Sep 14, 1987 - 0.5 ml. After 15 min of incubation at 50°C, 10 ,ul of 1 N ... The eluant was 0.01 N sulfuric ..... Kyriacou, A., C. R. MacKenzie, and R. J. Neufeld.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1987, 0099-2240/87/122835-05$02.00/0 Copyright © 1987, American Society for Microbiology
p. 2835-2839
Vol. 53, No. 12
Induction of Cellulolytic and Xylanolytic Enzyme Systems in
Streptomyces
spp.t
C. ROGER MAcKENZIE,* DORIS BILOUS, HENRY SCHNEIDER, AND KENNETH G. JOHNSON of Canada, Ottawa, Ontario, Canada KJA OR6
Division of Biological Sciences, National Research Council
Received 22 June 1987/Accepted 14 September 1987
flavogriseus
enzyme
cultures
and Streptomyces
grown on cellulose contained primarily cellulase activities, but similar preparations from cultures grown on xylan-containing materials possessed high levels of both cellulase and xylanase activities. Growth conditions that gave high endoxylanase levels also resulted in the production of enzymes involved in the
hydrolysis of the nonxylose components of xylan. Specific acetyl xylan
esterase
activitie's
were
identified in
enzyme preparations from both organisms. Both organisms also produced 4x-L-arabinofuranosidase a'ctivity that was not'associated with endoxylanase activity. Other activities produced were ot-L-O-methylglucuronidase
and ferulic acid esterase. The latter enzyme was produced only by S. olivochromogenes and is an activity which has not previously been identified as a component of hemicellulase preparations.
There has been considerable interest over the past 15 in the enzymatic degradation of lignocellulosic materials. The reasons for this interest have been varied and have included potential applications in waste treatment, fuel production, oxychemical' production, rumen digestion, and more recently, the pulp and paper industry (26, 30, 31). The two major carboyhydrate polymers found in lignocellulosic biomass are cellulose and xylan. Considerable information is available about enzyme systems that hydrolyze cellulose to monomers. Essentially only three enzyme activities need to be considered: those of endoglucanase, exoglucanase, and 3-glucosidase. Less is known, however, about the enzymology of xylan breakdown. It is possible that this has arisen because the structure of xylans is generally more complex than that of cellulose, as is the associated enzymology of hydrolysis to the monomeric components. Xylan is a ,-1,4-linked D-xylose polymer which can be highly substituted. The substituents include arabinosyl, O-acetyl, and uronyl groups (2) and arabinosecontaining side chains. Arabinose and uronic acids can appear as well at chain ends (40). Some of the arabinosyl substituents are esterified with ferulic and coumaric acids (27, 35). In addition, it has been suggested that xylans in plant cell walls may be cross-linked or complexed with lignin by diferulate bridges (15, 28). Breakdown of the backbone polymer chain of xylan requires endoxylanase and xylobiase activities, in analogy with cellulolytic enzyme systems. However, a complete xylanolytic system requires activities that hydrolyze the nonxylose components as well. Debranching activity is particularly important, as indicated by the reduced amount of sugar release by xylanases in its absence (3, 11). While considerable effort has been focused on enzymes that break glycosidic bonds between two Dxylose residues (2, 7), relatively few studies have appeared on the other enzymes that are involved. The most extensively studied cellulases and xylanases are those produced by efficient lignocellulose-degrading fungi,
particularly Trichoderma spp. Among the procaryotes, certain actinomycetes have been shown to efficiently degrade lignocellulosic biomass. They produce cellulases, xylanases, and ligninases (6, 14, 16, 18, 19, 22, 23, 25). However, only one thermophilic species, Thermomonospora sp. (14), and two mesophilic species, Streptomyces flavogriseus (16, 22, 23) and Streptomyces lividans (19), have been studied in any detail. While the cellulases produced by two of these organisms have been partially characterized, virtually no information is available about the nature of the xylanases, xylandebranching enzymes, and other activities involved in xylan breakdown. In the present study we examined cellulases, xylanases, and xylan-degrading enzymes produced by S. flavogriseus and Streptomyces olivochromogenes during growth on various substrates. The primary objective was to obtain information about the nature of xylan-degrading enzymes other than those which break xylose-xylose bonds, and the conditions which induce their synthesis. One cellulosic (Avicel) and two hemicellulosic (oat spelts xylan and wheat bran) substrates were used. Oat spelts xylan was chosen as a deacetylated and relatively simple hemicellulose (37), and wheat bran was chosen as a complex hemicellulose with acetyl, arabinosyl, 4-O-methylglucuronyl, and ferulic acid ester linkages (35).
years
*
MATERIALS AND METHODS Materials. Avicel PH105 was obtained from FMC Corp. (Food and Pharmaceutical Products Div., Philadelphia, Pa.). Oat spelts xylan, low-viscosity carboxymethyl cellulose (degree of substitution, 0.7), p-nitrophenylacetate, p-nitrophenylglycosides, 4-methylumbelliferyl esters, 4-methylumbelliferyl glycosides, and ferulic acid were obtained from Sigma Chemical Co. (St. Louis, Mo.). A wheat bran substrate was prepared by treatment of bran with 0.25% (wt/vol) potassium acetate at 95°C for 10 min, followed by extensive washing with water. This treatment was used to remove the starch that was present in the untreated material as a result of contamination with endosperm material. Analytical isoelectric focusing (IEF) gels were obtained from LKB Instruments, Inc. (Bromma, Sweden). Bradford protein reagent
Corresponding author.
t Issued
as
National Research Council of Canada paper no.
28308. 2835
2836
APPL. ENVIRON. MICROBIOL.
MAcKENZIE ET AL.
and Agarose Gel-Bond were obtained from Bio-Rad Laboratories (Richmond, Calif.). Acetyl xylan was prepared as described previously (K. G. Johnson, C. R. MacKenzie, and J. D. Fontana, Methods Enzymol., in press). Organisms and growth conditions. S. flavogriseus ATCC 33331 and S. olivochromogenes NRCC 2258 were maintained as described previously (22). For enzyme production, cultures were grown in 4-liter flasks containing 1 liter of IAF medium (16) supplemented with 1% (wt/vol) Avicel PH105, oat spelts xylan, or starch-free wheat bran. Following 96 h of incubation at 37°C in a gyratory shaker operated at 250 cycles/min, cultures were harvested by centrifugation, and the supernatants were retained. Enzyme preparations. Culture supernatants which were concentrated and desalted in an ultrafiltration cell (Amicon Corp., Lexington, Mass.) equipped with PM-10 membranes served as enzyme preparations. Protein concentrations were estimated by the method described by Bradford (5), using pooled immunoglobulin as the standard. Enzyme assays. One unit of enzyme activity was defined as the amount of enzyme that released 1 Rmol of reducing sugar (expressed as glucose), 1 ,umol of p-nitrophenol, 1 ,umol of 4-O-methylglucuronic acid, 1 Fmol of acetic acid, or 1 ,umol of ferulic acid per min. (i) Carboxymethylcellulase. Carboxymethylcellulase (CMCase) or endoglucanase assays were performed as described previously (21). Reducing sugar release was determined by the modification described by Somogyi (36) of the method described by Nelson (29), with glucose used as the standard. (ii) Xylanase. The xylanase assay was performed as described above for CMCase, except that the substrate was 1% (wt/vol) xylan. (iii) p-Nitrophenyllactopyranosidase. p-Nitrophenyllactopyranoside (pNPLase) is a substrate for ,-1,4-exoglucanase and ,B-glucosidase (8). Assays contained 0.83 mM pnitrophenyl-,B-D-lactopyranoside, enzyme at appropriate dilutions, and 50 mM MOPS (morpholinepropanesulfonic acid; pH 6.0) in a final volume of 1.5 ml. After incubation for 15 min at 50°C, 1 ml of 1 M sodium carbonate was added and p-nitrophenol release was determined from measurements of the A410(iv) Arabinosidase. Assays for arabinosidase were carried out as described above for pNPLase, but with 0.83 mM p-nitrophenyl a.-L-arabinofuranoside used as the substrate. (v) 4-O-Methylglucuronidase. Assays for 4-0-methylglucuronidase contained 2% (wt/vol) larchwood xylan (Sigma), 50 mM sodium acetate buffer (pH 4.5), and appropriate enzyme dilutions in a total volume of 0.5 ml. After a suitable incubation period at 50°C, usually 60 min, the reaction was terminated by the addition of 1.0 ml of methanol. Following 5 min of centrifugation at 10,000 x g, 0.5 ml of the supernatant fraction was withdrawn. The component sugars were converted to their silylated oxime derivatives
and analyzed by gas-liquid chromatography as described previously (J. D. Fontana, M. Gebara, M. Blumel, H. Schneider, C. R. MacKenzie, and K. G. Johnson, Methods Enzymol., in press). Phenyl-p-D-galactopyranoside served as the internal standard. (vi) Acetyl esterase. Assays for acetyl esterase contained 1 mM p-nitrophenylacetate, enzyme at appropriate dilutions, and 50 mM MOPS buffer (pH 6.0) in a final volume of 2.5 ml. The substrate was dissolved in 50% (vol/vol) methanol and was prepared immediately prior to use. After 15 min of incubation at 30°C, p-nitrophenol release was determined from measurements of the A410. (vii) Acetyl xylan esterase. Assays for acetyl xylan esterase contained 4% (wt/vol) acetyl xylan, enzyme at appropriate dilutions, and 100 mM MOPS (pH 6.0) in a final volume of 0.5 ml. After 15 min of incubation at 50°C, 10 ,ul of 1 N sulfuric acid was added to the reaction mixtures. Assay mixtures were then centrifuged at 10,000 x g for 2 min, and the acetic acid content of the supernatants was determined by high-pressure liquid chromatography by using a Polypore H column (Brownlee Labs). The eluant was 0.01 N sulfuric acid. (viii) Ferulic acid esterase. Assays for ferulic acid esterase contained 100 mg of starch-free wheat bran, enzyme at appropriate dilutions, and 100 mM MOPS (pH 6.0) in a final volume of 2.0 ml. After incubation for 30 min at 50°C, the assay mixtures were placed in a boiling water bath for 3 min. Following centrifugation at 10,000 x g for 2 min, the ferulic acid content of the supernatant fractions was determined by high-pressure liquid chromatography with a Bio-Sil ODS-5S column (250 by 4 mm; Bio-Rad). The mobile phase contained 17.5 mM potassium phosphate buffer (pH 7.0) and 5 mM tetrabutylammonium hydrogen sulfate in 20% (vol/vol) methanol (13). Analytical IEF. The protein composition of the various enzyme preparations was examined by analytical IEF. Protein bands in gels were visualized as described previously (22). Activity stains. Areas of CMCase and xylanase activity in IEF gels were located by the activity stain (zymogram) technique described by MacKenzie and Williams (24). Exoglucanase and a-L-arabinosidase activities were located by using 4-methylumbelliferyl-p-lactoside and 4-methylumbelliferylarabinofuranoside as substrates, respectively. 4-Methylumbelliferyl acetate was used as a substrate for acetyl esterase detection; and the 4-methylumbelliferyl ester of the chloride of p-(N,N,N,- trimethylammonium)cinnamic acid was used as substrate for ferulic acid esterase on the basis of the similarity of this compound (17) with esters of ferulic acid, which is a cinnamic acid derivative. The methylumbelliferyl substrates were incorporated into agar gels at concentrations of 5 mM, as described previously (20). The IEF and substrate gels were placed in contact for a suitable period of time, and the hydrolysis zones were visualized and photographed under UV light. Either the IEF or substrate gel
TABLE 1. Extracellular protein concentrations and enzyme activities of S. flavogriseus grown on various carbon sources Carbon
source
Cellulose Oat spelts xylan Wheat bran a
b
Enzyme concn (sp act)'
Protein
(mg/mi) 0.12 1.05 0.88
CMCase (U/ml)
pNPLase (mU/ml)
Xylanase
AXEase
(mU/ml)
MGase (mU/ml)
AEase
(U/ml)
(mU/ml)
(U/ml)
2.5 (20.8) 15.9 (15.1) 3.5 (4.0)
1.4 (0.01) 0 (0) 0 (0)
0.2 (1.7) 24.2 (23.0) 4.3 (4.9)
0 (0) 4.8 (0.005) 29.9 (0.034)
NDb 64 (0.06) 31 (0.035)
42 (0.35) 76 (0.07) 28 (0.03)
0.2 (1.7) 44.8 (42.7) 2.6 (3.0)
AFase
Abbreviations: AFase, arabinofuranosidase; MGase, 4-0-methylglucuronidase; AEase, acetyl esterase; AXEase, acetyl xylan esterase. ND, Not tested.
2837
ENZYME SYSTEMS IN STREPTOMYCES SPP.
VOL. 53, 1987
TABLE 2. Extracellular protein concentrations and enzyme activities of S. olivochromogenes grown on various carbon sources Enzyme concn (sp act)a
Carbon source
Protein (mg/ml)
Cellulose Oat spelts xylan
0.56 0.94 1.06
Wheat bran
CMCase
(U/ml) 20.0 (35.7) 13.0 (13.8) 10.4 (9.8)
pNPLase
(mU/ml) 14 (0.025)
5.9 (0.006) 0 (0)
Xylanase (U/mi) 0.4 (0.71) 28.9 (30.7) 15.0 (14.2)
MGase
AFase
(mU/mI) 1.2 (0.002) 67.1 (0.07) 10.2 (0.01)
AEase
AXEase
(mU/ml) NDb
(U/ml) 0.9 (1.6)
(U/ml) 0.2 (0.36)
61.8 (0.066) 89.9 (0.085)
1.8 (1.9) 1.1 (1.0)
20.2 (21.5) 5.2 (4.9)
FAEase
(mU/mI) 0.4 (0.001) 25.9 (0.027) 12.2 (0.01)
a Abbreviations: AF, arabinofuranosidase; MGase, 4-0-methylglucuronidase; AEase, acetyl esterase; AXEase, acetyl xylan esterase; FAEase, ferulic acid esterase. b ND, Not tested.
could be used for activity detection. The use of 4methylumbelliferyl derivatives as substrates for activity stains has been described by van Tilbeurgh et al. (39). RESULTS Enzyme levels and specific activities in S. flavogriseus cultures. Growth on cellulose resulted in substantial levels of only CMCase and pNPLase (Table 1). Cellulose was also the only growth substrate that resulted in pNPLase activity. In contrast, growth on xylan and wheat bran resulted in appreciably higher levels of extracellular protein coupled with the production of a wider range of enzyme activities. Those activities present included those for CMCase and xylanase and those for hydrolysis of acetyl, arabinosyl, and 4-0methylglucuronyl residues. Notably, growth on xylan and wheat bran resulted in higher CMCase levels than did growth on cellulose. The highest activity levels for xylanase, 4-0methylglucuronidase, and acetyl xylan esterase were observed in cultures grown on xylan. Acetyl esterase levels were low in all cultures and did not correlate with acetyl xylan esterase levels. Arabinosidase was produced maximally by growth on wheat bran; the levels were six times higher than those observed in xylan-supplemented cultures. Enzyme levels and specific activities in S. olivochromogenes cultures. Enzyme levels and specific activities in S. olivochromogenes cultures grown on various substrates resembled those of S. flavogriseus (Table 2). Growth on cellulose resulted largely in the production of CMCase and lower extracellular protein levels than did growth on xylan and wheat bran. Growth on the xylose-containing substrates resulted in the production of CMCase, xylanase, and other xylan-degrading activities. As with S. flavogriseus, higher levels of acetyl esterase and acetyl xylan esterase were found in cultures grown on xylan. Arabinosidase levels were also highest in xylan cultures, but 4-0-methylglucuronidase levels were highest in wheat bran cultures. Ferulic acid esterase was produced in appreciable amounts during growth on xylan and wheat bran, with the former resulting in higher activities. There was no correlation between acetyl esterase and acetyl xylan esterase activities.
Resolution of S. flavogriseus activities by IEF. Growth on cellulose and xylan resulted in distinctly different extracellular protein patterns, as analyzed by IEF (Fig. 1). While the major proteins produced during growth on cellulose were acidic (pl range, 4 to 5), those produced during growth on xylan were basic. The position of the major acidic proteins was in the region where endoglucanase activity was detected. The acidic region also contained a zone of methylumbelliferylcellobioside hydrolysis that was distinct from the endoglucanase zones. Four major extracellular proteins (pl range, 8 to 9) were found in xylan-grown cultures; three of these proteins were located in regions of
endoxylanase activity. The fourth, and most basic, protein corresponded to a zone of methylumbelliferylarabinofuranoside hydrolysis. Zones of methylumbelliferyl acetate hydrolysis could not be detected. Resolution of S. olivochromogenes activities by IEF. As described above for S. flavogriseus, the major proteins produced by S. olivochromogenes during growth on cellulose were acidic, with pl values ranging from 4 to 5 (Fig. 2). There were also several zones of endoglucanase activity in this region. A minor zone of methylumbelliferylcellobioside hydrolysis was present in the pH 4 to 5 range, with two more pronounced zones being visible in the central or neutral region of the gel. Electrofocusing showed that many acidic, neutral, and basic proteins were present in culture fluids of S. olivochromogenes grown on xylan. Also, in contrast to the preparations from cellulose-grown cultures, there was a predominance of higher-pI proteins. Several zones of endoxylanase activity were evident, with the major activity having a pl of approximately 6.8. Two major zones of methylumbelliferylarabinofuranoside hydrolysis were observed in the pl range of 7 to 8. Several acetyl esterases, as detected by methylumbelliferyl acetate hydrolysis, were also located in this region. At least one of the acetyl esterase bands was active against methylumbelliferyltrimethylammonium cinnamate chloride. Several major proteins (pI range, 8 to 9) were not obviously associated with any of the activities examined. -*
A o- 3.5
4-4.6 _-5.2
4-8.2 -8.7
I 2 3 FIG. 1. Analytical IEF of extracellular enzyme preparations obtained from S. flavogriseus cultures grown on Avicel (A) and xylan (B). Lanes: 1, Protein stain; 2, carboxymethyl cellulose hydrolysis; 3, methylumbelliferylcellobioside hydrolysis; 4, protein stain; 5, xylan hydrolysis; 6, methylumbelliferylarabinofuranoside hydrolysis. Positions of pl markers are indicated by arrows.
2838
MAcKENZIE ET AL.
*-4.6 0-5.2
FIG. 2. Analytical IEF of extracellular enzyme preparations obtained from S. olivochromogenes cultures grown on Avicel (A) and xylan (B). Lanes: 1, Protein stain; 2, carboxymethyl cellulose hydrolysis; 3, methylumbelliferylcellobioside hydrolysis; 4, protein stain; 5, xylan hydrolysis; 6, methylumbelliferylarabinofuranoside hydrolysis; 7, methylumbelliferyl acetate hydrolysis; 8, methylumbelliferyltrimethylammonium cinnamate chloride hydrolysis. Positions of pl markers are indicated by arrows.
DISCUSSION
Biosynthesis of activities for hydrolysis of the main chain bonds of cellulose and xylan was under separate control in S. flavogriseus and S. olivochromogenes. Growth of both organisms on cellulose induced cellulase activity, but failed to induce the synthesis of appreciable levels of endoxylanase. Growth of both organisms on xylan and wheat bran, which consist largely of xylose-containing material, induced high levels of endoxylanase. These results agree with those of Kluepfel and Ishaque (18). The results of this study also indicate that xylan-degrading enzymes, other than those which hydrolyze xylose-xylose linkages, were induced by growth on oat spelts xylan or wheat bran. Very low levels of these activities were produced in cultures grown on cellulose. These observations justify the use of the term xylanolytic system to include activities that cleave xylose-xylose bonds as well as other linkages. The xylanolytic systems of S. flavogriseus and S. olivochromogenes are complicated. S. flavogriseus produced two major endoxylanases, and S. olivochromogenes produced several such activities. Also, each organism produced activities for the hydrolysis of other xylan constituents, with multiple forms of these activities being produced in some instances. It was clear that the debranching activities can be highly specific for the linkages found in native xylans. Growth of S. flavogriseus on xylan induced very high levels of acetyl xylan esterase but not of nonspecific acetyl esterase, as measured by using p-nitrophenylacetate as the substrate. A similar but less dramatic effect was observed with S. olivochromogenes. These observations extend the findings of Biely et al. (3, 4) in fungal systems to procaryotic organisms. Five fungal enzyme preparations were shown to contain esterase activity, which demonstrated considerable specificity for acetyl xylan as compared with that for other miscellaneous esterases. It was also shown that fungal
APPL. ENVIRON. MICROBIOL.
esterases acted synergistically with xylanases in the hydrolysis of acetylated xylans (3). Relatively little is known about arabinosidases. There has been speculation that arabinosidase activity is an inherent property of some xylanases, but Biely (2) has suggested that this would be a highly unusual property of an endoglycanase. The results of the present study with S. flavogriseus indicate that the single a-L-arabinosidase band found on IEF was distinct from the endoxylanase zones. The arabinosidase pattern in S. olivochromogenes enzyme preparations was considerably more complicated, but again, the arabinosidase and xylanase bands were distinct and not identical. An a-L-arabinosidase from Ruminococcus albus, a rumen anaerobe, has been partially characterized and acts synergistically with xylanase in releasing reducing sugar from alfalfa cell wall material (11, 12). In addition, relatively little is known about the ox-4-0methyl-D-glucuronidase component of xylanolytic complexes. Two fungi, Agaricus bisporus and Pleurotus ostreatus, have recently been reported to be good producers of this enzyme (33). It was also found that the aglucuronidase produced by these organisms acts synergistically with xylanase in releasing xylose from xylans. The accumulation of free uronic acid during cultivation of organisms on xylan is indicative of the presence of this enzyme in other organisms (32, 37). S. olivochromogenes produces extracellular esterase, which releases ferulic acid from wheat bran. This is, to our knowledge, the first report of such an activity in hemicellulase preparations and of its coproduction with other enzymes that are specifically involved in xylan hydrolysis. Ferulic acid is esterified to arabinose in wheat bran cell walls. Treatment of wheat bran with Oxysporus cellulase releases
2-0-[5-0-(trans-feruloyl)-3-L-arabinofuranosylI-D-xylopy-
ranose (35). These linkages have also been identified in barley straw cell walls. In barley straw, trans-p-coumaric acid is also linked to arabinoxylans in the same way (27). In both wheat bran and barley straw, less than 1% of the pentose residues is esterified in this manner. Spinach and sugar beet pectins are also feruloylated plant cell wall polymers (9, 34). The function of phenolic acid substituents is unclear. They may play a role in cell wall growth or stabilization by providing a means of cross-linking xylans or forming lignin-hemicellulose complexes. In xylanolytic enzyme systems, the action of the esterase may lead to a rapid breakdown of cell wall integrity and the more rapid action of other enzymatic components present in the system. In this connection, it is noteworthy that the amount of phenolic materials released from cell wall material correlates with the amount of forage digested by ruminants (1, 10, 38). ACKNOWLEDGMENTS The technical assistance of Selwyn de Souza is gratefully acknowledged. We also thank J. D. Fontana for valuable advice.
LITERATURE CITED 1. Alibes, X., F. Munoz, and R. Faci. 1984. Anhydrous ammoniatreated cereal straw for animal feeding. Anim. Feed Sci. Technol. 10:239-246. 2. Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3:286-290. 3. Biely, P., C. R. MacKenzie, J. Puls, and H. Schneider. 1986. Cooperativity of esterases and xylanases in the enzymic degradation of acetyl xylan. Bio/Technology 4:731-773. 4. Biely, P., J. Puls, and H. Schneider. 1985. Acetyl xylan esterases in fungal cellulolytic systems. FEBS Lett. 186:80-84. 5. Bradford, M. 1976. A rapid and sensitive method for the
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6. 7. 8.
9. 10.
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quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72:248-254. Crawford, D. L. 1981. Microbial conversion of lignin to useful chemicals using a lignin degrading Streptomyces. Biotechnol. Bioeng. Symp. 11:275-291. Dekker, R. F. H. 1985. Biodegradation of the hemicelluloses, p. 505-533. In T. Higuchi (ed.), Biosynthesis and degradation of wood components. Academic Press, Inc., New York. Deshpande, M. U., K.-E. Eriksson, and L. G. Pettersson. 1984. An assay for selective determination of exo-1,4-p-glucanases in a mixture of cellulolytic enzymes. Anal. Biochem. 138:481-487. Fry, S. C. 1983. Feruloylated pectins from the primary cell wall: their structure and possible functions. Planta 157:111-123. Graham, H., and D. Aman. 1984. A comparison between degradation in vitro and in sacco of constituents of untreated and ammonia-treated barley straw. Anim. Feed Sci. Technol. 10:199-211. Greve, L. C., J. M. Labavitch, and R. E. Hungate. 1984. a-L-Arabinofuranosidase from Ruminococcus albus 8: purification and possible role in hydrolysis of alfalfa cell wall. Appl. Environ. Microbiol. 47:1135-1140. Greve, L. C., J. M. Labavitch, R. J. Stack, and R. E. Hungate. 1984. Muralytic activities of Ruminococcus albus 8. Appl. Environ. Microbiol. 47:1141-1145. Goycoolea, M., D. Seelenfreund, C. Ruttimann, B. Gonzalez, and R. Vicuna. 1986. Monitoring bacterial consumption of low molecular weight lignin derivatives by high performance liquid chromatography. Enzyme Microb. Technol. 8:213-216. Haigerdal, B. G. R., J. D. Ferchak, and E. K. Pye. 1978. Cellulolytic enzyme system of Thermoactinomyces spp. grown on microcrystalline cellulose. Appl. Environ. Microbiol. 36: 606-612. Hartley, R. D., and E. C. Jones. 1976. Diferulate as a component of cell walls of Lolium multiflorum. Phytochemistry 15: 1157-1160. Ishaque, M., and D. Kleupfel. 1980. Cellulase complex of a mesophilic Streptomyces strain. Can. J. Microbiol. 26:183-189. Jameson, G. W., D. V. Roberts, R. W. Adams, W. S. A. Kyle, and D. T. Elmore. 1973. Determination of the operational molarity of solutions of bovine a-chymotrypsin, trypsin, thrombin, and factor Xa by spectrofluorimetric titration. Biochem. J. 131:107-117. Kluepfel, D., and M. Ishaque. 1982. Xylan-induced cellulolytic enzymes in Streptomyces flavogriseus. Dev. Ind. Microbiol. 23:389-395. Kluepfel, D., F. Schareck, F. Mondou, and R. Morosoli. 1986. Characterization of cellulase and xylanase activities of Streptomyces lividans. Appl. Microbiol. Biotechnol. 24:230-234. Kyriacou, A., C. R. MacKenzie, and R. J. Neufeld. 1987. Detection and characterization of the specific and nonspecific endoglucanases of Trichoderma reesei: evidence demonstrating endoglucanase activity by cellobiohydrolase II. Enzyme Microb. Technol. 9:25-32. MacKenzie, C. R., and D. Bilous. 1982. Location and kinetic properties of the cellulase system of Acetivibrio celluloyticus.
Can. J. Microbiol. 28:1158-1164. 22. MacKenzie, C. R., D. Bilous, and K. G. Johnson. 1984. Purification and characterization of an exoglucanase from Streptomycesflavogriseus. Can. J. Microbiol. 30:1171-1178.
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