Biotransformation of lactose to galactitol

JOURNAL OF FERMENTATION"AND BIOENOI~Rn~G Vol. 77, NO. 1, 32-35. 1994

Biotransformation of Lactose to Galactitol SYED M U N I R U Z Z A M A N , H I R O M I C H I ITOH, AKIRA YOSHINO, TAKESHI KATAYAMA, ~a,rD KEN IZUMORI*

Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-07, Japan Received 10 June 1993/Accepted 10 September 1993

Myeobacterium smegmatis SMDU produced galactitol from a-galactose in a cultivation medium containing 1 ~ TSB (trypto-soya broth) and 1 ~ carbohydrate. The transformation rate was highest (70Yo or more) in the presence of D-glucose as a carbon source. Similar transformation results were obtained when ~-,-gaiactosidase treated whey was used in the medium instead of D-glucose and D-gaiactose. After the first transformation in the cultivation media, washed cells were used again for transformation, and a considerable amount of galactitoi was accumulated in the reaction mixture. This study revealed that after lactose is split by fl-D-gaiactosidase, whey can be used directly without further separation or purification for the production of gaiactitol.

Transformation conditions Transformation was carried out at 30°C in the presence of various carbohydrates in L-tubes aerobically with shaking in a medium of the following composition: 1% TSB powder, 1% D-galactose (substrate) and 1% carbohydrate. The transformation media was inoculated with a 100 pl preculture grown on 1% TSB. After determining the optimum conditions for the transformation of D-galactose to galactitol during growth in the L-tube, transformation was performed under the same conditions in a 500 ml Erlenmeyer flask containing 100 ml of medium. Analytical methods Samples were taken aseptically every 24 h from the transformation medium in order to monitor transformation. Accumulation of the product galactitol in the medium was detected by high performance liquid chromatography (HPLC, Nihonbunko H P L C 880 PU liquid chromatography, Shimadzu RID-6A refractive index detector and Shimadzu CR-6A chromatopac) using a Hitachi H P L C column GL-611. Separation was achieved at 60°C with 10 -4 M NaOH at a flow rate of 1.0 ml/min. Washed cell reaction M. smegmatis SMDU cells were grown aerobically at 30°C with shaking (170rpm) for 4 d in 1% TSB in the presence of either 1% D-galactose, 1% D-glucose, or 1% D-glucose+l% D-galactose. After growth was completed, the cells were harvested by centrifugation at 9,000 rpm for 10 min. The collected cells were washed twice with 0.05 M phosphate buffer (pH 7) for 10min at 9,000rpm. Transformation was carried out at 30°C with shaking in L-tubes containing a reaction mixture of the following composition: 0.05 M Na-phosphate buffer 5 ml (pH 7), washed cells 0.2 g (wet) and D-galactose 50 rag. Samples at different times were taken from the reaction mixture and the decrease in D-galactose was determined by the standard method (6, 7). Cleavage of whey lactose and D-galaetose transformation The cleavage of lactose in 4% o,/whey powder solution was accomplished by /~-D-galactosidase (15 units of enzyme per ml) at an acidic p H ( p H = 4 . 5 ) at room temperature. After 6h, lactose had been completely split into D-glucose and D-galactose. The D-glucose and Dgalactose were sterilized by millipore filtration and 3 ml of this solution were added to the L-tubes containing 3 ml of various concentrations of sterile TSB to yield a final 1% concentration of both D-glucose and D-galactose. The L-tubes were then inoculated with a 100 pl preculture of

Microorganisms can be considered as nature's mini factories that have tremendous potential to perform many transformation reactions at ambient temperature and pressure. We are exploiting this potential of microbes to produce rare sugars, which are not naturally abundant and at the same time difficult to produce by organic or chemical reactions. However, microbial production of expensive rare monosaccharides like D-tagatose, D-sorbose, and Dpsicose have already been reported by our laboratory (14). The practical application or usefulness of these rare sugars has not been well investigated untill now because of the high expense and unavailability. Some recent studies have revealed that rare sugars like D-tagatose can be used as a noncaloric sweetener. It has also been found that this rare sugar has almost the same sweetening effect and volume as sucrose (5). This sort of finding obviously increases the interest in studies regarding the production of rare sugars. For the microbial production of the rare monosaccharides mentioned above, galactitol, a relatively cheap sugar alcohol, is essential as a starting material. Commercial production of galactitol involves the chemical reduction of D-galactose which is obtained by the hydrolysis of lactose. During the course of the study on carbohydrate metabolism by Mycobacterium smegmatis SMDU, it was found that this strain can transform considerable amounts of D-galactose to gaiactitol. In the present paper, the transformation of lactose to galactitol using commercial/~-D-galactosidase and cells of M. smegmatis SMDU is described. MATERIALS A N D METHODS Microorganism M. smegmatis SMDU was used throughout this study. Cultures were maintained at 4°C on 2% trypto-soya agar slants. Chemicals Trypto-soya broth (TSB) was purchased from Nissui Seiyaku Co. (Tokyo). Carbohydrates were obtained from Wako Pure Chemical Industries (Osaka) and were certified as reagent grade. /3-D-Galactosidase of Aspergiilus oryzae (EC 3.2.1.23, grade 11) was purchased from Sigma Chemical Co. (St. Louis, USA). Dry whey powder was obtained from Meiji Milk Products Co. (Tokyo). * Corresponding author.

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Vow. 77, 1994

GALACTITOL PRODUCTION

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M. smegmatis SMDU grown on 1% TSB. The transformation was carried out at 30°C for 5 d with shaking. Samples from the transformation medium were aseptically removed every 24 h and subjected to H P L C for product analysis. Product recovery After transformation, the cells were removed by centrifugation at 9,000 rpm for 10 rain. The supernatant fluid was then treated with activated charcoal and filtered after centrifugation at 12,000rpm for 30min to remove the charcoal. The content was treated with a Microacilyzer (Asahi Kasei, Model G-l) for deionization. The microacilyzer treated sample was again deionized with a mixture of Diaion (SK1B, H +) and Amberlite (IRA-411, CO32-) ion exchange resins. The deionized content was then evaporated under vacuum until crystals appeared. The crystals were collected and dried in a dessicator after washing and their weights were measured.

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Utilization of the calls after the first transformation re action The cells were removed from the cultivation

media and washed twice with 0.05 M phosphate buffer (pH 7) as described above. 0.2 g of the washed cells were then used for the second transformation of D-galactose to galactitol in the presence or absence of I % D-glucose. The volume of the reaction mixture, buffer, and transformation conditions were the same as described above (washed cell reaction). Determination of product The product was identified by H P L C analysis infrared spectra and laC NMR spectra measurement with authentic galactitol. Infrared spectra and ~C NMR spectra were measured with a Nihonbunko infrared spectrophotometer (model A-302) using KBr tablets and with a NMR spectrophotometer (model JEOL A L P H A 400) at 400 MHz, respectively. RESULTS Effect of carbohydrates on D-galactose transformation

Since M. smegmatis SMDU grows slowly, an attempt to transform D-galactose to galactitol in the cultivation media during growth of the organism was made. In a previous report (8), it was found that D-galactose does not favorably support the growth of M. smegmatis. To support the growth, various carbohydrates at 1%, such as glycerol, D-mannitol, D-sorbitol, o-ribose, D-xylose, Larabinose, D-mannose, D-fructose and D-glucose, were added to a cultivation medium of 1% TSB containing 1% D-galactose as substrate. Figure 1 presents the rate of gaiactitol formation from D-galactose after 5 d of growth in media containing various carbohydrates in Ltubes. The highest degree of transformation (about 73%) Glycerol D.Xylose L-Arabinose D-Sorbitol D-Mannitol D-Ribose D-Mannose D-Glucose D-Fructose 0

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FIG. 2. Infrared spectra of the authentic galactitol and product. was found in the presence of D-glucose. Significant transformation (about 60%) also occured with D-fructose and D-ribose. The highest amount of product accumulated in the cultivation media during the 5th d of growth. A similar trend was observed for flask level transformation. In the flask level reaction, 0.6 g of crystals were obtained from 1 g of D-galactose, representing a net yield of 60%. Identification of the product The H P L C retention time and the infrared spectrum (Fig. 2) of the isolated crystals were indistinguishable from those of authentic galactitol. Figure 3 represents the 13C NMR spectrum of the isolated crystals. The spectrum was indistinguishable from that of authentic galactitol. The number of signals indicate the number of C-atoms of galactitol. From these findings, the product formed from D-galactose in the

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Transformation rate (%) FIG. 1. Galactitol production from D-galactose after 5-d growth in media containing different carbohydrates.

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FIG. 3. t~C NMR spectrum of the product. In D20, temperature 25°C, internal standard (I S, dioxane); 67.40ppm, C-3, C-4; 70.99 ppm, C-2, C-5; 70.18 ppm, C-I, C-6; 64.07 ppm (Ref. 12).

34

MUNIRUZZAMAN ET AL.

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FIG. 4. Galactose reduction by the cells of M. sraegraatis grown on v-galactose, v-glucose and o-galactose+v-glucose. Symbols: ©, D-glucose; C2, o-galactose; @, D-galactose+v-glucose.

FIG. 6. Galactitol production potential of the M. smegmatis cells after first transformation in the cultivation media. Symbols: t~, Dglucose 1%; @, D-glucose0%.

cultivation media was identified as galactitol.

creased on the 5th d. The variation in product accumulation for different concentrations of TSB could be attributed to the cell concentration in the media which is dependent on the TSB concentrations. It is apparent from the results that 1% TSB is suitable for the transformation of D-galactose to galactitol using the strain employed here. Second transformation After the first transformation in the cultivation media, the cells were reused for the second transformation in the presence or absence of Dglucose. Figure 6 represents the potential of the cells for the second transformation. The interesting feature of this figure is that when D-glucose was not added in the reaction mixture, transformation reached a peak on the 4th d, after which a decline of the product was observed. However, for the reaction mixture where 1% D-glucose was added, product accumulation increased until the 6th d of the reaction. Maximum transformation was 53.5% and 46.6% for the reactions with or without D-glucose, respectively.

To determine the inducer of transformation in the cultivation media, a washed cell reaction was performed using cells grown on either D-glucose, D-galactose or D-glucose+Dgalactose. Figure 4 shows that the cells grown on v-galactose can reduce the concentration of v-galactose and transform it to galactitol in the reaction mixture faster than the cells grown on v-galactose+v-glucose or D-glucose. However, the cells grown on o-galactose+v-glucose were found to have a higher reduction ability than those of cells grown on D-glucose. It is apparent from this finding that the substrate, v-galactose, induces the enzyme(s) responsible for the reduction of v-galactose to galactitol. Utilization of whey Since the combination of D-glucose and v-galactose in the medium was found to be the optimal condition for the production of galactitol from v-galactose (Fig. 1), whey was utilized as the source of lactose since the cleavage of lactose provides both Dglucose and o-galactose in equal amounts. Lactose in whey powder was enzymatically split to D-glucose and v-galactose which were used as growth carbon and substrate respectively in the presence of 0.5°~, 1% or 2% TSB powder to determine the optimum concentration of TSB for the transformation reaction. Maximum transformation (about 70%) was observed at 1% TSB followed by 2% TSB (Fig. 5). The highest product accumulation in these cases was observed on the 4th d of cultivation. However, in 0.5% TSB product accumulation also inInducer of the transformation reaction

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FIG. 5. Effectof TSB on galactitol formation from D-galactose obtained from whey. Symbols: ©, TSB 0.5°~; o, TSB 1%; •, TSB 2%.

DISCUSSION The bacterial strain used in this study has considerable potential for galactitol production. Onishi (9) reported a 42% yield of the sugar consumed in the first report of extraceUular dulcitol formation by microbial metabolism. In most systems, D-glucose has been found to be the principal effector of catabolite repression (10). Interestingly the transformation reported here was not repressed by Dglucose. Moreover, the transformation of D-galactose to galactitol was high in a medium containing D-glucose. The literature shows that about 50 million tons of whey are produced annually as waste by the world's milk product industries, of which 70% of the dry matter is lactose. 50% of this waste goes to municipal sewage, eventually creating water pollution (11). This waste can be effectively utilized for gaiactitol production by the present simple method since the splitting of whey lactose provides both growth carbon (D-glucose) and substrate (D-galactose) for the strain used here. The additional advantage of this method is that the cells of M. smegmatis can be reused after the first transformation in the cultivation media even though the rate of product formation is comparatively low and slow compared to that of the first reaction.

VoL. 77, 1994

GALACTITOL PRODUCTION REFERENCES

1. Izumori, K., Mlyoshl, T., Tokuda, S., and Yamabe, K.: Production of D-tagatose from dulcitol by Arthrobacter glob~formis. Appl. Environ. Microbiol., 46, 1055-1057 (1984). 2. Izumori, K. and Tsuzaki, K.: Production of D-tagatose from D-galactitol by Mycobacterium smegmatis. J. Ferment. Technol., 66, 225-227 (1988).

3. Khan, A.R., Takahata, S., Okaya, H., Tsumura, T., and Inlmori, K.: D-Sorbose fermentation from galactitol by Pseudomonas sp. ST 24. J. Ferment. Bioeng., 74, 149-152 (1992). 4. Izumorl, K., Yamaldta, M., Tsumura, T., and Kobayashi, H.: Production of D-psicose from D-talito1, D-tagatose or D-galactitol by Alcaligenes sp. 70lB. J. Ferment. Bioeng., 70, 26-29 (1990). 5. Zehner, L. R.: D-Tagatose as a low-calorie carbohydrate sweetener and bulking agent. Eur. Pat. Appl. EP 257626 (C1. A23LI/236), (1988). 6. Nelson, N.: A photometric adaptation of the Somogyi method

7. 8. 9. 10. 11. 12.

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for the determination of glucose. J. Biol. Chem., 153, 375-380 (1944). Somogyi, M.: Notes on sugar determination. J. Biol. Chem., 195, 19-23 (1952). Izumorl, K., Yamanaka, K., and Elheln, A. D.: Pentose metabolism in Mycobacterium smegmatis: specificity of induction of pentose isomerases. J. Bacteriol., 128, 587-591 (1976). Onlshl, H. and Suzuki, T.: Formation of dulcitol in aerobic dissimilation of D-galactose by yeast. J. Bacteriol., 95, 1745-1749 (1968). Palgen, g. and Williams, B.: Catabolite repression and other control mechanism in carbohydrate utilization. Adv. Microbiol. Physiol., 4, 251-324 (1970). Cruege, A. and Cruege, W.: Carbohydrates, p. 448. In Kieslich, K. (ed.), Biotechnology, 6a (1984). Voelter, W. and BreRmaler, E.: The influence of methylation of 13C chemical shifts of galactose derivatives. Tetrahedron., 29, 3845-3848 (1973).