Xylan degradation bythe anaerobic bacterium Bacteroides xylanolyticus.
Philippe Schyns
Promotor: Co-promotor:
Dr.A.J.B.Zehnder Hoogleraar indeMicrobiologie Dr.ir.A.J.M. Stams universitair docentbij devakgroep microbiologie
PJ.Y.M.J. Schyns
Xylan degradation bytheanaerobic bacterium Bacteroides xylanolyticus.
Proefschrift terverkrijging vandegraadvandoctor opgezagvanderector magnificus vandeLandbouwuniversiteit Wageningen, dr.C.M.Karssen, inhetopenbaarte verdedigen opmaandag 9juni 1997 desnamiddagstevieruurindeAula
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This research was carried out at the Department of Microbiology, Wageningen Agricultural University, the Netherlands. It was supported by a grant of the Program Committee onAgricultural Biotechnology, established bythe ministry of Agriculture, Nature Conservation andFisheries.
-nitrophenyl-jS-D-xyloside and showed low activity towards p-nitrophenyl-a-L-arabinofuranoside. Very low levels of a-L-arabinofuranosidase activity were also found in several other bacterial^-xylosidase
87
Chapter3
preparations (1,14,18). p-Nitrohenyl-a-L-arabinofuranosidase activity has been reported for a Trichoderma reesei and Butyrivibrio fibrisolvens ^-xylosidase (28,35) and p-nitrophenyl-a-L-arabinopyranosidase has been reported for the Pénicilliumwortmanni y?-xylosidase (7). The ^-xylosidase of Thermomonospora ethanolicus showed both a-arabinosidase activities (33). The general properties of the ^-xylosidase from B.xylanolyticusarecomparabletothosereported for otherbacteria(Table4).
Table4. Generalpropertiesofthepurified^-xylosidasesfrom severalmicroorganisms. v„„
Topt (°C)
K„(mM pNPX)
5.8-6.2
40-45
0.125
33
6.3
16
6-6.5
45
3.7
19.6
5.85
18
70,000
3.4
7
2.4
3.4
4.4
26
150,000
75,000
34.2
6
70
1.2
4.2
23
Thermomonospora pjsca
165,000
56,000
8
5-9
40-60
0.89
4.37
1
Thermoanaeroba der ethanolicus
165,000
85,000
66
5-5.2 5.8-6
82 93
0.018 0.038
122 183
4.6
33
53,000
49.2
5.7
70
10
64
4.3
14
2.8
2.6
4.86
36
30.9
4.3
24,34
Organism
Mol wt (Da)
Mol wt of subunits (Da)
Spact (uinol. iniif'.mg" 1 )
pH opt
Bacteraides xylanolyticus
165,000
85,000
31
Clostridium acetohutilicum
224,000
85,000 63,000
Bacillus pumilus
130,000
Bacillus sieamthermophilus
Caldoceltum saccharolylicum Chaetomhtm trilaterale
240,000
Aspergillus
niger
Aspergillus
niger
118,000
78,000
4.5
Pi
Ref.
(Mmol, min'mg"')
5.2
6.7-7
4.2
3-4
42 0.362 0.12
5
7
15
Pénicillium wortmanni
100,000
11.4
3.3JI
Trichoderma viride
101,000
10.8
4.5
55
5.8
445
20
Trichoderma reesei
100,000
28.2
4
60
0.08
4.7
28
Kmericella nidulatvi
240,000
62.9"
4.5-5
55
1
3.25
21
116,000
The j3-xylosidases from Bacillus pumilus, Bacillus stearothermophilus and Thermoanaerobacter ethanolicus arealsodimericenzymesofcomparable size.TheKMof 0.125 mM forp-nitrophenyl-jS-D-xyloside is relatively low. A big difference between the ^-xylosidaseofB.xylanolyticusandtheenzymesfrom different sources istheextreme low
88
ß-Xylosidase
thermostability ofthepurified protein.Invivo,theenzyme wouldprobably bemore stable when located in the cytosol. The pH optimum of B. xylanolyticus like that of most bacterial^-xylosidases is around 6, whereas the optimal pH of fungal enzymes is usually below4.5 (Table4).The pi ofthe^-xylosidase of B. xylanolyticuswas near pH 6, aswas found for the enzyme of Clostridium acetobutylicum (18). Most other ^-xylosidases characterized thus far haveapi closeto4(Table4).
References 1.
Bachmann, S.L., and A.J. McCarthy. 1989.Purification and characterization of a thermostable yS-xylosidase from Thermomonospora fusca. J. Gen. Microbiol. 135:293-299. 2. Bachmann, S.L., and A.J. McCarthy. 1991.Purification and cooperative activity of enzymes constituting the xylan-degrading system of Thermomonosporafusca. Appl.Environ. Microbiol.57:2121-2130. 3. Biely, P., M. Vrsanska, and Z. Kratky. 1980. Xylan degrading enzymes of the yeast Cryptococcus albidus-iéevA\ï\ç,dXion and cellular location. Eur. J. Biochem. 108:313-321 4. Biely,P. 1985.Microbial xylanolytic systems.TrendsBiotechnol. 3:286-290 5. Biesterveld,S.M.D.Kok, CDijkema,A.J.B.Zehnder, andA.J.M. Stams. 1994. D-Xylose catabolism in Bacteroides xylanolyticus X5-1. Arch. Microbiol. 161:521-527 6. Bradford M.M. 1976. A rapid and sensitive method for the quantification of microgram quantities ofprotein utilizing theprinciple of protein dye binding. Anal. Biochem. 72:248-254 7. Deleyn,F.,M. Cleassens,J. VanBeelmen, and CK. De Bruyne. 1978. Purification and properties ofy?-xylosidase from Pénicilliumwortmanni.Can. J. Biochem. 56:43-50. 8. Dong, X., P.J.Y.M.J. Schyns, and A.J.M. Stams. 1991. Degradation of galactomannan by a Clostridium butyricum strain. Antonie van Leeuwenhoek 60:109-114. 9. Hespell,R.B.,R.Wolf,R.J.Bothast. 1987.Fermentation ofxylansbyButyrivibrio fibrisolvens and otherruminai bacteria.Appl.Environ.Microbiol. 53:2849-2853 10. Hespell, R.B. 1992. Fermentation of xylans by Butyrivibrio fibrisolvens and Thermoanaerobacter strain B6A: utilization of uronic acids and xylanolytic activities. Curr.Microbiol.25:189-195. 11. Howard, B.H., G. Jones, and M.R. Purdom. 1960. The pentosanases of some rumenbacteria.Biochem.J.74:173-180 12. Hrmova, M., Biely, P., and M. Vrsanska. 1986. Specificity of cellulase and ß-xylanase induction in Trichoderma reesei QM 9414. Arch. Microbiol. 144:307-311
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Hrmova,M.,E.Petrakova, and P.Biely. 1991.Induction of cellulose- andxylandegrading enzyme systems inAspergillusterreusbyhomo-and heterodisaccharides composed ofglucoseandxylose.J.Gen.Microbiol. 137:541-547. Hudson R.C., L.R. Schofield, T. Coolbear, R.M. Daniel, and H.W. Morgan. 1991. Purification andproperties ofanaryiy?-xylosidasefrom acellulolytic extreme thermophile expressed inEscherichia coli.Biochem. J.273:645-650. John, M., B. Schmidt, and J. Schmidt. 1979.Purification and some properties of five endo-l,4-ß-D-xylanases and aß-D-xylosidaseproduced by a strain ofAspergillusniger.Can.J. Biochem. 57:125-134 Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the headofbacteriophage T4.Nature.227:680-685 Lee, S.F., C.W. Forsberg, and L.N. Gibbins. 1985. Xylanolytic activity of Clostridium acetobutylicum. Appl.Environ.Microbiol. 50:1068-1076 Lee, S.F., and C.W. Forsberg. 1987. Isolation and some properties of a^-xylosidase from Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 53:651-654. Lee, Y.E., S.E. Lowe and J.G. Zeikus. 1993. Regulation and characterization of xylanolytic enzymes of Thermoanaerobacterium saccharolyticumB6A-RI. Appl. Environ. Microbiol. 59:763-771 Matsuo, M., and T. Yasui. 1984. Purification and some properties of^-xylosidase from Trichodermaviride.Agric.Biol.Chem.48:1845-1852. Matsuo, M., and T. Yasui. 1984.Purification and someproperties of/?-xylosidase from Emericellanidulans. Agric.Biol.Chem.48:1853-1860. Nakanishi, K., T. Yasui, and T. Kobayashi. 1976. Inducers for xylanase productionbyStreptomycessp.J.Ferment.Technol.54:801-807. Nanmori, T., T. Watanabe, R. Shinke, A. Kohno, and Y. Kawamura. 1990. Purification andproperties ofthermostable xylanaseand^-xylosidaseproduced bya newly isolated Bacillusstearothermophilus strain.J.Bacteriol. 172:6669-6672. Oguntimein, G.B., and P.J. Reilly. 1980. Properties of soluble and immobilized Aspergillusw'ger^-xylosidase.Biotechnol.Bioeng.22:1143-1154. O'Farrell, P.H. 1975.Highresolution two-dimensional electrophoresis ofproteins. J.Biol. Chem.250:4007-4021. Panbangred, W., O. Kawaguchi, T. Tomita, A. Shinmyo, and H. Okada. 1984. Isolation of two^-xylosidase genes of Bacilluspumilus and comparison of their geneproducts.Eur.J.Biochem. 138:267-273 Pou-Llinas, J., and H. Driguez. 1987.D-Xylose as inducer of thexylan-degrading enzyme system in the yeast Pullularia pullulons. Appl. Microbiol. Biotechnol. 27:134-138 Poutanen, K., and J. Puis. 1988. Characteristics of Trichoderma reesei^-xylosidase and its use in the hydrolysis of solubilized xylans. Appl. Microbiol. Biotechnol.28:425-432.
ß-Xylosidase
29.
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31.
32.
33. 34.
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36. 37. 38.
39.
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41.
42. 43.
Reilly, P.J. 1981.Xylanases: structure and function, pli1-129. In A. Hollaender (ed.), Trends inthe biology of fermentations for fuels and chemicals. Plenum Press, NewYork. Scholten-Koerselman, I., F. Houwaard, P. Janssen, and A.J.B. Zehnder. 1986. Bacteroidesxylanolyticussp.nov.,axylanolytic bacterium from methane producing cattlemanure.AntonievanLeeuwenhoek J.Microbiol.46:523-531 Schyns,P.J.Y.M.J., and A.J.M. Stams. 1992.Xylan degradation bythe anaerobic bacterium Bacteroides xylanolyticus X5-1. p. 295-299. In J.Visser, G. Beldman, M.A. Kusters-van Someren and A.G.J. Voragen (Eds.), Xylans and Xylanases: Progress inBiotechnology vol.7.Elsevier SciencePubl.,TheNetherlands. Sewell, G.W., H.C. Aldrich, D. Williams, B. Mannarelli, A. Wilkie, R.B. Hespell, P.H. Smith, L.O. Ingram. 1988.Isolation and characterization of xylandegrading strains of Butyrivibrio fibrisolvens from Napier grass-fed anaerobic digester. Appl.Environ. Microbiol. 54:1085-1090 Shao,W., and J. Wiegel. 1992.Purification and characterization of a thermostable ^-xylosidase from Thermoanaerobacterethanolicus. J.Bacteriol. 174:5848-5853. Takenishi, S., Y. Tsujisaka, and J. Fukumoto. 1973. Studies on hemicellulases IV. Purification and properties of the^-xylosidase produced by Aspergillus niger vanTieghem.J.Biochem.73:335-343. Utt, E.A., CK. Eddy, K.F. Keshav, and L.O. Ingram. 1991. Sequencing and expression of the ButyrivibriofibrisolvensxylB gene encoding a novel bifunctional protein with^-D-xylosidase and a-L-arabinofuranosidase activities. Appl. Environ. Microbiol. 57:1227-1234. Uziie, M., M. Matsuo, and T. Yasui. 1985. Purification and some properties of Chaetomium trilateraleß-xylosidase. Agric.Biol.Chem.49:1159-1166. Uziie, M., M. Matsuo, and T. Yasui. 1985.Possible identity ofjS-xylosidase and ^-glucosidaseofChaetomium trilaterale. Agric.Biol.Chem.49:1167-1173. Whitehead, T.R., and R.B. Hespell. 1990. The genes for three xylan-degrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region. J. Bacteriol. 172:2408-2412. Williams, A.G., and S.E. Withers. 1982. The production of plant cell wall polysaccharide-degrading enzymes by hemicellulolytic rumen bacterial isolates grown onarangeofcarbohydrate substrates.J.Appl.Bacteriol. 52:377-387 Williams A.G.,and S.E. Withers. 1982. The effect of the carbohydrate growth substrate on the glycosidase activity of hemicellulose-degrading rumen bacterial isolates.J.Appl.Bacteriol. 52:389-401 Williams, A.G., and S.E. Withers. 1992. The regulation of xylanolytic enzyme formation by Butyrivibrio fibrisolvens NCFB 2249. Lett. Appl. Microbiol. 14:194-198 Wong, K.K.Y., L.U.L. Tan, andJ.N. Saddler. 1988.Multiplicity ofß-xylanasein microorganisms:functions andapplications.Microbiol.Rev.52:305-317 Yasui,T.,B.T.Nguyen,and K.Nakenishi. 1984.Inducersfor xylanase production by Cryptococcusflavus.J.Ferment. Technol.62:353-359.
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Chapter 4
Production, Purification and characterization ofan a-L-arabinofuranosidase from BacteroidesxylanolyticusX5-1.
PhilippeJ.Y.M.J.Schyns, JackdeFrankrijker, Alexander J.B.Zehnder, andAlfons J.M. Stams
93
Chapter4
Abstract Cell-free extracts of L-arabinose- and D-xylose- grown cells of the mesophylic anaerobic bacterium Bacteroidesxylanolyticus X5-1 contained high activities (2 U/mg) of an aL-arabinofuranosidase (EC 3.2.1.55). The enzyme was also produced during growth on xylan, but not during growth on glucose or cellobiose. The enzyme was mainly extracellularly attached to the cell when the organism was grown on xylan and was not released into the medium. The enzyme was purified 41-fold to apparent homogeneity. The native enzyme had an apparent molecular mass of 364 kDa and was composed of six polypeptide subunits of 61 kDa. The enzyme displayed a pH optimum of 5.5 to 6.0, and a pHstability of5.5to9.0.Thetemperature optimumwas50°Candtheenzymewasstableup to50°C.Thiol groupswereessential for activity,buttheenzymeactivity wasnot dependent ondivalent cations.TheKmandVmax forp-nitrophenyl-a-L-arabinofuranoside were 0.5mM and 155U/mgofprotein,respectively.Theenzymewasspecific forthe a-linkedarabinoside in the furanoside configuration. The enzyme displayed activity with arabinose-containing xylo-oligosaccharides with a polymerization degree of 2-5, but not with the polymeric substrates oat spelt xylan or arabinogalactan. The enzyme belongs to theStreptomyces purpurascens-type of a-L-arabinofuranosidase.
Introduction Xylanisawidelydistributed typeofhemicellulose ofplantcellwalls.Thiscomplexpolymer consists of a ß-D-l,4-linked xylopyranoside backbone substituted with arabinosyl, acetyl, uronyl and glucosyl side chains. The nature of the substituents is dependent on the source from whichthexylanhasbeenisolated.Xylansofsoftwoods andmonocotylssuchasgrasses and cereals are generally characterized by the presence of L-arabinose residues, linked a-glycosidicallyto0-3 positionsofD-xylose(Timell 1967;Wilkie 1979;Biely 1985).Many cellulolytic rumen bacteria have been shown to degrade xylan, but only a few are able to grow on this substrate (Dehority 1965; 1967; Williams and Withers 1982; Hespell et al.
94
a-L-arabinofuranosidase
1987). Anaerobic bacteria able to grow on xylan predominantly belong to the genera of Bacteroides(Scholten-Koerselman 1986; Salyers et al. 1982), Butyrivibrio(Hespell et al. 1987;Sewelletal. 1988)andClostridium(Madden 1983;Berengeretal.1985). The complete microbial degradation of branched xylan involves the action of several hydrolytic enzymes; endo-ß-l,4-xylanases which hydrolyse the internal ß-l,4-xylosic linkages of the xylan backbone, ß-D-xylosidases which release xylose residues from small oligomeric substrates, and several enzymes capable of hydrolysing substituents from the xylan backbone such as arabinofuranosidases, acetyl esterases, uronidases and glucosidases (Biely 1985). a-L-arabinofuranosidases (AF), which remove L-arabinose side chains from polymeric or oligomeric substrates, have been purified from fungi (Kaji et al. 1970) and bacterialikestreptomyces (Kaji etal. 1981;Komaeetal. 1982),Bacillussubtilis(Weinstein and Albersheim 1979),Ruminococcus albus8,Clostridium acetobutylicum and Butyrivibrio fibrisolvens(Grèveetal. 1984;Leeetal. 1987;HespellandO'Brian 1992). Bacteroides xylanolyticus X5-1, a predominant strain isolated from fermenting cattle manure, grows efficiently on xylan. Unlike many other anaerobic xylanolytic bacteria, it is unable to use cellulose or other hemicelluloses for growth (Scholten-Koerselman et al. 1986).Severalxylanolyticenzymeactivitiescould bedetected inculturesfrom B. xylanolyticusgrownonxylan (Schyns and Stams 1992).Theseenzyme activities includexylanase, ßD-xylosidase, acetyl-esterase and a-L-arabinofuranosidase activities. Here we report the purification and characterization of the a-L-arabinofuranosidase from B. xylanolyticus, expressed inhighamountsduringgrowthonxyloseandarabinose.
Materials and methods Chemicals. Unless stated otherwise, chemicals used were of analytical grade. L-arabinose, D-cellobiose, oat spelt xylan, larch wood arabinogalactan, Tris(hydroxymethyl)aminomethane, all /7-nitrophenyl-glycosides, except />nitrophenyl-jß-D-glucopyranoside, and amyloglucosidase from Rhizopus(lot no.48B-2186)were purchased from Sigma Chemical Co. (St. Louis, MO,USA).p-nitrophenyl-^-D-glucopyranoside was obtained from Boehrin-
95
Chapter4
ger GmbH (Mannheim, FRG). Xylanase from Trichoderma viride (lot no. 95595) was obtainedfromFlukaBiochemika (Buchs, Switzerland). Sodium dodecylsulfate, acrylamide, hydroxylapatite andBiogelP2werefromBiorad (Veenendaal,theNetherlands). Q-Sepharose Fast Flow, Sepharose CL-6B,Mono-Q HR 5/5, Superose 6 HR 10/30, molecular mass markers for SDS-PAGE and for gel filtration were purchased from Pharmacia LKB Biotechnology (Uppsala, Sweden).All other chemicals werepurchasedfromMerck (Darmstadt,FRG). Media and cultivation. BacteroidesxylanolyticusX5-1 (DSM 3808) was isolated and described by Scholten-Koerselmanetal.(1986).Theorganismwasculturedinabicarbonate buffered medium containing (g/1): Na2HP04.2H20, 0.60; KH2P04, 0.45; NH4C1, 0.3; CaCl2.2H20, 0.11; MgCl2.6H20, 0.11;NaCl, 0.3; NaHC03, 4.0; Na2S.9H20, 0.24; yeast extract, 0.5; trypton, 0.4; resazurin, 0.0005; 1ml of a trace element solution according to Wolinetal.(1963)perliterand 1 mlofavitaminsolutionaccordingtoZehnderetal.(1980) per liter.Thevitamin solution was filter sterilized. Xylan wasaddedtothe medium prior to autoclaving. Sugarswereaddedfrom2Mfilter-sterilizedstocksolutions. B.xylanolyticus was cultivated routinely at 37°C inthe dark in 120-ml serum vials with 50 ml of medium. Vials were sealed with butyl rubber stoppers (Rubber BV, Hilversum, Holland) and aluminium caps. The gas phase in the vials consisted of N 2 /C0 2 (80:20 v/v; 170 kPa.). Mass-cultivation was performed in 8-1 bottles at 37°C. Cells were harvested aerobically at the late logarithmic phase by continuous centrifugation (Heraeus Sepatech Biofuge 28RS,Osterode,FRG). Preparation of cell-free extract Cells were suspended in 50 mM Tris-HCl pH 7.6 (0.2 g wet cells/ml buffer) and disrupted by sonification at 0°C (5 times 20 s, at 40 W, using a Branson Sonic Sonifier, Danbury, CT,USA).Thebroken cellswere centrifuged for 20min at 10,000 xg and the supernatant was centrifuged again at 100,000xg for 1h at 4°C.The supernatantcontained about20mgproteinpermlandwasdesignatedascellfreeextract. Enzyme purification. Unless otherwise stated allprocedureswerecarried outaerobically at roomtemperature.Thecrude extractwasappliedto aQ-sepharose fast flow column (3.2by 12 cm) equilibrated with 50 mM Tris/HCl pH 7.6 (buffer A). The column was developed
96
a-L-arabinofuranosidase
with 350 ml of a linear gradient of 0 to 0.6 M NaCl in buffer A. The active a-L-arabinofuranosidase eluted at 0.45 MNaCl. The AF-containing fractions were concentrated by ultrafiltration with an Amicon ultrafiltration cell (Grace, Rotterdam, the Netherlands) equipped with a PM 30 filter. The ultrafiltration retentate was adjusted to 0.25 M (NH^(S04) by slow addition of granular ammonium sulfate and loaded onto a column (2.2by 10 cm) packed with phenylsepharose. The column was equilibrated with 0.25 M ammonium sulfate inbuffer Aandeluted withalineargradientof0.25Mto0Mammonium sulfate ata flow rate of 1ml/min. AF activity-containing fractions were pooled, diluted with sodium phosphate buffer (10 mM, pH 7), and then concentrated and desalted by repeated dilution andultrafiltration. Thisenzymepreparation wasappliedtoahydroxylapatite column (2.2by 18cm)equilibrated withsodiumphosphatebuffer (10mM,pH7).Theadsorbed proteinwas eluted from the column ina220ml-lineargradientof 10mMto 150mM sodium buffer pH 7. TheAF eluted at 100mM sodium phosphate. Fractions withactivity were combined and appliedtoaMono-QHR5/5column,equilibratedwith50mMsodiumphosphatebuffer (pH 6.5) ataflow rate of 1 ml/min.Theenzymeeluted from thecolumn at0.35 MNaCl ina20 ml-lineargradientof0to0.5MNaCl.FractionswithAFactivitywerecombined and250 ul was injected onto a Superose 6 HR 10/30 gelfiltration column (V0=7.9 ml; Vt=23.4 ml) equilibrated in buffer A plus 100mMNaCl. The column was developed at a flow rate of 0.25ml/min.ThecolourlessAFelutedasonesymmetricalpeakat 13.8ml. a-L-arabinofuranosidase assay. Routinely a-L-arabinofuranoside (EC 3.2.1.55) was assayedusingp-nitrophenyl-a-L-arabinofuranoside (ArafaNp) asanartificial substrate(aryla-arabinofuranosidase). En2yme activity was determined by measuring the amount of pnitrophenol (pNP) released from the substrate.Theassaymixturecontained 1 mM substrate in 50mM sodium phosphate (pH6.5) inafinalreaction volume of0.25ml.The incubation temperature was 50°C unless indicated otherwise. The reaction was terminated by the addition of 0.5 ml of 0.1 M sodium carbonate. The released pNP was determined spectrophotometrically at405ran,withpH? asa standard. Oneunit of activity was defined astheamountofenzymereleasing 1umolofpNP permin.AFwasalsoassayedusingxylan or xylan oligomers as substrates. The assay mixture contained 0.5% (w/v) substrate in 50
97
Chapter4
mM Tris/HCl (pH 7.0) containing 100 mM NaCl in a final volume of 1ml. The assay mixturewasincubated at 37°C during 30min. Assayscontaining xylanwere incubated for 2 hours with 10times as much enzyme.Thereaction was stopped by boiling during 1min. Thereactionwasfollowed bymeasuringthereleaseof arabinoseby HPLCequipped with a Chrompack organic acid column (30 cmx 6.5 mm).Themobilephase was0.005 M H 2 S0 4 at a flow rate of 0.6 ml/min. The column temperature was 60°C. Samples (20 ul) were injected usingaSpectraPhysicsautosampler (SP8775).Compoundselutingwere quantified bydifferential refractometry using aLKB2142refractometer. Oneunit ofAFactivity isthe amountofenzymereleasing 1.0 umol ofarabinosepermin. Protein determination. Protein was determined with Coomassie brilliant blue G250 as describedbyBradford (1976).Bovineserumalbuminwasusedasastandard. Polyacrylamide gel electrophoresis (PAGE). SDS PAGE was performed on 12.5%(w/v) gels according to the method of Laemmli (1970). Protein was stained with Coomassie brilliant blue. Molecular weight of the subunits was estimated by comparison to protein standards. Preparation of xylo-oligosaccharide mixture. Oat spelt xylan was pretreated with amyloglucosidase to remove the contaminating starch. After ethanol precipitation (3 volumes), the xylan was resuspended (50 g/1)in20 mM acetate buffer pH 5.0 and partially hydrolysed with axylanasepreparation from Trichoderma viride at 37°C. Thereactionwas stopped by boiling for 5minutes. The mixture was centrifuged at 5000xg for 10min and thesupernatantwasconcentratedbyrotaryevaporation.Theoligosaccharideswereseparated by a repeated gel filtration on a column (100 x 2.2 cm) of Bio-gel P2 (fractionation range 100-1800). Sugars were analyzed by HPLC equipped with an Aminex HPX-87P column (Biorad) maintained at 80°C with degassed water as the mobile phase. The degree of polymerization oftheoligosaccharideswasestimatedasdescribedbyLeeetal. 1985.
98
a-L-arabinofuranosidase
Results Growth conditions and enzyme activities. When BacteroidesxylanolyticusX5-1 was grown on glucose or cellobiose only low AF activities were detected in the cultures (Table 1).CellsgrownonL-arabinose orD-xylosedisplayed AFactivitiesofupto2.5U/mg.When oatspeltxylanwasusedasasubstratethespecificAFactivitieswereabout0.35U/mg.
Table1. Theeffect ofthegrowthsubstrateonthespecificactivityandthetotal activity per culture volume of the a-L-arabinofuranosidase from Bacteroidesxylanolyticus X5-1. a-L-arabinofuranosidase (U/mg)
(U/ nl culture)
Glucose
0.05
0.01
Cellobiose
0.08
0.01
L-Arabinose
2.4
0.3
Xylose
1.9
0.25
0.35
0.05
Carbon source
Xylan (oat spelt)
Dataarethemeanvaluesoftriplicatedeterminations. During batch growth on L-arabinose or D-xylose, the enzyme was produced throughout the logarithmic growth phase until all the substrate had been consumed. Only about 5%of the totalAFactivity wasfound inthecell free growthmedium attheendofgrowth.Eveninthe latestationary phaseno increase inAFactivity intheculture supernatant could be observed. The AF activity could readily be measured with intact whole cells. When grown on L-arabinose the AF activity of whole cells was about 50%of the activity measured in cell extracts, suggesting that 50 % of the enzyme resides intracellularly and the rest is extracellularly bound to the cells. The extracellular AF activity could not be released from thecellsby anosmotic shocktreatment (NeuandHeppel 1965).After disruption ofthecells by sonification themajor partoftheAFactivity wasreleased from the cells and cell debris. The activity did not remain membrane bound as was shown by fractionation of crude
99
Chapter4
extracts of cells grown on L-arabinose by ultra-centrifugation at 100,000 x g (1h). About 95% of the activity remained in the supernatant. When grown on xylan the whole cells exhibited approximately the same AF activities as cell free extracts. This suggests that the AFactivitywasmainly located extracellularly andcellboundwhentheorganismwasgrown onxylan. Table2.Purification ofa-L-arabinofuranosidase fromBacteroidesxylanolyticus X5-1. Total protein
Total activity
Specific activity
Recovery
Purification
(mg)
(U)
(U/mg)
(%)
(fold)
Cell extract
1045
2758
2.6
100
1
Q-sepharose
137
1290
9.4
46
3.6
Phenyl-sepharose
50
821
16.4
29.8
6.2
Hydroxyapatite
15
414
27.5
15
10.4
MonoQ
1.7
138
78.6
5
29.8
Superose6
0.9
97
108
3.5
41.5
Purification. Thepurification of the oxygen stable AF was performed aerobically at room temperature.Theenzymepurification issummarized inTable2.Thecellfree extractofcells grown on 20 mM L-arabinose was fractionated by Q-sepharose anion exchange column chromatography (Fig. 1).The AF activity eluted in a major peak at 0.45 MNaCl and in a minor peak at 0.5 M NaCl from column. The minor peak coeluted with a peak containing high ß-xylosidase activity. The major peak, which accounted for 80% of the total activity was selected for further purification. The hydroxylapatite column was used to separate the AF from contaminating ß-xylosidase activity. When chromatographed on superose-6, the enzyme eluted as one symmetrical peak. The volume at which the enzyme eluted corresponded to a size of 364000 dalton. The enzyme was purified about 40 fold with a recoveryof3.5%.
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