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Characterization of β-xylosidase enzyme from a Pichia stipitis mutant
Bioresource Technology 99 (2008) 38–43
Characterization of b-xylosidase enzyme from a Pichia stipitis mutant Pervin Basaran b
a,b,*
, Meltem Ozcan
q
b
a Institute for Prospective Technological Studies, Joint Research Center, Sevilla, Spain Department of Food Engineering, College of Agriculture, Suleyman Demirel University, Cunur/Isparta, Turkey
Received 10 October 2006; received in revised form 26 November 2006; accepted 27 November 2006 Available online 8 March 2007
Abstract b-Xylosidase production was maximal for the mutant Pichia stipitis NP54376 grown on xylan as the sole carbon source. b-Xylosidase was purified from culture supernatant by (NH4)2SO4 precipitation and a hydrophobic interaction chromatography on phenyl sepharose. Optima of pH and temperature were 5.0 and 50 °C, respectively. The enzyme was inhibited by 2-mercaptoethanol (100%) and Fe3+ 2+ (80%), and moderately affected by Cu2+, Ag+, NHþ and SDS. The purified xylosidase hydrolyzed xylobiose and xylo-oligo4 and Mg saccharides and it did not exhibit activity against cellulose, starch, maltose and cellobiose. 2.5 g l1 glucose repressed b-xylosidase activity in the NP54376 strain. The Km and Vmax values on p-nitrophenyl-b-xylopyranoside were 1.6 mM and 186 lmol p-nitrophenyl min1 mg1 protein, respectively. Analysis of the hydrolysis products by HPLC indicated that the major hydrolysis product is xylobiose in all the carbon sources tested. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Mutant; b-Xylosidase; Pichia stipitis
1. Introduction Plant cell walls are composed of three major polymers cellulose, hemicellulose (xylan, mannans, and gluconase) and lignin (Coughlan, 1992). Xylan is one of the major heteropolysaccharides present in plant cell walls, and its full hydrolysis requires coordinated action of a number of enzymatic activity including xylanase, xylulokinase, acetyl xylan esterase arabino-furanosidase and b-xylosidase (Passoth and Hagerdal, 2000; Basaran et al., 2001; Basaran and Hang, 2000; Basaran et al., 2000). Xylosidase enzyme or 1,4-beta-D xylohydrolase (EC 3.2.1.37) is an exoglycosidase that belongs to an enzyme complex which releases
q Disclaimer: The views expressed in this study do not necessarily reflect those of the European Commission (EC). * Corresponding author. Present address (On sabbatical leave): Institute for Prospective Technological Studies (IPTS), Sustainability in Agriculture, Food and Health Unit, European Commission, Joint Research Center Edificio EXPO-Calle Inca Garcilaso, s/n-E 41092 Sevilla, Spain. Tel.: +90 542 431 0413; fax: +90 246 211 1634. E-mail address: [email protected] (P. Basaran).
0960-8524/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.11.056
xylosyl residues by endwise attack of xylo-oligosaccharides and xylobiose. Xylosidase is important in xylan degradation, considering that xylans are not completely hydrolyzed by xylanases alone. Many microorganisms can produce b-xylosidase, however only few yeast species and limited number of strains can produce xylosidase enzyme (Linden and Hahn-Hagerdal, 1989). Environmental concerns and increasing oil prices made ethanol as an alternative energy source, and therefore isolating the yeast strains capable of fermenting xylose to maximize ethanol production from xylose from agricultural wastes is becoming of great importance. Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose (Ho et al., 1998). Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose; whereas only some strains of the yeast P. stipitis is capable of effective co-fermentation of glucose and xylose (Ho et al., 1998). The cost of enzymes is one of the factors determining the economics of a biocatalytic process, and can be reduced by finding optimum conditions for their production, by isolating hyperproducing strains through screening and selection
P. Basaran, M. Ozcan / Bioresource Technology 99 (2008) 38–43
of induced mutants (Rodriguez and Alea, 1992; PonceNoyola and de la Torre, 1995) and by constructing efficient producers using genetic engineering (Basaran et al., 2001; Bajpai, 1997). Mutant NP54376 isolated in our previous study (Basaran et al., 2001) has a high xylanolytic activity when it grows on various xylan sources, and here we describe a detailed description of characterization on one of the xylanolytic enzymes crucial for the fermentation of xylose to ethanol. The objective of this study was to characterize b-xylosidase enzyme in the Pichia mutant NP54376 in detail. 2. Methods 2.1. Culturing conditions To provide inocula, cells were grown at 30 °C on an orbital shaker at 150 rpm in Erlenmeyer-flasks containing xylose medium and Yeast Nitrogen Base (YNB). The growth was monitored by recording absorbance at 600 nm at regular intervals. Cells were collected in the exponential phase, and separated aseptically from the supernatant solution by centrifugation (4 °C, 5000g, 20 min) and washed twice with 0.85% peptone water. Cells were resuspended in xylan medium and used as inoculum (3% (v/v)) for the enzyme production experiments. The cultivation medium for enzyme production consisted of hemicellulosic hydrolysate (birchwood, oat, beechwood xylan and corn cob) supplemented with 0.7% YNB in medium. The cultivation was carried out for 96 h in Erlenmeyer flasks (125 ml) containing 50 ml of medium at 30 °C and initial pH 5.5 (uncontrolled). Agricultural by product (corn cob) were chopped and dried at 100 °C for overnight and either directly added in the range of 1–3% to the medium or after steam hydrolysis at 121 °C for 30 min. Corn cob concentrations higher than 3% were not used due to the increase in medium viscosity. In all cases, samples were taken every 24 h, and pH, extracellular protein content, growth and enzyme activity were determined. 2.2. Enzyme production and purification Enzyme production experiments were conducted in 300ml Erlenmeyer flasks each containing 100 ml of 0.67% YNB and 0.5% xylan. After sterilization, each flask was inoculated with 3 ml of inoculum and cultivated at 30 °C in a rotary shaker operated at 140 rpm for 48 h. They were centrifuged at 14,000g for 20 min. (NH4)2SO4 was added to the culture supernatant to make 1 M concentration and mixed well, whereupon the solution was loaded onto a phenyl sepharose (Sigma) HIC column that had been equilibrated with 1.2 M (NH4)2SO4. Elution was performed as a step gradient: First with 3–4 column volumes of 0.5 M (NH4)2SO4 10 M NaCl in 5 mM (NH4)2SO4; next 2–3 volumes of 0.25 M (NH4)2SO4 10 mM NaCl in 5 mM (NH4)2SO4; and finally 5 mM K2PO4 (pH 6.0). Fractions containing xylosidase activity were pooled. The partially
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purified enzyme was dialyzed against 0.5 mM Tris–HCl buffer (pH 7.0). The dialysate was centrifuged at 7000g for 15 min, then loaded onto a Superdex 200 26/10 (Pharmacia) gel filtration column running 50 mM Tris–HCl buffer pH 7.0 and 0.02% NaN3. One peak showing xylosidase activity was pooled. The fraction was concentrated 15 fold using Biomax-30 high flux polysulfane membrane (Millipore) with a cut off of 20 kDa. The enzyme fraction was further concentrated with a 2 ml Centricon concentrator (Amicon). This purification procedure resulted in about 30 mg of nearly homogeneous enzyme from 3 l culture, with an overall yield of 40% and 5-fold purification. Cell extracts were obtained as follow. The overnight culture was harvested at exponential phase in the respective medium, resuspended in buffer (60 ml of 50 mM Tris–HCl and 100 mM NaCl pH 7.0) and disrupted by two passages through a French press. After centrifugation in the cold at 8000g for 15 min, the supernatants were used for enzyme assays and determination of protein concentration. The entire procedure was carried out at 4 °C. 2.3. Analytical methods The reaction medium containing 250 ll of 2 mM pnitrophenyl-b-xylopyranoside (pNPX) (50 mM phosphate buffer (pH 5)) and suitably diluted enzyme was incubated in a total volume of 0.5 ml at 45 °C for 30 min. The reaction was stopped by the addition of 1 ml of 2 M Na2CO3 solution, and the amount of p-nitrophenyl released was determined by measuring the absorbance at 405 nm and compared with a standard reference. One unit of b-xylosidase is defined as the amount of enzyme required to release of nmol p-nitrophenyl per hour under the conditions described above (Biely et al., 1980). The initial trials showed that the enzyme was stable under the assay conditions and the hydrolysis rates that were measured behave linear during assay. The kinetic constants (Km and Vmax) of the purified enzyme for the hydrolysis of pNPX were estimated by the method of Lineweaver and Burk (1934). Protein was determined by the dye-binding method (Bradford, 1976) using serum albumin as the standard and specific activity was defined as U mg1 of protein. Growth rate was determined by direct cell count in a Neubauer chamber (Basaran et al., 2001). 2.4. Effect of pH and temperature on activity The effect of pH on xylosidase enzymatic activity was measured as follow. Reactions were carried out in 12 ml glass tubes placed in a water bath at 45 °C with pNPX as a substrate. The mixtures contained 500 ll of 5 mM substrate and appropriately diluted enzyme in 50 mM of the buffer (pH 3.0 and 8.3, dimethyl glutamic acid (pH 3.0– 5.0); phosphate buffer (pH 6.0–8.0) and Tris–HCl buffer (pH 7.5–9.0). The reactions were stopped by the addition of Na2CO3 (final concentration of 0.3 M), and the released
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P. Basaran, M. Ozcan / Bioresource Technology 99 (2008) 38–43
p-nitrophenyl was determined. To analyze the effect of pH on xylosidase stability, the enzyme solution was incubated for 2 h at 45 °C in buffers at the above pHs, and residual enzyme activity was assayed as described above. The effect of temperature on xylosidase activity was measured by incubating the enzyme reaction mixture 500 ll containing xylosidase, 50 mM sodium acetate (pH 6.0) buffer at various temperatures from 20 to 80 °C. The thermal stability of xylosidase was measured by incubating the enzyme solution at various temperatures for 2 h, then cooling it in cold water. The remaining activity was assayed as described before at 45 °C. 2.5. Effect of chemicals on xylosidase activity Different concentrations of Tween 80 and Triton X100 (0.1–0.5%) were tested into the medium containing birchwood xylan, and its effect on xylosidase enzyme production was examined. In order to determine the effect of various chemicals, the enzyme solution (2.5 U) was incubated with various chemicals (10 mM or 1 mM in acetate buffer pH 5.0) in a total volume of 1 ml at 45 °C for 1 h. The remaining xylosidase activity was determined as described earlier. 2.6. Analysis of enzymatic hydrolysis products by HPLC Sugars and other fermentation products resulting from enzymatic hydrolysis of various xylan sources were analyzed by HPLC (Hewlett Packard). The culture was grown to late exponential phase and the supernatant fluid was removed by centrifugation at 15,000g frozen and lyophilized to dryness and kept refrigerated at 20 °C until analysis. Dried supernatant was suspended in 50 mM Tris–HCl buffer (pH 5) to give a 1% final concentration. The enzymatic hydrolysis was terminated by adding 1 ml of 0.3 N zinc sulfate (Sigma), and 1 ml 0.3 N barium hydroxide (Sigma). The samples to test were centrifuged for 45 min, at 10,000g at 4 °C. Then they were conserved on ice and filtered through a cellulose nitrate membrane filter (Gelman), to remove unhydrolyzed xylan and other insoluble contaminants. Analysis was performed with Aminex HPX-87 H column (BioRad) with water MilliQ as mobile phase at a flow rate 0.5 ml min1, at 65 °C. The peaks were identified by comparison of the relative retention time of pure standards, glucose, xylose (X1), xylobiose (X2), xylotriose (X3), and other xylo-oligosaccharide mixtures (X4, X5) (Sigma). Waters Millennium Chromatography Manager Software was used for quantification. 2.7. Amino acid analysis The xylosidase band was excised and hydrolyzed for 90 min with 6 N HCl at 150 °C prior to column fractionation and subjected to amino acid analysis using an Applied Biosystems Mode 420 amino acid derivatizer system. The N-terminal amino acid sequence of the purified xylosidase was compared with protein sequence data avail-
able at National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and European Bioinformatics Institute (EBI, http://srs.ebi.ac.uk/) databases using the on-line BLAST network service. 3. Results and discussion P. stipitis NP54376 is a high-growth-rate mutant obtained after NTG mutagenic treatment (Basaran and Hang, 2000). The mutant NP54376 assimilates xylose faster than the wild type; is able to use a larger portion of xylan sources than the wild strain, nevertheless, the mechanism of this mutational change is unclear (Basaran et al., 2001). Other strains of P. stipitis (Y-11544, Y-11545, Y-7124) tested were only in the range of 80–85% of the maximal activity produced by P. stipitis mutant. For P. stipitis, bxylosidase activities found in the culture supernatants of 3-day xylose- and xylan-grown cells were 25 and 29 mU/ ml/h, respectively (Ozcan et al., 1991), that is in good agreement with our mutant data for extracellular bxylosidase. The production of xylosidase by NP54376 in shake-flask culture was dependent on culturing conditions. Maximum growth as well as xylanase and xylosidase production were found to be around pH 5.0, pH values above or below lessened the enzyme production (Basaran, 1999). During the cultivation the pH dropped from the initial pH of 5.0 to pH 3.0–3.5. A study by Haltrich et al., 1994) indicated that low pH was essential since regulating the pH at a constant value of 5.0 resulted in excretion of largely oxalic acid which led to decrease in growth of Sclerotium rolfsii (Haltrich et al., 1994). Furthermore, there have been reports for several fungi where cultivation of the organism at an unfavorable pH resulted in limited grow rate and increased yields of xylanolytic enzyme activity (Haltrich et al., 1994). The reason for this might be the reduced accessibility of the hemicellulose substrate due to the unfavorable conditions. Enzyme production by NP54376 was enhanced by increasing the shaking speed up to 180 rpm but thereafter higher values inhibited the enzyme production. Reduced enzyme activity associated with the high shaking speed could be caused by shear forces, which may cause leakage of intracellular material and probably disrupting enzyme synthesis (Warzywoda et al., 1992). The xylosidase activity was observed maximum at pH 4.8–5.0. Only a few bacterial xylanases were reported to have a high level of activity at pH values higher than 7, most xylanolytic enzymes fall pH values between 4.0 and 6.7 (Shao et al., 1995). The Pichia xylosidase was stable in a pH range of 3.5–7.2. The optimum temperature for enzyme activity was 45 °C, which was similar to many of the yeast proteins (Haltrich et al., 1994; Warzywoda et al., 1992). Xylosidase from NP54376 was stable for 30 min at 60 °C. However, it lost 20% of its activity above 50 °C. The thermostability of the xylosidase from mutant NP54376 falls into the previously reported ranges (25– 60 °C) (Du Preez et al., 1987).
P. Basaran, M. Ozcan / Bioresource Technology 99 (2008) 38–43
b-Xylosidase production by the mutant NP54376 was similar when grown on beechwood, birchwood, oatspelt xylan and xylose (max of 0.018 U/ml). When cultivated on pretreated corn cob hydrolysate, a 40% lower b-xylosidase activity was observed. Addition of peptone to corn cob medium did not affect xylosidase production, whereas yeast extract increased enzyme production by 10%. Several other researchers have reported microbial inhibition effect when utilizing hydrolysate from lignocellulosic material (Perttunen et al., 1996; Preziosi-Belloy et al., 1997). Xylosidase production was repressed upon addition of different sugars. At a concentration of 3% (w/w), lactose showed significant repression of xylosidase production (50%), whereas the effect of sucrose was mild (20%). Xylosidase was repressed by the addition of glucose (1.0; 1.5; 2.0 or 2.5 g l1) to the culture medium containing 1% birchwood xylan. Xylosidase and xylanase activities from wild strain and mutant NP54376 were sharply decreased when glucose (2.5 g l1) was added into the birchwood medium. Xylose, an end-product of xylosidase activity, has been reported as an inhibitor of enzymatic activity in some cases (Biely et al., 1991). In order to determine the minimum xylose concentration required to inhibit the xylanolytic systems on P. stipitis wild type and its mutant NP54376, they were grown on beechwood xylan supplemented with xylose. At a concentration of 5 gl1, xylose completely repressed xylosidase production. The crude xylosidase was active on xylooligosaccharides but was not active on starch, cellulose, maltose and cellobiose. The effect of different concentrations of additives on the thermostability of xylosidase was measured in terms of protective effect, which is defined as the ratio of xylosidase half-life in the presence and absence of an additive. The addition of Tween 80 (0.1%) did not prevent the loss in activity at 80 °C. Similarly, COCl2, CaCl2, KCl and NaCl, at a concentration of 10 mM did not have any effect on the stabilization of xylosidase. Xylosidase activity was assayed with and without the presence of several metals (FeSO4, CuSO4, MgSO4, and AgNO3), a metal chelator (EDTA) and protein disulphide reducing agents, DTT and 2-mercaptoethanol, at 1 mM. The metal ions that inhibit the xylosidase activity were: strong inhibition Fe3+ (80%), and moderate (less than 20%) effect Cu2+, Ag+, and Mg2+. When 2-mercaptoethanol at 1 mM concentration was tested, a total loss of the activity was observed. 2-Mercaptoethanol, counteract the oxidative effects of S-S linkages, presumably from cysteine residues. A number of fungal and bacterial xylan hydrolyzing enzymes have been reported to poses cysteine residues essential for activity (Coughlan, 1992). When the amino acid composition of xylosidase is analyzed, it was observed that the enzyme is rich in Cysteine (10%) and Methionine (6%) residues. Substrate specificity of the purified b-linked saccharide derivatives. The b-xylosidase showed no action on phenyl b-D galactoside and p-nitrophenyl a-arabinoside. The effect of different polyols (2 M) (Ethylene glycol (C2), glycerol (C3), sorbitol and mannitol
41
(C4)) on xylosidase deactivation was studied at 45 °C. An increase in the concentration of sorbitol resulted in an increase in its protective effect on xylosidase, and sorbitol provided maximum protection at a concentration of 2 M. The stabilizing effect of polyols depends on hydrophilic and hydrophobic characteristics of the enzyme; polyols have the capability to form hydrogen bonds that play the key role in supporting the native conformation of the protein and aid in protein stabilization (Wong et al., 1988). With P. stipitis 60% of the b-xylosidase activity was detected in culture supernatant and about 20% of the activity was found in the crude extracts (Ozcan et al., 1991). Stimulation of growth as well as enzyme or protein secretion of different microorganisms by surfactants has been reported in earlier studies (Konopka and Zakharova, 2002). Addition of Triton X100 at 0.6–1 g l1 to the culture broth after termination of its cultivation resulted in approximately 2.2–2.4-fold increase in xylanase yields in Rhodothermus marinus (Hregvidsson et al., 1996; Manelius et al., 1994). From the known membrane bound status of b-xylosidase, we have tested the stimulative effect of Triton X100, and when the cells were treated with the surfactant, no significant effect on the enzyme production was observed. Xylooligosaccharides, the potential inducers, should be produced by through a consequential reaction, and the rate of product generation is predicted to decrease when the concentration of xylan increases. To determine the hydrolytic pattern, the degradation products released from birchwood, oatspelt and beechwood xylans were analyzed. The composition of the hydrolysis products of P. stipitis xylosidase from various xylans is shown in Fig. 1. Results presented here demonstrated that the source of the xylan significantly effected the product composition. The composition of the oligosaccharides from hydrolysis in all three xylan sources consisted of mainly xylobiose. The production of xylobiose was found to be the fastest with 90% production within 24 h compared to 50–60% of xylotriose after 24 h. The xylobiose concentration increased by the time with birchwood reaching maximum of amount of 0.94 mg/ml after three days of incubation. Oat spelt and beechwood produced similar amounts of xylobiose 0.53– 0.5 mg/ml that was half of the xylobiose concentration produced with birchwood (Fig. 1b and c). With oatwood (Fig. 1b) and birchwood (Fig. 1c) xylans, xylotriose concentration remained same throughout the fermentation, 0.2 mg/ml and 0.5 mg/ml, respectively. In all three xylan sources similar amounts of xylose were detected. However, the relatively fast release and conservation of the amount of xylose released indicated that limiting sugar for hydrolysis was the monomer xylose. Our results were in good agreement with b-xylosidase of Malbranchea pulchella that was acting on xylan in limited rate and other xylosidases such as those from Trichoderma viride, Emericella nidulans had almost no effect on the direct hydrolyses of xylan (Matsuo et al., 1977a,b).
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P. Basaran, M. Ozcan / Bioresource Technology 99 (2008) 38–43
clone the hydrolytic enzymes coding sequences into yeasts to create new enzyme production systems with the possible improvement of enzyme production and activity for commercial purposes (Akada, 2002). Pichia yeast strains may fit to fulfill requests of industrial production of enzymes and fermentation. However, the mechanism of action and properties of the enzymatic system needs to be studied in detail.
Birchwood Xylan Hydrolysis
1 0.8 0.6 mg/ml
0.4 0.2
Xylobiose Xylotriose Xylose
0 Day 1 Day 2
Day 3 Day 4 DAYS
1 0.8 mg/ml 0.6 0.4
0 Day 1 Day 2 Day 3 Day 4 DAYS
Xylobiose Xylotriose Xylose
Beechwood Xylan Hydrolysis 1 0.8 0.6 mg/ml 0.4 0.2 0 Day 1 Day 2 Day 3 Day 4 DAYS
We would like to thank A. Artac and M. Selcuk for their contribution for the manuscript. References
Oat Spelt Xylan Hydrolysis
0.2
Acknowledgements
Xylobiose Xylotriose Xylose
Fig. 1. HPLC analysis of hydrolysis products of NP54376 when incubated in various xylan sources (a) Birchwood xylan, (b) Oat spelt xylan, (c) Beechwood xylan. The values are means of at least three measurements (above bars are standard deviations). Xylan incubated without enzyme was taken as 0 time and its sugar contents were deduced from each oligosaccharide.
The amino acid sequence of the N-terminal of P. stipitis xylosidase was determined to be Ala-Iso-Met-Gly-Val. The N-terminal sequencing data of 37 kDa b-xylosidase has shown no homology with the N-terminal amino acid sequences of any of the xylosidase proteins available in the SWISS-Prot database (data not shown). Recently developed genetic techniques promise opportunities to
Akada, R., 2002. Review: Genetically modified industrial yeast ready for application. J. Biosci. Bioeng. 94, 536–544. Bajpai, P., 1997. Microbial xylanolytic enzyme system: properties and applications. Adv. Appl. Microbiol. 43, 141–194. Basaran, P., 1999. Production, characterization, cloning and sequencing of yeast xylanase. Ph.D. Thesis. Cornell University. Basaran, P., Hang, Y.D., 2000. Purification and characterization of acetyl esterase from Candida guilliermondii. Lett. Appl. Microbiol. 30, 167– 171. Basaran, P., Basaran, N., Hang, Y.D., 2000. Isolation and characterization of Pichia stipitis mutants with enhanced xylanase activity. World J. Microb. Biot. 16, 545–550. Basaran, P., Hang, Y.D., Worobo, R.W., Basaran, N., 2001. Cloning, sequencing and heterogeneous expression of a xylanase gene from Pichia stipitis in E. coli. J. Appl. Microbiol. 90, 248–255. Biely, P., Vrsanska, M., Kratky, Y., 1980. Xylan-degrading enzymes of the yeast Cryptococcus albidus. Eur. J. Biochem. 108, 313–321. Biely, P., Vrsanska, M., Kucar, S., 1991. Identification and mode of endob1 leads to greater than 4 b-beta-xylanases. In: Progress in Biotechnology, 7. Elsevier, pp. 81–95. Bradford, M.M., 1976. A rapid sensitive method for the quantitation of protein utilization the principle of protein–dye binding. Anal. Biochem. 72, 248–252. Du Preez, J.C., Bosch, M., Prior, B.A., 1987. Temperature profiles of growth and ethanol tolerance of the xylose-fermenting yeasts Candida shehatae and Pichia stipitis. Appl. Microbiol. Biotechnol. 25, 521–525. Coughlan, M.P., 1992. Towards the understanding of the mechanism of action of main chain-hydrolzing xylanases. In: Visser, J., Beldman, G., Kusters-van Someren, MAR., Voagen, A.G.J. (Eds.), Xylans and xylanases, . In: Progress in Biotechnology, 7. Elsevier, pp. 111–141. Haltrich, D., Laussamayer, B., Steiner, W., Nidetzky, B., Kulbe, K.D., 1994. Cellulolytic and hemicellulolytic enzymes of Sclerotium rolfii: optimization of the culture medium and enzymatic hydrolysis of lignocellulosic material. Biores. Technol. 50, 43–50. Ho, N.W.Y., Chen, Z., Brainard, A.P., 1998. Genetically engineered Saccharomyces yeast capable of effective cofermentation of glucose and xylose. Appl. Environ. Microb. 64, 1852–1859. Hregvidsson, G.O., Kaiste, E., Holst, O., Eggertsson, G., Palsdottir, A., Kristjansson, J.K., 1996. An extremely thermostable cellulase from the thermophilic eubacterium Rhodothermus marinus. Appl. Environ. Microb. 62, 3047–3049. Konopka, A., Zakharova, T., 2002. Evaluation of methods to solubilize and analyze cell-associated ectoenzymes. J. Microbiol. Meth. 53, 273– 282. Linden, T., Hahn-Hagerdal, B., 1989. Fermentation of lignocellulose hydrolysates with yeasts and xylose isomerase. Enzyme Microb. Techn. 11, 583–589. Lineweaver, H., Burk, D., 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56, 658–666.
P. Basaran, M. Ozcan / Bioresource Technology 99 (2008) 38–43 Manelius, A., Dahlberg, L., Holst, O., 1994. Some properties of a thermostable b-xylosidase from Rhodothermus marinus. Appl. Biochem. Biotech. 44, 39–48. Matsuo, M., Yasui, T., Kobyashi, T., 1977a. Purification and some properties of b-xylosidase from Malbranchea pulchella var. sulferea. Agr. Biol. Chem. 41, 1601–1607. Matsuo, M., Yasui, T., Kobyashi, T., 1977b. Purification and some properties of b-xylosidae from Trichoderma viride. Agr. Biol. Chem. 48, 1845–1852. Ozcan, S., Kotter, P., Ciriacy, M., 1991. Xylan-hydrolysing enzymes of the yeast Pichia stipitis. Appl. Microbiol. Biotech. 36, 190–195. Passoth, V., Hagerdal, B., 2000. Production of a heterologous endo 1,4-bxylanase in the yeast Pichia stipitis with O2 regulated promoter. Enzyme Microb. Tech. 26, 781–784. Perttunen, J., Sohlo, J., Maentausta, O., 1996. The fermentation of birch hemicellulose liquor to lactic acid. In: Proceedings of the 6th Conference on Biotechnology in the Pulp and Paper Industry Chemical Abstracts CAN 683172, pp. 295–298.
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Ponce-Noyola, T., de la Torre, M., 1995. Isolation of a high-specificgrowth-rate mutant of Cellulomonas flavigena on sugar-cane bagasse. Appl. Microbiol. Biot. 42, 709–712. Preziosi-Belloy, J., Nolleau, V., Navarro, J.M., 1997. Fermentation of hemicellulosic sugars and sugar mixtures to xylitol by Candida parapsilosis. Enzyme Microb. Tech. 21, 124–129. Rodriguez, H., Alea, F., 1992. Regulation of xylanase activity in Cellulomonas sp. libc. Acta Biotechnol. 12, 337–343. Shao, W., Deblois, S., Wiegel, J., 1995. A high molecular weight cell associated xylanase isolated from exponentially growing Thermoanaerobacterium sp. Strain JW/SL-YS 485. Appl. Environ. Microb. 61, 937–940. Warzywoda, M., Larbre, E., Pourquie, J., 1992. Production and characterization of cellulolytic enzymes from Trichoderma reesei grown on various carbon sources. Biores. Technol. 9, 125–130. Wong, K.K.Y., Tan, L.U.L., Saddler, J.N., 1988. Multiplicity of b-1,4xylanases in microorganisms: functions and applications. Microbiol. Rev. 52, 305–317.
Characterization of b-xylosidase enzyme from a Pichia stipitis mutant Pervin Basaran b
a,b,*
, Meltem Ozcan
q
b
a Institute for Prospective Technological Studies, Joint Research Center, Sevilla, Spain Department of Food Engineering, College of Agriculture, Suleyman Demirel University, Cunur/Isparta, Turkey
Received 10 October 2006; received in revised form 26 November 2006; accepted 27 November 2006 Available online 8 March 2007
Abstract b-Xylosidase production was maximal for the mutant Pichia stipitis NP54376 grown on xylan as the sole carbon source. b-Xylosidase was purified from culture supernatant by (NH4)2SO4 precipitation and a hydrophobic interaction chromatography on phenyl sepharose. Optima of pH and temperature were 5.0 and 50 °C, respectively. The enzyme was inhibited by 2-mercaptoethanol (100%) and Fe3+ 2+ (80%), and moderately affected by Cu2+, Ag+, NHþ and SDS. The purified xylosidase hydrolyzed xylobiose and xylo-oligo4 and Mg saccharides and it did not exhibit activity against cellulose, starch, maltose and cellobiose. 2.5 g l1 glucose repressed b-xylosidase activity in the NP54376 strain. The Km and Vmax values on p-nitrophenyl-b-xylopyranoside were 1.6 mM and 186 lmol p-nitrophenyl min1 mg1 protein, respectively. Analysis of the hydrolysis products by HPLC indicated that the major hydrolysis product is xylobiose in all the carbon sources tested. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Mutant; b-Xylosidase; Pichia stipitis
1. Introduction Plant cell walls are composed of three major polymers cellulose, hemicellulose (xylan, mannans, and gluconase) and lignin (Coughlan, 1992). Xylan is one of the major heteropolysaccharides present in plant cell walls, and its full hydrolysis requires coordinated action of a number of enzymatic activity including xylanase, xylulokinase, acetyl xylan esterase arabino-furanosidase and b-xylosidase (Passoth and Hagerdal, 2000; Basaran et al., 2001; Basaran and Hang, 2000; Basaran et al., 2000). Xylosidase enzyme or 1,4-beta-D xylohydrolase (EC 3.2.1.37) is an exoglycosidase that belongs to an enzyme complex which releases
q Disclaimer: The views expressed in this study do not necessarily reflect those of the European Commission (EC). * Corresponding author. Present address (On sabbatical leave): Institute for Prospective Technological Studies (IPTS), Sustainability in Agriculture, Food and Health Unit, European Commission, Joint Research Center Edificio EXPO-Calle Inca Garcilaso, s/n-E 41092 Sevilla, Spain. Tel.: +90 542 431 0413; fax: +90 246 211 1634. E-mail address: [email protected] (P. Basaran).
0960-8524/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.11.056
xylosyl residues by endwise attack of xylo-oligosaccharides and xylobiose. Xylosidase is important in xylan degradation, considering that xylans are not completely hydrolyzed by xylanases alone. Many microorganisms can produce b-xylosidase, however only few yeast species and limited number of strains can produce xylosidase enzyme (Linden and Hahn-Hagerdal, 1989). Environmental concerns and increasing oil prices made ethanol as an alternative energy source, and therefore isolating the yeast strains capable of fermenting xylose to maximize ethanol production from xylose from agricultural wastes is becoming of great importance. Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose (Ho et al., 1998). Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose; whereas only some strains of the yeast P. stipitis is capable of effective co-fermentation of glucose and xylose (Ho et al., 1998). The cost of enzymes is one of the factors determining the economics of a biocatalytic process, and can be reduced by finding optimum conditions for their production, by isolating hyperproducing strains through screening and selection
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of induced mutants (Rodriguez and Alea, 1992; PonceNoyola and de la Torre, 1995) and by constructing efficient producers using genetic engineering (Basaran et al., 2001; Bajpai, 1997). Mutant NP54376 isolated in our previous study (Basaran et al., 2001) has a high xylanolytic activity when it grows on various xylan sources, and here we describe a detailed description of characterization on one of the xylanolytic enzymes crucial for the fermentation of xylose to ethanol. The objective of this study was to characterize b-xylosidase enzyme in the Pichia mutant NP54376 in detail. 2. Methods 2.1. Culturing conditions To provide inocula, cells were grown at 30 °C on an orbital shaker at 150 rpm in Erlenmeyer-flasks containing xylose medium and Yeast Nitrogen Base (YNB). The growth was monitored by recording absorbance at 600 nm at regular intervals. Cells were collected in the exponential phase, and separated aseptically from the supernatant solution by centrifugation (4 °C, 5000g, 20 min) and washed twice with 0.85% peptone water. Cells were resuspended in xylan medium and used as inoculum (3% (v/v)) for the enzyme production experiments. The cultivation medium for enzyme production consisted of hemicellulosic hydrolysate (birchwood, oat, beechwood xylan and corn cob) supplemented with 0.7% YNB in medium. The cultivation was carried out for 96 h in Erlenmeyer flasks (125 ml) containing 50 ml of medium at 30 °C and initial pH 5.5 (uncontrolled). Agricultural by product (corn cob) were chopped and dried at 100 °C for overnight and either directly added in the range of 1–3% to the medium or after steam hydrolysis at 121 °C for 30 min. Corn cob concentrations higher than 3% were not used due to the increase in medium viscosity. In all cases, samples were taken every 24 h, and pH, extracellular protein content, growth and enzyme activity were determined. 2.2. Enzyme production and purification Enzyme production experiments were conducted in 300ml Erlenmeyer flasks each containing 100 ml of 0.67% YNB and 0.5% xylan. After sterilization, each flask was inoculated with 3 ml of inoculum and cultivated at 30 °C in a rotary shaker operated at 140 rpm for 48 h. They were centrifuged at 14,000g for 20 min. (NH4)2SO4 was added to the culture supernatant to make 1 M concentration and mixed well, whereupon the solution was loaded onto a phenyl sepharose (Sigma) HIC column that had been equilibrated with 1.2 M (NH4)2SO4. Elution was performed as a step gradient: First with 3–4 column volumes of 0.5 M (NH4)2SO4 10 M NaCl in 5 mM (NH4)2SO4; next 2–3 volumes of 0.25 M (NH4)2SO4 10 mM NaCl in 5 mM (NH4)2SO4; and finally 5 mM K2PO4 (pH 6.0). Fractions containing xylosidase activity were pooled. The partially
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purified enzyme was dialyzed against 0.5 mM Tris–HCl buffer (pH 7.0). The dialysate was centrifuged at 7000g for 15 min, then loaded onto a Superdex 200 26/10 (Pharmacia) gel filtration column running 50 mM Tris–HCl buffer pH 7.0 and 0.02% NaN3. One peak showing xylosidase activity was pooled. The fraction was concentrated 15 fold using Biomax-30 high flux polysulfane membrane (Millipore) with a cut off of 20 kDa. The enzyme fraction was further concentrated with a 2 ml Centricon concentrator (Amicon). This purification procedure resulted in about 30 mg of nearly homogeneous enzyme from 3 l culture, with an overall yield of 40% and 5-fold purification. Cell extracts were obtained as follow. The overnight culture was harvested at exponential phase in the respective medium, resuspended in buffer (60 ml of 50 mM Tris–HCl and 100 mM NaCl pH 7.0) and disrupted by two passages through a French press. After centrifugation in the cold at 8000g for 15 min, the supernatants were used for enzyme assays and determination of protein concentration. The entire procedure was carried out at 4 °C. 2.3. Analytical methods The reaction medium containing 250 ll of 2 mM pnitrophenyl-b-xylopyranoside (pNPX) (50 mM phosphate buffer (pH 5)) and suitably diluted enzyme was incubated in a total volume of 0.5 ml at 45 °C for 30 min. The reaction was stopped by the addition of 1 ml of 2 M Na2CO3 solution, and the amount of p-nitrophenyl released was determined by measuring the absorbance at 405 nm and compared with a standard reference. One unit of b-xylosidase is defined as the amount of enzyme required to release of nmol p-nitrophenyl per hour under the conditions described above (Biely et al., 1980). The initial trials showed that the enzyme was stable under the assay conditions and the hydrolysis rates that were measured behave linear during assay. The kinetic constants (Km and Vmax) of the purified enzyme for the hydrolysis of pNPX were estimated by the method of Lineweaver and Burk (1934). Protein was determined by the dye-binding method (Bradford, 1976) using serum albumin as the standard and specific activity was defined as U mg1 of protein. Growth rate was determined by direct cell count in a Neubauer chamber (Basaran et al., 2001). 2.4. Effect of pH and temperature on activity The effect of pH on xylosidase enzymatic activity was measured as follow. Reactions were carried out in 12 ml glass tubes placed in a water bath at 45 °C with pNPX as a substrate. The mixtures contained 500 ll of 5 mM substrate and appropriately diluted enzyme in 50 mM of the buffer (pH 3.0 and 8.3, dimethyl glutamic acid (pH 3.0– 5.0); phosphate buffer (pH 6.0–8.0) and Tris–HCl buffer (pH 7.5–9.0). The reactions were stopped by the addition of Na2CO3 (final concentration of 0.3 M), and the released
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p-nitrophenyl was determined. To analyze the effect of pH on xylosidase stability, the enzyme solution was incubated for 2 h at 45 °C in buffers at the above pHs, and residual enzyme activity was assayed as described above. The effect of temperature on xylosidase activity was measured by incubating the enzyme reaction mixture 500 ll containing xylosidase, 50 mM sodium acetate (pH 6.0) buffer at various temperatures from 20 to 80 °C. The thermal stability of xylosidase was measured by incubating the enzyme solution at various temperatures for 2 h, then cooling it in cold water. The remaining activity was assayed as described before at 45 °C. 2.5. Effect of chemicals on xylosidase activity Different concentrations of Tween 80 and Triton X100 (0.1–0.5%) were tested into the medium containing birchwood xylan, and its effect on xylosidase enzyme production was examined. In order to determine the effect of various chemicals, the enzyme solution (2.5 U) was incubated with various chemicals (10 mM or 1 mM in acetate buffer pH 5.0) in a total volume of 1 ml at 45 °C for 1 h. The remaining xylosidase activity was determined as described earlier. 2.6. Analysis of enzymatic hydrolysis products by HPLC Sugars and other fermentation products resulting from enzymatic hydrolysis of various xylan sources were analyzed by HPLC (Hewlett Packard). The culture was grown to late exponential phase and the supernatant fluid was removed by centrifugation at 15,000g frozen and lyophilized to dryness and kept refrigerated at 20 °C until analysis. Dried supernatant was suspended in 50 mM Tris–HCl buffer (pH 5) to give a 1% final concentration. The enzymatic hydrolysis was terminated by adding 1 ml of 0.3 N zinc sulfate (Sigma), and 1 ml 0.3 N barium hydroxide (Sigma). The samples to test were centrifuged for 45 min, at 10,000g at 4 °C. Then they were conserved on ice and filtered through a cellulose nitrate membrane filter (Gelman), to remove unhydrolyzed xylan and other insoluble contaminants. Analysis was performed with Aminex HPX-87 H column (BioRad) with water MilliQ as mobile phase at a flow rate 0.5 ml min1, at 65 °C. The peaks were identified by comparison of the relative retention time of pure standards, glucose, xylose (X1), xylobiose (X2), xylotriose (X3), and other xylo-oligosaccharide mixtures (X4, X5) (Sigma). Waters Millennium Chromatography Manager Software was used for quantification. 2.7. Amino acid analysis The xylosidase band was excised and hydrolyzed for 90 min with 6 N HCl at 150 °C prior to column fractionation and subjected to amino acid analysis using an Applied Biosystems Mode 420 amino acid derivatizer system. The N-terminal amino acid sequence of the purified xylosidase was compared with protein sequence data avail-
able at National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and European Bioinformatics Institute (EBI, http://srs.ebi.ac.uk/) databases using the on-line BLAST network service. 3. Results and discussion P. stipitis NP54376 is a high-growth-rate mutant obtained after NTG mutagenic treatment (Basaran and Hang, 2000). The mutant NP54376 assimilates xylose faster than the wild type; is able to use a larger portion of xylan sources than the wild strain, nevertheless, the mechanism of this mutational change is unclear (Basaran et al., 2001). Other strains of P. stipitis (Y-11544, Y-11545, Y-7124) tested were only in the range of 80–85% of the maximal activity produced by P. stipitis mutant. For P. stipitis, bxylosidase activities found in the culture supernatants of 3-day xylose- and xylan-grown cells were 25 and 29 mU/ ml/h, respectively (Ozcan et al., 1991), that is in good agreement with our mutant data for extracellular bxylosidase. The production of xylosidase by NP54376 in shake-flask culture was dependent on culturing conditions. Maximum growth as well as xylanase and xylosidase production were found to be around pH 5.0, pH values above or below lessened the enzyme production (Basaran, 1999). During the cultivation the pH dropped from the initial pH of 5.0 to pH 3.0–3.5. A study by Haltrich et al., 1994) indicated that low pH was essential since regulating the pH at a constant value of 5.0 resulted in excretion of largely oxalic acid which led to decrease in growth of Sclerotium rolfsii (Haltrich et al., 1994). Furthermore, there have been reports for several fungi where cultivation of the organism at an unfavorable pH resulted in limited grow rate and increased yields of xylanolytic enzyme activity (Haltrich et al., 1994). The reason for this might be the reduced accessibility of the hemicellulose substrate due to the unfavorable conditions. Enzyme production by NP54376 was enhanced by increasing the shaking speed up to 180 rpm but thereafter higher values inhibited the enzyme production. Reduced enzyme activity associated with the high shaking speed could be caused by shear forces, which may cause leakage of intracellular material and probably disrupting enzyme synthesis (Warzywoda et al., 1992). The xylosidase activity was observed maximum at pH 4.8–5.0. Only a few bacterial xylanases were reported to have a high level of activity at pH values higher than 7, most xylanolytic enzymes fall pH values between 4.0 and 6.7 (Shao et al., 1995). The Pichia xylosidase was stable in a pH range of 3.5–7.2. The optimum temperature for enzyme activity was 45 °C, which was similar to many of the yeast proteins (Haltrich et al., 1994; Warzywoda et al., 1992). Xylosidase from NP54376 was stable for 30 min at 60 °C. However, it lost 20% of its activity above 50 °C. The thermostability of the xylosidase from mutant NP54376 falls into the previously reported ranges (25– 60 °C) (Du Preez et al., 1987).
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b-Xylosidase production by the mutant NP54376 was similar when grown on beechwood, birchwood, oatspelt xylan and xylose (max of 0.018 U/ml). When cultivated on pretreated corn cob hydrolysate, a 40% lower b-xylosidase activity was observed. Addition of peptone to corn cob medium did not affect xylosidase production, whereas yeast extract increased enzyme production by 10%. Several other researchers have reported microbial inhibition effect when utilizing hydrolysate from lignocellulosic material (Perttunen et al., 1996; Preziosi-Belloy et al., 1997). Xylosidase production was repressed upon addition of different sugars. At a concentration of 3% (w/w), lactose showed significant repression of xylosidase production (50%), whereas the effect of sucrose was mild (20%). Xylosidase was repressed by the addition of glucose (1.0; 1.5; 2.0 or 2.5 g l1) to the culture medium containing 1% birchwood xylan. Xylosidase and xylanase activities from wild strain and mutant NP54376 were sharply decreased when glucose (2.5 g l1) was added into the birchwood medium. Xylose, an end-product of xylosidase activity, has been reported as an inhibitor of enzymatic activity in some cases (Biely et al., 1991). In order to determine the minimum xylose concentration required to inhibit the xylanolytic systems on P. stipitis wild type and its mutant NP54376, they were grown on beechwood xylan supplemented with xylose. At a concentration of 5 gl1, xylose completely repressed xylosidase production. The crude xylosidase was active on xylooligosaccharides but was not active on starch, cellulose, maltose and cellobiose. The effect of different concentrations of additives on the thermostability of xylosidase was measured in terms of protective effect, which is defined as the ratio of xylosidase half-life in the presence and absence of an additive. The addition of Tween 80 (0.1%) did not prevent the loss in activity at 80 °C. Similarly, COCl2, CaCl2, KCl and NaCl, at a concentration of 10 mM did not have any effect on the stabilization of xylosidase. Xylosidase activity was assayed with and without the presence of several metals (FeSO4, CuSO4, MgSO4, and AgNO3), a metal chelator (EDTA) and protein disulphide reducing agents, DTT and 2-mercaptoethanol, at 1 mM. The metal ions that inhibit the xylosidase activity were: strong inhibition Fe3+ (80%), and moderate (less than 20%) effect Cu2+, Ag+, and Mg2+. When 2-mercaptoethanol at 1 mM concentration was tested, a total loss of the activity was observed. 2-Mercaptoethanol, counteract the oxidative effects of S-S linkages, presumably from cysteine residues. A number of fungal and bacterial xylan hydrolyzing enzymes have been reported to poses cysteine residues essential for activity (Coughlan, 1992). When the amino acid composition of xylosidase is analyzed, it was observed that the enzyme is rich in Cysteine (10%) and Methionine (6%) residues. Substrate specificity of the purified b-linked saccharide derivatives. The b-xylosidase showed no action on phenyl b-D galactoside and p-nitrophenyl a-arabinoside. The effect of different polyols (2 M) (Ethylene glycol (C2), glycerol (C3), sorbitol and mannitol
41
(C4)) on xylosidase deactivation was studied at 45 °C. An increase in the concentration of sorbitol resulted in an increase in its protective effect on xylosidase, and sorbitol provided maximum protection at a concentration of 2 M. The stabilizing effect of polyols depends on hydrophilic and hydrophobic characteristics of the enzyme; polyols have the capability to form hydrogen bonds that play the key role in supporting the native conformation of the protein and aid in protein stabilization (Wong et al., 1988). With P. stipitis 60% of the b-xylosidase activity was detected in culture supernatant and about 20% of the activity was found in the crude extracts (Ozcan et al., 1991). Stimulation of growth as well as enzyme or protein secretion of different microorganisms by surfactants has been reported in earlier studies (Konopka and Zakharova, 2002). Addition of Triton X100 at 0.6–1 g l1 to the culture broth after termination of its cultivation resulted in approximately 2.2–2.4-fold increase in xylanase yields in Rhodothermus marinus (Hregvidsson et al., 1996; Manelius et al., 1994). From the known membrane bound status of b-xylosidase, we have tested the stimulative effect of Triton X100, and when the cells were treated with the surfactant, no significant effect on the enzyme production was observed. Xylooligosaccharides, the potential inducers, should be produced by through a consequential reaction, and the rate of product generation is predicted to decrease when the concentration of xylan increases. To determine the hydrolytic pattern, the degradation products released from birchwood, oatspelt and beechwood xylans were analyzed. The composition of the hydrolysis products of P. stipitis xylosidase from various xylans is shown in Fig. 1. Results presented here demonstrated that the source of the xylan significantly effected the product composition. The composition of the oligosaccharides from hydrolysis in all three xylan sources consisted of mainly xylobiose. The production of xylobiose was found to be the fastest with 90% production within 24 h compared to 50–60% of xylotriose after 24 h. The xylobiose concentration increased by the time with birchwood reaching maximum of amount of 0.94 mg/ml after three days of incubation. Oat spelt and beechwood produced similar amounts of xylobiose 0.53– 0.5 mg/ml that was half of the xylobiose concentration produced with birchwood (Fig. 1b and c). With oatwood (Fig. 1b) and birchwood (Fig. 1c) xylans, xylotriose concentration remained same throughout the fermentation, 0.2 mg/ml and 0.5 mg/ml, respectively. In all three xylan sources similar amounts of xylose were detected. However, the relatively fast release and conservation of the amount of xylose released indicated that limiting sugar for hydrolysis was the monomer xylose. Our results were in good agreement with b-xylosidase of Malbranchea pulchella that was acting on xylan in limited rate and other xylosidases such as those from Trichoderma viride, Emericella nidulans had almost no effect on the direct hydrolyses of xylan (Matsuo et al., 1977a,b).
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clone the hydrolytic enzymes coding sequences into yeasts to create new enzyme production systems with the possible improvement of enzyme production and activity for commercial purposes (Akada, 2002). Pichia yeast strains may fit to fulfill requests of industrial production of enzymes and fermentation. However, the mechanism of action and properties of the enzymatic system needs to be studied in detail.
Birchwood Xylan Hydrolysis
1 0.8 0.6 mg/ml
0.4 0.2
Xylobiose Xylotriose Xylose
0 Day 1 Day 2
Day 3 Day 4 DAYS
1 0.8 mg/ml 0.6 0.4
0 Day 1 Day 2 Day 3 Day 4 DAYS
Xylobiose Xylotriose Xylose
Beechwood Xylan Hydrolysis 1 0.8 0.6 mg/ml 0.4 0.2 0 Day 1 Day 2 Day 3 Day 4 DAYS
We would like to thank A. Artac and M. Selcuk for their contribution for the manuscript. References
Oat Spelt Xylan Hydrolysis
0.2
Acknowledgements
Xylobiose Xylotriose Xylose
Fig. 1. HPLC analysis of hydrolysis products of NP54376 when incubated in various xylan sources (a) Birchwood xylan, (b) Oat spelt xylan, (c) Beechwood xylan. The values are means of at least three measurements (above bars are standard deviations). Xylan incubated without enzyme was taken as 0 time and its sugar contents were deduced from each oligosaccharide.
The amino acid sequence of the N-terminal of P. stipitis xylosidase was determined to be Ala-Iso-Met-Gly-Val. The N-terminal sequencing data of 37 kDa b-xylosidase has shown no homology with the N-terminal amino acid sequences of any of the xylosidase proteins available in the SWISS-Prot database (data not shown). Recently developed genetic techniques promise opportunities to
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