1.1.10), D-iditol 2-dehydrogenase (EC 1.1.1.15), or D- reaction mixture. arabinitol 2-dehydrogenase (EC 1.1.1.250), reacts with D-threo configuration, such as D-talitol, L-glucitol, galactitol, D-iditol, D-arabitol, xylitol, and D-threitol, References and transforms them to their corresponding ketoses (Fig. 1d).9) Although the favored substrates of polyol 1) Granstro¨m, T. B., Takata, G., Morimoto, K., Leisola, M., dehydrogenases vary according to the different sources and Izumori, K., L-Xylose and L-lyxose production from of organisms in the same group as above, the substrate xylitol using Alcaligenes 701B strain and immobilized specificity of these enzymes is limited. We conclude that L-rhamnose isomerase enzyme. Enz. Microbiol. Tech- nol., 36, 976–981 (2005). B. pallidus possesses two polyol dehydrogenases, ribitol 2) Mizanur, M. R., Takeshita, K., Moshino, H., Takada, 2-dehydrogenase and xylitol 4-dehydrogenase, because G., and Izumori, K., Production of L-erythrose via it was specific for these polyols, which have D-erythro L-erythrulose from erythritol using microbial and enzy- and D-threo configurations respectively (Fig. 1a and d). matic reactions. J. Biosci. Bioeng., 92, 237–241 (2001). In the case of B. pallidus, the favorite substrate of ribitol 3) Menavuvu, B. T., Poonperm, W., Takeda, K., Morimoto, 2-dehydrogenase was allitol, followed by L-arabitol, and K., Granstro¨m, T. B., Takada, G., and Izumori, K., Novel unfavorable substrates were L-glucitol and L-talitol; substrate specificity of D-arabinose isomerase from while the favorite substrate of xylitol 4-dehydrogenase Klebsiella pneumoniae and its application for the was xylitol, followed by D-threitol, and unfavorable production of D-altrose from D-psicose. J. Biosci. Bioeng., 101, 340–345 (2006). substrates were L-glucitol and D-talitol. Attempts to acquire information by crystal structure analysis of these 4) Poonperm, W., Takata, G., Morimoto, K., Granstro¨m, T. B., and Izumori, K., Production of L-xylulose from enzyme are ongoing. xylitol by a newly isolated, Bacillus pallidus Y25 and We have reported that crude extracts of B. pallidus characterization of its relevant enzyme, xylitol dehydro- contain significant activity for D-arabinose isomerase genase. Enz. Microbiol. Technol., 40, 1206–1212 (2007). and L-rhamnose isomerase, that L-xylose is produced 5) Poonperm, W., Takata, G., Lumyong, S., Lumyong, from xylitol via L-xylulose, and that D-allose and D- P., and Izumori, K., Efficient conversion of allitol to 4,5) altrose are produced from allitol via D-psicose. In this D-psicose by Bacillus pallidus Y25. J. Biosci. Bioeng., study, we observed the production of elusive rare sugars, 103, 282–285 (2007). including L-talose, L-glucose, and L-mannose from 6) Izumori, K., Bioproduction strategies for rare hexose L-talitol, L-mannitol, and L-mannitol respectively, and sugars. Naturwissenschaften, 89, 120–124 (2002). 7) Dishe, Z., and Borenfreud, E., A new spectrophotometric L-talitol and L-mannitol were produced by reverse method for the detection of keto sugars and trioses. reaction from L-tagatose and L-fructose respectively J. Biol. Chem., 192, 583–587 (1951). (Fig. 2). L-Rhamnose isomerase derived from Pseudo- 8) Nelson, N., A photometric adaptation of the Somogyi monas stutzeri showed broad substrate specificity for method for the determination of glucose. J. Biol. Chem., various aldoses and ketoses, while the same enzyme 153, 375–381 (1944). derived from Escherichia coli showed high substrate 9) Richard, P., Putkonen, M., Vaananen, R., specificity, and it does not act on D-allose.10) In this Londesborough, J., and Penttila, M., The missing link study, we found four reactions that were catalyzed by in the fungal L-arabinose catabolic pathway, identifica- this enzyme from B. pallidus (Fig. 3). This result tion of the L-xylulose reductase gene. Biochemistry, 41, suggests that the substrate specificity of L-rhamnose 6432–6437 (2002). isomerase from this bacterium is higher than that of the 10) Leang, K., Takada, G., Fukai, Y., Morimoto, K., enzyme from P. stutzeri and lower than that of the Granstro¨m, T. B., and Izumori, K., Novel reactions of L-rhamnose isomerase from Pseudomonas stutzeri and enzyme from E. coli. D-Arabinose isomerase was active its relation with D-xylose isomerase via substrate on L-fucose, D-arabinose, L-altrose, L-galactose, L-xy- 3,11) specificity. Biochim. Biophys. Acta, 1674, 68–77 (2004). lose, and L-glucose. The enzyme from B. pallidus 11) Sultana, I., Mizanur, R., Takeshita, K., Takada, G., and showed almost the same substrate specificity, i.e., D- Izumori, K., Direct production of D-arabinose from arabinose, D-altrose, L-galactose, L-xylose, and L-glu- D-xylose by a coupling reaction using D-xylose isomer- 3,11) cose, as was found in previous studies (Fig. 3). ase, D-tagatose 3-epimerase and D-arabinose isomerase. The benefits of using B. pallidus biotransformation in J. Biosci. Bioeng., 95, 342–347 (2003).