Oxidation and Reduction of D-Xylose by Cell-Free Extract of Hansenula polymorpha

Australian Journal of Basic and Applied Sciences, 5(12): 95-100, 2011 ISSN 1991-8178 Oxidation and Reduction of D-Xylose by Cell-Free Extract of Hansenula polymorpha Ahmed, Y.M., Ibrahim, I.H., Khan, J.A. and Kumosani, T.A., Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. Abstract: The mechanism of oxidation and reduction of D-xylose by cell-free extract of Hansenula polymorpha was studied in medium contain xylose as carbon source. Xylose dehydrogenase which NADP was detected dependent in the cell free extract. Both the oxidation and reduction were coupled in this enzyme and proved by HPLC as xylose; xylitol and xylonic acid. Xylose reductase, NADPH dependent in cell free extract that reduce xylose to xylitol. Both D-xylose and L-arabinose can be used as substrate Xylitol dehydrogenase was also found in the cell free extract which is NAD dependent and xylulose produced in the reaction was identified and it was found to oxidize a variety of polyol sugars. Key words: Hansenula polymorpha, Xylose dehydrogenase- cell free extract- Xylose reductase- Xylitol dehydrogenase- NADP-NADPH-NAD-NADH-Xylose. INTRODUCTION Lignocellulosic material containing cellulose, hemicellulose, and lignin is an abundant renewable organic resource that can be used for the production of energy and biochemicals (Weber et al., 2010). The conversion of both the cellulose and hemicellulose fractions for producing biochemicals is being intensively studied. Between 23% to 40% of the lignocellulosic biomass consists of hemicelluloses, the main component being xylose in most hardwoods and annual plants (Lee et al., 1979). Although abundant, D-xylose is not considered to be fermentable (to ethanol) by yeasts (Barnett, 1976). Numerous studies have been carried out on various aspects of D-xylose bioconversion (Winkelhausen and Kuzmanova, 1998; Townsend and Howarth, 2010). In some yeasts and fungi, conversion of D-xylose to D-xylulose more often occurs by two enzymatic steps. First, D- xylose is reduced by a NADPH/NADH-linked xylose reductase (XR) to xylitol, whereupon the latter is oxidized to xylulose by an NAD-linked xylitol dehydrogenase (XDH) (Bruinenberg and Van Dijken, 1983). D-xylulose is subsequently phosphorylated to D-xylulose-5-phosphate by D-xylulokinase before entering the pentose phosphate, Embden-Meyerhof, and phosphoketolase pathways (Skoog and Hahn-Hagerdal, 1988). Some yeasts as in Candida shehatae and Pichia stipitis (Dupreez et al., 1986); and Pachysolen tannophilus (Morimoto et al., 1986) an unusual pathway for xylose oxidation to xylonic acid by xylose dehydrogenase enzyme found in cell free extract of Pichia quercuum described by (Suzuki and Onishi, 1975). Recently, recombinant Saccharomyces cerevisiae expressing a fungal pentose gen able to ferment all sugars present, including the pentose sugars L- arabinose and D-xylose (Bettiga et al., 2009) to produce ethanol. The aim of this work is to study the mechanism of formation of xylitol, xylonic and xylulose from corn cops (as a source of xylose) by cell free extract of H. polymorpha. MATERIALS AND METHODS Hansenula polymorpha was obtained from Microbial and Natural Products Chemistry Lab National Research Centre, Cairo, Egypt. Maintenance Medium: The organism was maintained on YPD medium containing 10g of yeast extract, 20g of peptone, 50g of D- glucose and 10g of agar per liter of distilled water. Preparation of Inocula: A loopful of H. polymorpha cells from a YPD medium agar slant was transferred to 20ml of medium containing 0.67% (w/v) yeast nitrogen base (YNB; Difco) without amino acids and 2% (w/v) xylose. The culture was incubated at 30 C in a loosely capped 125ml Erlenmeyer flask which was agitated at 200 rpm in a New Brunswick shaker for 48h (Lee et al., 1986). Growth Medium: Inoculums 2ml of H. polymorpha culture for each was transferred to 50ml of medium containing the following gradient (g/l): (NH 4 ) 2 SO 4, 0.1; yeast extract, 4.0; NaCl, 2.5; xylose, 10.0; KH 2 PO 4, 1.5 and K 2 HPO 4, 1.0 and ph was adjusted to 6.0. The culture was incubated at 30 C in a loosely capped 125ml Erlenmeyer flask Corresponding Author: Yousry Ahmed, Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia. E-mail:youssri01@yahoo.com 95

which was agitated at 200 rpm in a New Brunswick shaker for 48 h After 24 hr of growth the cells were harvested, [in case of different the cells were harvested after 1, 2, 3, 5 day respectively). Cells grown for 24h on glycerol alone served as the control for the experiments (Bicho et al., 1988). Preparation of Cell Extracts: Upon termination of incubation, H. polymorpha cells were harvested by centrifugation at 5000 rpm with a Sorvall refrigerated centrifuge (GSA rotor). The cell pellet was suspended in 2 to 4 ml of ice-cold 50mM potassium phosphate buffer (ph 7.0) containing 1mM EDTA. Cells were broken by sonication (Maleszka et al., 1983). Enzyme Assays in the Cell Free Extract of H. Polymorpha: 1- D-Xylose dehydrogenase (EC 1.1.1.175) (Suzuki and Onishi, 1973). Assay mixtures (4.0ml) contained 0.2ml 1M phosphate buffer ph 7.0, 0.5M, D-xylose, 0.5ml and cell free extract 1ml + 2.15ml H 2 0. At zero time, 0.15ml NAD or NADP 3.4mM was added. The change of absorbance at 340nm. was determined at room temperature. The increasing of absorbance by the time indicate the reduction of NAD or NADP to NAD(P)H One unit of enzyme was defined as the quantity of enzyme required to reduce 1umol of NAD(P) to NAD(P)H. 2- D-Xlose reductase (EC 1.1.1.21) (Sunzki and Onishi, 1973). D-Xylose reductase enzyme in the cell free extracte was examined by the same method as cited above in (1) except that 0.15 mm of NADH or NADPH was added. In all experiments, controls reading were made without adding D-xylose. One unit of enzyme was defined as the quantity of enzyme required to oxidize 1umol of NADPH per minute under the experimental conditions. 3- Xylitol dehydrogenase (EC. 1.1.1.10) (Ahmed, 1990). The reaction mixture contained 0.3ml of 0.5M xylitol, 0.1ml of 3.4mM NADP or NAD and 8ul of 1M MgCl 2.6H 2 0, 1ml of 0.15M Tris buffer (ph, 8.0) and cell free extract 1ml + H2O in total volume of 4.0ml. The reaction was started by the addition of xylitol, and the increase in optical density at 340 nm was measured. One enzyme units was defined as the quantity of enzyme that reduce1umol of NAD or NADP per minute under condition of the assay. Protein was estimated by the method of lowry et al., (1951). D-xylulose was identified by the cysteine carbazol test of Dische and Barenfreund (1951). Product Identification: The reaction product was identified by HPLC using a Shimadzu HPLC LC(10). The used column was Shim-pack SCR -10(7.9mm i.d. x30cm), the mobile phase: water with flow rate 0.6/min. Column temperature was 80 C, in detector: Refractive index. Calcium D-xylonate was prepared by oxidation of D-xylose with periodic acid (Moore and Link, 1940) and used as a standard. RESULTS AND DISCUSSION H. polymorpha was found to produce many enzymes which convert xylose to xylitol xylonic and xylulose. The presence of such enzymes was detected in the cell free extract of H. polymorpha. Xylose Dehydrogenase (EC 1.1.1.175): The presence of xylose dehydrogenase was detcted in the crude and dialyzed cell free extract of H. polymorpha, the results are reported in Table (1) the dialyzed and crude enzyme contained dehydrogenase activity which oxidized xylose to xylonic acid in the presence of NADP as coenzyme but no enzyme activity was noticed with NAD as coenzyme. Table 1: D-Xylose dehydrogenase activity in the crude and dialyzed cell-free extract prepared from H. polymorpha. Cell free D-xylose dehydrogenase * Protein Specific activity Substrate extract NADP Unit NAD Unit (mg/ml) (u/mg) Crude D-xylose 0.6 0.0 1.4 0.43 Dialyzed D-xylose 1.1 0.0 1.8 0.60 A reversible reaction was also noticed by measuring the decrease in wave length which indicated the oxidation of NADPH to NADP. This means that both the oxidation and reduction were coupled. Our results are in accordance with those found by some yeasts as in Candida shehatae and Pichia stipitis (Dupreez et al., 1986); Pachysolen tannophilus (Morimoto et al., 1986) and Pichia quercuum described by (Suzuki and Onishi, 1975) 96

that an unusual pathway for xylose oxidation to xylonic acid by xylose dehydrogenase enzyme found in cell free extract. In our study for the substrate specificity of D-xylose-dehyrogenase different monosugars were tested as substrate in Table (2). We can see that both D-xylose and L-arabinose were fit for the dehydrogenase enzyme, while the other sugars showed no activity. Table 2: substrate specificity of D-xylose dehydrogenase in cell-free extract of H. polymorpha. Substrate Coenzyme Activity (µ/ml) Relative Activity D-xylose NADP 0.29 100 L-arabinose NADP 0.0.13 44 D-arabinose NADP 0.0 0.0 D-glucose NADP 0.0 0.0 D-Mannose NADP 0.0 0.0 D-galactose NADP 0.0 0.0 The effect of the growth time on the enzyme production was proved by determination of enzyme activity after 1, 2, 3 and 5 days. No activity was observed after 3 days; while the activity decreased to about to 1/3 after 2 days as compared with the first day as indicated in Table (3). Table 3: Effect of time of growth on the D-xylose-dehydrogenase activity. Dehydrogase activity (Days) Activity (U/ml) Relative activity (%) 1 0.09 100 2 0.03 33 3 0.0 0.0 5 0.0 0.0 Proved Of Oxidation-Reduction Reaction: Coupling reaction was fund as oxidation and reduction of D- xylose with cell free extract of H. polymorpha. This result was confirmed through the analysis of the enzymatic reaction products, xylose, xylitol and xylonic acid were detected by HPLC. D-Xylose Reductase (EC 1.1.1.21): D-xylose reductase activity in cell-free extract H. polymorpha was measured spectrophotometrically by following the decrease in the optical density at 340 mm due to the oxidation of NADPH (Fig. 1) in the presence of D-xylose as substrate. Minimal oxidation of NADPH was observed in the control experiment which was run without D-xylose. Fig. 1: Reduction of xylose by xylose reductase in cell free extract of H. polymorpha. In agreement with the obtained results of xylose reductase enzyme which, catalyzes reduction of D-xylose to xylitol, such results were found with some microorganisms such as Pachysolen tannophilus (Samiley and Boien, 1982; Ligthelm et al., 1988a); Candida guillermondii and Candida Parapsilosis (Nolleau et al., 1993) Kluyveromyces marxianuse (Mueller, 2011). 97

The substrate specificity of xylose reductase activity in the cell free extract of H. polymorpha was measured using either D-xylose, D-arabinose, L-arabinose, D-glucose, D-Mannose and D-galactose at concentration of 0.5M as described in Materials and Methods. The results in Table (4) show that both D-xylose and L-arabinose are substrate for this enzyme. No activity was observed with either D-arabinose or D-galactose. Low activity was observed when D-glucose or D-mannose was the substrate. In general; the best substrate were those with hydroxyl group at carbon-2 in the threo-configuration as reported by Ahmed (1990) for Rhodotorula rubra and Candida Pseudotropicalis, it also reported by Verduyn et al. (1985) in contrary to these results D-xylose reductase of Enterobacter liquefaciens showed activity with most monosaccharides with only little differences (Yoshitake et al., l976). Table 4: Substrate Specificity of D-xylose reductase of cell free extract H. polymorpha. Substrate Co enzyme Activity (u/ml) Relative activity (%) D-xylose NADPH 0.4 100 D-Arabinose NADPH 0.00 0.00 L-Arabinose NADPH 0.15 37.5 D-glucose NADPH 0.06 15.0 D-Mannose NADPH 0.04 10.0 D-galactose NADPH 0.0 0.0 The cell free extract of H. polymorpha was allowed to react with D-xylose in the presence of two cofactors. As Table (5) shows the enzyme activity was NADPH dependent, but has slight activity with NADH. Table 5: Coenzyme specificity of D-Xylose reductase in cell free extract of H. polymorpha. Substrate Co enzyme u/ml Relative activity D-Xylose NADPH 0.14 100 D-Xylose NADH 0.02 14 Xylitol Dehydrogenase (EC. 1.1.1.10): Xylitol dehydrogenase activity in the cell free extract of H. polymorpha was examined with either NADP or NAD as cofactor (Fig. 2). NAD was gradually reduced by xylitol, while, NADP shows very low activity. A control experiment was done without xylitol. Fig. 2: Xylitol dehydrogenase activity in cell free extract of H. polymorpha. The product of the reaction of xylitol dehydrgenase with xylitol is xylulose which gave blue-violet color, an absorption at 540nm (Dische and Barenfreund, 1951). The obtained results are in accordance with those found by Lighthelm el al., (1988b) and Toyler (1993). The substrate specificity of xylitol dehydrogenase on several polyalcohols such as D-mannitol; D-sorbitol; D-xylitol, arabitol; and erythritol was studied and the results are reported in Table (6). The results show that the enzyme could oxidize a variety of polyols in the presence of NAD. Xylitol was the best substrate for the enzyme. Different yeasts were described to have xylitol dehydrogenase enzyme which oxidize xylitol to xylulose, then xylulose phosphorelated and enters the pentose phosphate cycle and is eventually converted to ethanol. Our previous founding are confirmed by (Lighthelm et al., 1988b) for Pichia stipitis. Also, a different pathway was found in Zynomonas mobilis bacterial Feldmann et al. (1992) and Rhadotorula gracilis (Hofer et al., 1971) where D-xylose is directly isomerized to xylulose. 98

Table 6: Polyols as substrates of Xylitol dehydrogenase in cell free extract of H. polymorpha. Polyols test U/ml protein (mg/ml) Specific activity (u/mg) Relative activity (%) Xylitol Arabitol Sorbitol Mannitol Erthritol 0.4 0.15 0.1 0.08 0.02 2.22 0.83 0.56 0.44 0.11 100 37.5 25 20 05 Conclusion: The utilization of D-xvlose by Kbulgaricus appears to involve the pathways showed in the following scheme: The presence of such enzyme in H. polymorpha may be of high importance for the utilization of agricultural wastes containing xylose such as corn cobs. For this reason further studies have to be conducted in this field. REFERENCES Ahmed, Y.M., 1990. Biochemical studies on the use of farm-ketosugars and sugar-alcohols by microorganisms. Ph.D. Thesis, Cairo, Univ. Barnett, J.A., 1976. Utilization of sugars by yeasts. Adv. Carbohydr. Chem. Biochem., 32: 125-234. Bettiga, M., O. Bengtsson, B. Hahn-Hägerdal and M.F. Gorwa-Grauslund, 2009. Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway.microbial Cell Factories, 8: 40-51. http://www.microbialcellfactories.com/content/8/1/40. Bicho, P.A., P.L. Runnals, J.D. Cunningham and H. Lee, 1988. Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars. Appl. Environ. Microbiol., 54: 50-54. Bruinenberg, P.M. and J.P. Van Dijken, 1983. An enzymatic analysis of NAPDH production and consumption in Candida utilis. J. Gen. Microbiol., 129: 965-971. Dische, Z. and E. Barenfreund, 1951. A new spectrophotometric method for the detection and determination of ketosugars and trioses. J. Biol. Chenx., 192: 583-589. Dupreez, J.C., M. Bosth and B.A. Prior, 1986. Xylose fermentation by Candida shehatae and Pichia stipitis: effect of ph, temperature and substrate concentration. Enzyme Microbiol. Technol., 8: 360-364. Feldmann, S.D., H. Sahm and G.A. Sprenger, 1992. Pentose metabolism in Zymomonas mobilis wild ype and recombinant strains. Appl. Microbiol. Biotechnol., 38: 354-361. Hofer, M.L., A. Betz and A. Kotyk, 1971. Metabolism of the obligatory aerobic yeast Rhodotorula gracilis IV: Induction of enzyme Biophys. Acta., 252: 1-12. Lee, H., A.P. James, D.M. Zahab, G. Mahmourides, R. Maleszka and H. Schneider, 1986. Mutants of Pachysolen tannophilus with improved production of ethanol from D-Xylose. Appl. Environ. Microbiol., 51: 1252-1258. Lee, Y.Y., C.M. Lin, T. Johnson and R.P. Chambers, 1979. Selective hydrolysis of hardwood hemicellulose by acids. BiotechnolBioeng Symp., 8: 75-88. Ligthelm, M.E., B.A. Prior and J.C. Dupreez, 1988a. The induction of D- xylose catabolizing relationship to anaerobic D-xylose fermentation. Biotechnol. Lett., 10: 207-212. 99

Ligthelm, M.E., B.A. Prior, J.C. Du Preez and V. Brandt, 1988b. An investigation of D-[1-'3C]xylose metabolism in Pichia stipitis under aerobic and anaerobic conditions. Appl. Microbiol. Biotechnol., 28: 293-296. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Maleszka, R., L.G. Neirinck, A.P. James, H. Rutten and H. Schneider, 1983. Xylitol dehydrogenase mutants of Pachysolen tannophilus and the role of xylitol in D-xylose catabolism. FEMS Microbiol. Lett., 17: 227-229. Moore, S. and K.P. Link, 1940. Carbohydrate characterization. I. The oxidation of aldoses by hypoiodide in Methanol II. The identification of seven aldo-monosaccharides as as benzimidazole derivatives. J. Biol. Chem., 133: 293-311. Morimoto, S., M. Matsuo, K. Azuma and A.J. Sinskey, 1986. Purification and Properties of D-Xylose reductase from Pachysolen tannophillus. J. Ferment. Technol., 64: 219-25. Mueller, M., M.R. Wilkins and I.M. Banat, 2011. Production of Xylitol by the Thermotolerant Kluyveromyces marxianus. J. Bioprocess Biotechniq., 1: 1-5. http://dx.doi.org/10.4172/2155-9821.1000102e. Nolleau, V., L. Preziasi-Belloy, J.P. Delgenes and J.M. Novarro, 1993. Xylitol Production from xylose by two yeast strains: Sugar tolerance. Curr. Microbiol., 27: 191-197. Samiley, K.L. and P.L. Bolen, 1982. Demonstration of D-Xylose reductase and D-Xylitol dehydrogenase in Pachysolen tannophilus. Biotech. Lett., 4: 607. Skoog, K. and B. Hahn-Hagerdal, 1988. Xylose fermentation. Enzyme Microbiol. Technol., 10: 66-80. Suzuki, J. and H. Onishi, 1973. Oxidation adn reduction of D-xylose by cell-free extract of Pichia quercuum. Appl. Microbiol., 25: 850-852. Suzuki, T. and H. Onishi, 1975. Purification and properties of polyol: NADP oxidoreductase from Pichia quercuum. Agric. and Biol. Chemist., 39: 2389-2397. Townsend, A.R. and R.W. Howarth, 2010. Fixing the global nitrogen problem. New Scientific American Inc: 64-71. Toyler, J., 1993. Characterization of ethanol production from xylose and xylitol by a cell free Pochysolen tannophillus. Appl. Environ. Microbiol., 59: 231-235. Verduyn, C., R. Vankleef, J.J. Frand, H.J. Schreuden, J.P. Van Dijken and W.A. Scheffers, 1985. Properties of the NADP (H) dependent Xylose reductase from the xylose fermenting yeast. Pichia stipitis. Biochem J., 226: 669-677. Weber, C., A. Farwick, F. Benisch, D. Brat, H. Dietz, T. Subtil and E. Boles, 2010. Trends and challenges in the microbial production of lignocellulosic bioalcohol fuels. Appl. Microbiol. Biotechnol., 87: 1303-1315. Winkelhausen, E. and S. Kuzmanova, 1998. Review: Microbial conversion of D-xylose to xylitol. J. Ferment. Bioeng., 86: 1-14 Yoshitake, I., M. Shimamura, H. Ishizuki and Y. Irie, 1976. Xylitol Production by Enterobacter liquefaciens. Agric. Biol. Chem., 40: 1493-1503. 100