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Cellulase-free xylanase from Thermomyces lanuginosus: Optimization of production in submerged and solid-state culture
Cellulase-free xylanase from
Thermomyces lanuginosus:
Optimization of production in submerged and solid-state culture Heinz Purkarthofer,* Michael Sinner? and Walter Steiner* * I n s t i t u t e J b r B i o t e c h n o l o g y , T e c h n i c a l U n i w . r s i t y Graz, Graz, A u s t r i a a n d + V O E S T - A L P I N E l n d u s t r i e a n l a g e n b a u G m b H , Linz, A u s t r i a
Cultural conditions fi)r the production o f thermostable xylanase (EC 3.2.1.8) by the thermophilic Jitngus Thermomyces lanuginosus were investigated in both submerged and solid-state culture. Under submerged growth conditions in shake flasks, the C and N sources mainly influenced the enzyme yield. The xylanase of T. lanuginosus was found to be inducible by xylan containing C sources as well as by xylose. The enzyme spectrum in culture filtrates after growth on d~fferent C-sources was compared by means o f sodium dode~TI sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) . A central composite design was used to optimize the fermentation medium with respect to medium components. The medium f o r the optimal production (31.2 g I I corn cobs, 30.2 g I- i yeast extract, and 5.0 g I- i KI~PO4) yielded 36,200 nkat m l i within 7 days o f cultivation. The prod.orion o f other li~,nocelhdolytic enzymes (~-xylosidase, [3-glucosidase, acetyl esterase, and a-arabino.fitranosidase) using the optimized medium was vel3' low. The optimal p t f values o f all enzymes were determined. Filter paper celhdase, CMCase, [3-mannosidase. and mannanase activities were absent. Solid-state cultures using corn cobs as C source were carried oat with a water content o f 70% and with yeast extract between 1.4 attd 9.6% (w/w). The optimized medium containing 1.7.5% yeast extract yielded 125,000 nkat ml- t xylanase activity (337,000 nkat g I dr3' solid) within 9 days o f cultivation.
Keywords:Thermomy¢'es lanuginowts: xylanase; central composite design: medium optimization: solid-state culture: submerged culture Introduction Endo-l,4-~-D-xylanases (EC 3.2.1.8) are produced by numerous microorganisms, among which the fungi are the most potent producers.~ Most publications dealing with xylanases concentrate on the purification and characterization, whereas much less has been reported on the production, application, and process economy of utilizing xylanolytic enzymes.: Since it is possible to reduce the lignin content of hardwood kraft pulp and to lessen the amount of chlorine used for bleaching by employing x y l a n a s e s J they represent a promising chance to reduce the emission of toxic compounds from the bleaching process. With several large-scale trials
in progress. 4-5 xylanases will in all probability be one of the first major applications of biotechnology in the pulp and paper industry. H o w e v e r . for a broad application, the cost of enzymes is one of the main factors determining the economics of a process. 6 In view of the basic research for industrial application, the aim of these studies was to reduce the costs of e n z y m e production by optimizing the fermentation medium. The effect of mcdia components on the production of xylanase by T h e r m o m y c e s l a n u g i n o s t t s was studied in both submerged and solid-state cultures. A central composite design was used to find an optimal medium composition.
Materials and methods Organism
Address reprint requests to Dr. Steiner at the Institutefor Biotechnology, Technical University Graz. Petersgasse 12, A-8010 Graz. Austria Received 22 September 1992; revised 26 November 1992
© 1993
Butterworth-Heinemann
T. lanuginosus was isolated from decomposed jute stacks by I. Gomes at the Jute Research Institute, Dhaka, Bangladesh. It is deposited at the German type culture collection (DSM Braunschweig) under the number DSM 5826.
Enzyme Microb. Technol., 1993, vol. 15, August
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Papers Stock cultures were maintained on potato dextrose agar (PDA) at 4~C and transferred every 6-7 weeks. PDA-plates were incubated at 50°C for 3-4 days.
Medic+ Submerged culture. A mineral medium containing 4.0 g 1 (NH+),SQ, 10.0 g l -L KH2P() 4.0.3 g l i CaCIz, 0.3 g l -I FeSO4, and 0.3 g I ~ M g S Q , in addition to carbon and organic nitrogen sources, was used unless otherwise stated. Media were generally autoclaved once (30 rain, 121°C) except those containing corn cobs. After a tirst sterilization (45 rain, 121°C), every medium containing corn cobs was incubated at 50°C for 2 days, followed by the addition of organic N source and a second sterilization (45 rain. 121"C). This procedure was necessary to avoid growth ofthermophilic bacteria. by which the corn cobs were contaminated. The initial pH value of the media was adjusted to 6.5. Additional sterile water was added to compensate for losses during autoclaving. Solid-state culture. The basal medium contained 71) g corn cobs (milled). 0.40 g (NH4):SO.~. 2.1 g KH:POa, 0.11 g FeSO~, 0.311 g M g S Q . and 0.30 g CaCI,. The initial water content of all media was adjusted to 71Y~ iw/w). Autoclaving was carried out at 121°C for 51) rain. lnoculum
and cultivation
Submerged culture, A piece of potato dextrose agar ( 1 cm z) from an actively growing 3- to 4-day-old colony of T. lanugittostt,s was used as an inoculum for shake flask cultures in 300-ml unbaffled Erlenmeyer flasks containing 100 ml of medium. The inoculated flasks werc shaken continuously (Infors HT RBI-112 Benchtop Incubator Shaker "Aerotron") with humidified air at 1517re',' rain L(stroke 12.5 ram) and at 50°C for 3-7 days. For the evaluation of the carbon source effect, a rotary shaker (Infors AG. Bottmingen, Switzerland. stroke 25 ram)at 121) rcv rain-i was used. The culture broth was centrifuged for the estimation of enzyme activitics. Solid-state culture. Five pieces (each 1 cm-') of an actively growing colony on potato dextrose agar werc used as an inoculum. Solid-state cultures were carried out in 1000-ml Erlcnmeyer flasks. The media were incubated statically at 50°C in a H,O-saturated atmosphere. Every 24 h the flasks were shaken by hand to prevent solidification of the medium. The soluble enzyme preparation was obtained by pressing the mycelium and medium by hand using a nylon cloth and centrifuging for further analysis 115 rain, 4000 rev rain ~).
2 ml 1 M Na,CO~ solution. Absorbance was read at 405 nm. B-Glucosidase, /3-mannosidase, and o~-arabinofuranosidase activities were assayed as described for/3-xylosidase using substrate solutions of 4 mg ml - i p-nitrophenyl ,B-D-glucopyranoside (NPG). 4 mg m l i p-nitrophenyl-/3-D-mannopyranoside (NPM), and 3 mg ml i p-nitrophenyl-~-L-arabinofuranoside (NPA), respectively. Filter paper activity was assayed according to IUPAC recommendations at a pH value of 6.5. ~o CMCase activity was determined analogously using a solution of 1.0% (w/v) carboxymethylcellulose with a hydrolysis time of 30 rain. Acetyl esterase was determined according to Poutanen e t a / . ~ at a pH of 6.5 using c~-naphthylacetate as substrate.
Eiectrophoresis of protein. Electrophorcsis was carried out under denaturing conditions as described by Laemmli. t-~The gels and running buffer contained 0.1% sodium dodecyl sultate (SDS). Twelve percent acrylamide gels were used and electrophoresis was carried out at pH 8.3 in Tris-glycine buffer 11).05 M Tris. 0.38 M glycine). The proteins in the gels were stained using a 0.5c2~ solution of Coomassie brilliant blue prepared in methanol : acetic acid : water 20 : 7 : 73 (v/v/v). Optimization. The optimization of the production medium was carried out within the framework of a central composite design for the two variables corn cobs and yeast extract. In this design an axial spacing of c~ = 1.5 was chosen in order to obtain greater precision for the estimates of the quadratic effects. This value for the axial distance o~does not fulfill the conditions for orthogonality for all coefficients. Four center points were used to estimate the experimental error. A commercially available program STATGRAPHICS (Statistical Graphics Corp.. USA) was used for the contour plotting.
Results and discussion
Effect +~ carbon sources T h e effect of c a r b o n s o u r c e s o n the e n z y m e yield was tested by the use of the basal m e d i u m with a d d i t i o n of 28.5 g I ~ yeast extract. T h e c o n c e n t r a t i o n of each C source was 25 g 1 ~. Table I s h o w s the e n z y m e titers o b t a i n e d from different C s o u r c e s after 5 d a y s of cultivation. Significant d i f f e r e n c e s in the rate of x y l a n a s e
Table 1 Effect of carbon sources on xylanase production
Carbon source
Enzyme activity assays. Endo-1.4-,0-D-xylanase was assayed according to Bailey et a/.: by incubating the diluted enzyme solution at a pH of 6.5 and a temperature of 50°C for 5 rain using a substrate solution of 1.0% (w/v) birchwood xylan (Roth, Karlsruhe, FRG). Reducing sugars were assayed by adding 3 ml of DNS (2-hydroxy-3,5 dinitrobenzoic acid) reagent, boiling for 5 rain, cooling, and measuring the absorbance at 5411nm. s Mannanase was determined in an analogous manner using a solution of 0.5% (w/v) carob seed galactomannan as substrate. /3-Xylosidase was determined using a modified method according to Lachke et al. ~ Onehalf milliliter 11.05 M citrate buffer of adjusted pH, 0.25 ml diluted sample, and 0.25 ml of a solution of 3 mg mlp-nitrophenyl-/3-n-xylopyranoside(NPX) were incubated at 50°C. After 10 rain the reaction was stopped by addition of
678
E n z y m e M i c r o b . T e c h n o l . , 1993, vol. 15, A u g u s t
Alfalfa Birch pulp (bleached) Corn cobs (powdered) Corn cobs (coarse) Glucose Jute fiber (steamed) Poplar wood chips Rice husk Soya oil Oat spelts Starch (soluble) Wheat straw (steamed) Xylan (beechwood) Xylose
Xylanase activity (nkat m1-1) 545 1,900 7,952 26,700 8 1,600 338 1,630 25 1,020 28 3,870 17,300 4,270
Xylanase from Thermomyces lanuginosus: H. Purkarthofer et al. production occurred by variation of the C sources. Xylan or a xylan-containing C source is necessary for a satisfactory production of xylanase by T. lanuginosus, not only because it is the main C source, but probably also because its hydrolysis products act as inducers. Xylosc also had an inducing effect on the formation of xylanolytic enzymes. With media that did not contain inducers, xylanase liters of only about 25 nkat ml-~ were obtained. From these data it seems to be clear that a low level of xylanase formation is constitutive. Culture filtrates were found to bc frec of filter paper activity. The S D S - P A G E pattern of the excreted enzymes from cultivation with several different carbon sources is shown in Figure 1. When corn cobs and xylan were used as C sources, xylanasc was the main component in the culture filtrate, as can be judged from the good correlation between xylanasc activity and intensity of the major band. Xylanasc occurs as a single band with a molecular wcight of 26.0 kD, a value that slightly differs from those molecular weights of 22.5 and 21.5 kD previously determined by S D S - P A G E using different isolates of T. lanttginosus, a'''14 Taking into consideration the low costs of corn cobs, which are a cheap agricultural waste material, further steps of the optimization process were carried out using this C source. In the following experiments the particle size of corn cobs was found to have a strong influence on xylanase production. A particle size of 2-7 mm was optimal. These data indicate that a slower solubilization of reducing sugars is beneficial for xylanase production. Steam treatment of the corn cobs to possibly attain a better accessibility of the substrate and hot water extraction for a reduction of the initial concentration of soluble sugars in the medium did not affect enzyme production.
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Table 2 Effect of organic nitrogen sources on xylanase production during utilization of xylan and corn cobs (2-7 ram) as C source Xylanase activity (nkat ml 1) N source
Corn cobs
Xylan
24190 22000 19370 18750 18090 17820 17770 16540 16400 13620 1600
16670 10990 15100 12650 13300 11300 16940 19670 10270 11150 2450
Yeast extract Casein peptone Meat extract Pha rmamedia Cornsteep powder Cottonseed Fish peptone Fish peptone a Meat peptone Gelitaflex Soya meal a 30% yeast blend
Effect o f nitrogen sources The effect of various organic nitrogen compounds on the formation of xylanase by T. lanuginosus was tested by the use of the basal medium together with corn cobs or xylan (25.0 g 1-~) and the organic N source. The concentrations of the N sources used were based on their total nitrogen content as estimated by the Kjeidahl method. The concentration of organic nitrogen was fixed at 3.0 g N 1- ~. The xylanase activities after 5 days of cultivation using different N sources are shown in
Table 2. All N sources promoted growth of the fungus. In all but one medium containing corn cobs, the pH value after 5 days of incubation (pH 7.5-8.4) was higher than in those containing xylan as C source (pH 6.0-6.5), which seems to be more favorable for xylanase production. With soya meal as N source, the final pH value was 5.5 (corn cobs) and 6.0 (xylan). With all N sources except for fish peptone, e n z y m e production using corn cobs was higher than with xylan. The protein pattern of the culture filtrates did not change by varying the N source. Yeast extract was chosen as the best N source for further optimization.
Optimization o1" the m e d i u m
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6
7
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8
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9
10
Figure 1 SDS-PAGE of culture filtrates of T. lanuginosus grown on different carbon sources. Lane 1, MW standards; lane 2, wheat bran; lane 3, alfalfa; lane 4, starch; lane 5, oat spelts; lane 6, wheat straw; lane 7, corn cobs (coarse); lane 8, soya oil; lane 9, xylan; lane 10, MW standards
Using two-level factorial designs, we found that addition of calcium, iron, and magnesium ions as well as several trace elements (Zn, Mn, Co, Cu, Ni, B) had no influence on xylanase production when tap water was used for media preparation (data not shown). Therefore, these supplements as well as ammonium sulfate were omitted in the following experiments. An increase in the concentrations of C and N sources up to a level of 30 to 35 g 1- ~led to a prolonged production phase. The highest activities of xylanase at these concentrations were reached after 7 days of cultivation. Realizing that the C and N sources were by far the most important factors for xylanase production, the Enzyme Microb. Technol., 1993, vol. 15, August
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Papers
optimal concentrations o f media components were determined using a central composite design with the two variables C and N source. The media consisted of 5.0 g l - ~ KH2PO ~ and different concentrations of corn cobs and yeast extract. The levels of the two variable factors are presented in Table 3. The results were evaluated by the response surface statistical method similar to that used by McDaniel et al. ~ Some modifications had to be made taking into account that the design was not completely orthogonal. A regression analysis was run to fit a second-order model to the experimental data. It yielded the least squares estimated coefficients: b,: 36390; b~: - 9 3 2 : b,: - 4 9 2 : b ~
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: I
X\XI
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-2372:
.........
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b_,2" - 1574; b 1 2 : - 7 8 8 X
The model is an adequate approximation to the data at the 99% probability level. The coefficients b~ and bl2 were found not to be significantly different from zero at a significance level of c~ = 0.10. Nevertheless. these coefficients were not dismissed as unnecessary in the regression equation, since there was no reason for making the hypothesis b, = 0 and b,2 = 0, respectively, and they were the best estimates for this effect. The optimum was determined at x, = - 0 . 1 8 and x, = - 0 . I0, which correlates to 31.2 g I ~ corn cobs and 30.2 g I ~ yeast extract. The contour plot (Fi~,,ure 2) shows a rather broad plateau region with the highest xylanase activity, in which the activities changed relatively little with changes in nutrient levels. In order to determine the accuracy of the optimization procedure, shake-flask experiments with nutrient media combinations representing the estimated optimum were repeatedly conducted yielding 36,160 -'- 1,880 nkat ml (n = 6), which is one of the highest xylanase activities ever reported. The addition of 0. I% polypropyleneglycol (PPG) as an antifoaming agent did not influence the rate of xylanase production, whereas soya oil in the same concentration reduced the xylanase yield by 25%. The final pH of the media to which soya oil was added was 6.1, as compared to a pH value of 7.9 in the media without soya oil.
Time course o f e n z y m e production in s u b m e r g e d culture The time course of the production of various lignocellulolytic enzymes which could play a major role in bleaching of pulp was determined in shake flasks using the optimized medium. For all detected enzyme activi-
Table3
Range of values for the central composite design
- 1.5 21.3 19.0
- 1.0 25.0 23.0
0 32.5 31.0
- 1.0 40.0 39.0
- 1.5 43.8 43.0
Concentrations of corn cobs and yeast extract are given in g I
680
Enzyme
Microb.
Technol.,
/
-
/ •
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z
i
F i g u r e 2 Contour plot of the equation calculated to fit the data of the central composite design. Concentrations of corn cobs and yeast extract are s h o w n in Table 3. Xylanase activity is given in/2kat m1-1
ties (/3-xylosidase,/3-glucosidase, acetyl esterase, and o~-L-arabinofuranosidase), the pH optima were determined. These data are shown in Fi~,,ure 3. The pH dependency of acetyl esterase activity suggests that it consists of at least two different enzymes with different pH optima. The culture filtrate did not show any filter paper or CMCase activity. Mannanase and [3-mannosidase activities were also not detectable. As can be seen in Table 4, the excretion of the enzymes ~-xylosidase, /3-glucosidase, tx-arabinofuranosidase, and acetyl esterase was very low as compared to that of xylanase. The optimum of/.3-xylosidase and o~-arabinofuranosidase activities was reached at the sixth day of cultivation, whereas/3-glucosidase and acetyl esterase activity increased with pronounced autolysis of the fungus. Since the pH optima of the other enzymes differ from the pH optimum of 6.5 found for xylanase, the actual ratio of xylanase to the activity of these other enzymes is even greater, as indicated in Table 4 when the culture filtrate is used for bleaching purposes at this pH value.
Solid-state cultivation
Levels Variables Corn cobs Yeast extract
j
1 9 9 3 , v o l . 15, A u g u s t
The effect of nitrogen concentration on the production of xylanase by T. lanuginosus in solid-state cultivation was studied using media with an initial water content of 70% (w/w) at a temperature of 50°C, which has proved to be optimal for production. These values were
Xylanase from Thermomyces lanuginosus: H. Purkarthofer et al. 120
140(300
----a~
100
JB-Glucosidase
=
~-Xylosidase
8o
120000
'
~m 100000
-
80000 -"
•~
•
60
60000 -
~ 40
40000 -'
211
20000 "
~
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----o~
J
i
i
ct-Arabinofuranosidase
•
i
200
100
i
Acetylesterase
1.75 % 21% 5.7% 7.0%
300
Hours Of cultivation
Figure 4 Time course of xylanase production in solid-state cultures by T. lanuginosus at different yeast extract concentrations
80
20
3
i
i
i
!
i
4
5
6
7
8
9
pH
Figure 3 Effect of pH on the activity of acetyl esterase, B-glucosidase,/3-xylosidase, and (~-arabinosidase of T. lanuginosus
somewhat higher than the values of 45°C and 65% moisture content found by Kitprcevanavich et al.~6 Yeast extract was added in the amounts of 1.4, 1.75, 2. I, 2.4, 2.9, 4.4, 5.7, 7.0, 8.3, and 9.6% (w/w), respectively. Strong growth of the fungus occurred on the surface of the corn cob particles, with an average particle size of 5 mm after a lag of I day, except in the case of the two media containing the highest amounts of yeast extract. The concentration of the nitrogen source had a strong influence on the production of xylanase. Surprisingly, low amounts of nitrogen lead to a higher e n z y m e production. The 1.75% yeast extract was found to be optimal. After 9 days of cultivation, an activity of 125,000 nkat ml-~ culture centrifugate (336,700 nkat g ~ dry solid) was determined. Selected time courses of xylanase production with media containing low amounts of yeast extract arc prcsented in Figure 4. The final xylanase titers after 179 h of cultivation using the media not shown in Figure 4 were 105,000 nkat ml - t (1.4%), 95,600 nkat ml i (2.9%), 85,600 nkat ml - i
Table 4 Time course of various lignocellulolytic enzymes using the optimized medium in shake-flask experiments Activity (nkat m1-1) Enzyme
3 days
Xylanase 5380 /3-Xylosidase 1.8 /3-Glu cosidase 1.3 a-Arabinosidase 0.1 Acetyl esterase --
4 days
5 days
6 days
7 days
11880 20630 32170 36150 4.1 6.5 7.5 5.2 1.2 1.6 2.1 3.0 0.1 0.8 1.0 0.3 0.6 0.9 1.3 1.5
(4,4%), 59,300 nkat m l ~ (7.0%), 45,000 nkat ml -~ (8,3%), and 11,300 nkat ml ~(9.6%), respectively. Agitating the solid-state culture continuously at low speed (15 rev rain -~) in a rolling bottle (20 1) reduced the production of xylanase drastically to a value of 14% in comparison to that in static culture. This could be explained by shear stress sensitivity of the fungal mycelium. The molecular weight of the xylanase was identical to that produced in submerged culture. The xylanase yield based on the nitrogen source which was the most expensive medium compound in solid-state cultivation (43, I00 nkat mg t N) was superior to that in submerged culture ( 11,300 nkat mg t N). Furthermore the productivity was much higher (solid state: 553 nkat ml ~ h j" submerged: 215 nkat ml ~h ~). Although these data are promising, a scaleup of this solid-state process seems to be difficult due to the generally known problems of heat transfer, inhomogeneity of media, and aeration. These problems are exacerbated by the shear sensitivity of T. lanuginosus. The thermophilic nature of the fungus represents an advantage with regard to heat transfer within the medium. The culture filtrates of T. lanuginosus did not show any filter paper or CMCase activities, which is of tremendous importance for pulp bleaching, since treatment of pulp with xylanase preparations containing cellulases has resulted in a reduction of the polymerization degree of the cellulose fibers and a drop in product quality. J7 Therefore, it is possible to use these filtrates for pulp bleaching without previous purification, which dramatically lowers the costs of application. The lack oflignocellulolytic enzymes other than xylanase makes this strain very promising, not only for bleaching purposes, but also for processes where a relatively pure xylanase without expensive purification procedure would be needed.
Acknowledgements The skillful technical assistance of Ms. Sabine Mauler is gratefully acknowledged. This work was financially supported by the V O E S T A L P I N E lndustrieanlagenbau GesmbH, Linz, Austria.
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8 9
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Senior, J. S., Mayers, P. R. and Saddler, J, N. Production and purification of xylanases. In Plant (.'ell Wall Polymers. Bio~,enesis and Biodek, radation (l,cwis, N. G. and Paice, M. G., eds.) ACS Symposium Series 399, American Chemical Society. Washington, I)C, 1989, pp. 641-654 Poutanen, K., Ratio6, M., Puls. J. and Viikari, L. Evaluation of different microbial xylanolytic systems. J. Biotectmol. 1987, 6, 49-60 Paice, M. G., Bernier. R., Jr. and Jurasek. I,. Viscosityenhancing bleaching of hardwood kraft pulp with xylanase from a cloned gene. Biotechnol. Bioeng. 1988, 32, 235-239 Wizani, W., Esterbauer. H., Steiner, W. and Gomes, J. Xylanase manufacture with Thermomyces. Ear. Pat. Appl. 1991, 10 Sinner, M., Ditzelmtiller, G., Wizani, W., Steiner, W. and Esterbauer. H. Papier. 1991, 45, 403-410 Biely, P. Microbial xylanolytic enzymes. 7)'end,~ Biotechnol. 1985, II, 286-290 Bailey, M. J., Biely, P. and Poutanen, K. lnterlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 1992, 23, 257-270 Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31,426-428 I,achke, A. H.. Deshpande. M. V. and Srinivasan. M. C. Extra-
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cellular/3q~-xylosidase of Sclerotium rolfsii. Enzyme Microb. lechnol. 1985, 7, 445-448 IUPAC. Measurement ofcellulase activities. Pure Appl. Chem. 1987, 59, 257-268 Poutanen. K. and Sundberg, M. An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans. Appl. Microbiol. Biotechnol. 1988, 2,8, 419-424 Lacmmli, U. K. Nuture 1970. 227, 680-685 Anand. L.. Krishnamurthy. S. and Vithayathil, P. J. Purification and properties of xylanasc from the thcrmophilic fungus. tltmlic~la lanu,l,,ino.~a (Griffon and Maublanc) Bunce. Arch. Biochem. Biophys. 1990, 276, 546-553 Kitpreechavanich, V.. Hayashi, M. and Nagai, S. Purification and properties of cndo-l,4-/:t-xylanase from Humicola lanuk, ino.~a. J. Ferment. Technol. 1984, 62, 415-420 McDaniel, L. E., Bailey. E. G., Erhiraj, S. and Andrews, H. P. Application of response surface optimization technique to polyene macrolide fermentation studies in shake flask. Det,. Ind. Mi('rohiol. 1976, 17, 91-94 Kitprcechavanich, V. Production of xylan degrading enzymes by thermophilic fimgi, A,~pergillus .fimfigatus and Humicola lanu~,,ino.sa. J. Ferment. Tec'hm~/. 1984, 62, 63-69 Senior, D. J., Mayers. P. R.. Miller, D.. Sutcliffe, R.. Tan. 1,. and Saddler. J. N. Selective solubilization of xylan in pulp using a purified xylanase from Trichoderma harzianum. Biotechnol. l.ett. 1988, 10, 907-912
Thermomyces lanuginosus:
Optimization of production in submerged and solid-state culture Heinz Purkarthofer,* Michael Sinner? and Walter Steiner* * I n s t i t u t e J b r B i o t e c h n o l o g y , T e c h n i c a l U n i w . r s i t y Graz, Graz, A u s t r i a a n d + V O E S T - A L P I N E l n d u s t r i e a n l a g e n b a u G m b H , Linz, A u s t r i a
Cultural conditions fi)r the production o f thermostable xylanase (EC 3.2.1.8) by the thermophilic Jitngus Thermomyces lanuginosus were investigated in both submerged and solid-state culture. Under submerged growth conditions in shake flasks, the C and N sources mainly influenced the enzyme yield. The xylanase of T. lanuginosus was found to be inducible by xylan containing C sources as well as by xylose. The enzyme spectrum in culture filtrates after growth on d~fferent C-sources was compared by means o f sodium dode~TI sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E ) . A central composite design was used to optimize the fermentation medium with respect to medium components. The medium f o r the optimal production (31.2 g I I corn cobs, 30.2 g I- i yeast extract, and 5.0 g I- i KI~PO4) yielded 36,200 nkat m l i within 7 days o f cultivation. The prod.orion o f other li~,nocelhdolytic enzymes (~-xylosidase, [3-glucosidase, acetyl esterase, and a-arabino.fitranosidase) using the optimized medium was vel3' low. The optimal p t f values o f all enzymes were determined. Filter paper celhdase, CMCase, [3-mannosidase. and mannanase activities were absent. Solid-state cultures using corn cobs as C source were carried oat with a water content o f 70% and with yeast extract between 1.4 attd 9.6% (w/w). The optimized medium containing 1.7.5% yeast extract yielded 125,000 nkat ml- t xylanase activity (337,000 nkat g I dr3' solid) within 9 days o f cultivation.
Keywords:Thermomy¢'es lanuginowts: xylanase; central composite design: medium optimization: solid-state culture: submerged culture Introduction Endo-l,4-~-D-xylanases (EC 3.2.1.8) are produced by numerous microorganisms, among which the fungi are the most potent producers.~ Most publications dealing with xylanases concentrate on the purification and characterization, whereas much less has been reported on the production, application, and process economy of utilizing xylanolytic enzymes.: Since it is possible to reduce the lignin content of hardwood kraft pulp and to lessen the amount of chlorine used for bleaching by employing x y l a n a s e s J they represent a promising chance to reduce the emission of toxic compounds from the bleaching process. With several large-scale trials
in progress. 4-5 xylanases will in all probability be one of the first major applications of biotechnology in the pulp and paper industry. H o w e v e r . for a broad application, the cost of enzymes is one of the main factors determining the economics of a process. 6 In view of the basic research for industrial application, the aim of these studies was to reduce the costs of e n z y m e production by optimizing the fermentation medium. The effect of mcdia components on the production of xylanase by T h e r m o m y c e s l a n u g i n o s t t s was studied in both submerged and solid-state cultures. A central composite design was used to find an optimal medium composition.
Materials and methods Organism
Address reprint requests to Dr. Steiner at the Institutefor Biotechnology, Technical University Graz. Petersgasse 12, A-8010 Graz. Austria Received 22 September 1992; revised 26 November 1992
© 1993
Butterworth-Heinemann
T. lanuginosus was isolated from decomposed jute stacks by I. Gomes at the Jute Research Institute, Dhaka, Bangladesh. It is deposited at the German type culture collection (DSM Braunschweig) under the number DSM 5826.
Enzyme Microb. Technol., 1993, vol. 15, August
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Papers Stock cultures were maintained on potato dextrose agar (PDA) at 4~C and transferred every 6-7 weeks. PDA-plates were incubated at 50°C for 3-4 days.
Medic+ Submerged culture. A mineral medium containing 4.0 g 1 (NH+),SQ, 10.0 g l -L KH2P() 4.0.3 g l i CaCIz, 0.3 g l -I FeSO4, and 0.3 g I ~ M g S Q , in addition to carbon and organic nitrogen sources, was used unless otherwise stated. Media were generally autoclaved once (30 rain, 121°C) except those containing corn cobs. After a tirst sterilization (45 rain, 121°C), every medium containing corn cobs was incubated at 50°C for 2 days, followed by the addition of organic N source and a second sterilization (45 rain. 121"C). This procedure was necessary to avoid growth ofthermophilic bacteria. by which the corn cobs were contaminated. The initial pH value of the media was adjusted to 6.5. Additional sterile water was added to compensate for losses during autoclaving. Solid-state culture. The basal medium contained 71) g corn cobs (milled). 0.40 g (NH4):SO.~. 2.1 g KH:POa, 0.11 g FeSO~, 0.311 g M g S Q . and 0.30 g CaCI,. The initial water content of all media was adjusted to 71Y~ iw/w). Autoclaving was carried out at 121°C for 51) rain. lnoculum
and cultivation
Submerged culture, A piece of potato dextrose agar ( 1 cm z) from an actively growing 3- to 4-day-old colony of T. lanugittostt,s was used as an inoculum for shake flask cultures in 300-ml unbaffled Erlenmeyer flasks containing 100 ml of medium. The inoculated flasks werc shaken continuously (Infors HT RBI-112 Benchtop Incubator Shaker "Aerotron") with humidified air at 1517re',' rain L(stroke 12.5 ram) and at 50°C for 3-7 days. For the evaluation of the carbon source effect, a rotary shaker (Infors AG. Bottmingen, Switzerland. stroke 25 ram)at 121) rcv rain-i was used. The culture broth was centrifuged for the estimation of enzyme activitics. Solid-state culture. Five pieces (each 1 cm-') of an actively growing colony on potato dextrose agar werc used as an inoculum. Solid-state cultures were carried out in 1000-ml Erlcnmeyer flasks. The media were incubated statically at 50°C in a H,O-saturated atmosphere. Every 24 h the flasks were shaken by hand to prevent solidification of the medium. The soluble enzyme preparation was obtained by pressing the mycelium and medium by hand using a nylon cloth and centrifuging for further analysis 115 rain, 4000 rev rain ~).
2 ml 1 M Na,CO~ solution. Absorbance was read at 405 nm. B-Glucosidase, /3-mannosidase, and o~-arabinofuranosidase activities were assayed as described for/3-xylosidase using substrate solutions of 4 mg ml - i p-nitrophenyl ,B-D-glucopyranoside (NPG). 4 mg m l i p-nitrophenyl-/3-D-mannopyranoside (NPM), and 3 mg ml i p-nitrophenyl-~-L-arabinofuranoside (NPA), respectively. Filter paper activity was assayed according to IUPAC recommendations at a pH value of 6.5. ~o CMCase activity was determined analogously using a solution of 1.0% (w/v) carboxymethylcellulose with a hydrolysis time of 30 rain. Acetyl esterase was determined according to Poutanen e t a / . ~ at a pH of 6.5 using c~-naphthylacetate as substrate.
Eiectrophoresis of protein. Electrophorcsis was carried out under denaturing conditions as described by Laemmli. t-~The gels and running buffer contained 0.1% sodium dodecyl sultate (SDS). Twelve percent acrylamide gels were used and electrophoresis was carried out at pH 8.3 in Tris-glycine buffer 11).05 M Tris. 0.38 M glycine). The proteins in the gels were stained using a 0.5c2~ solution of Coomassie brilliant blue prepared in methanol : acetic acid : water 20 : 7 : 73 (v/v/v). Optimization. The optimization of the production medium was carried out within the framework of a central composite design for the two variables corn cobs and yeast extract. In this design an axial spacing of c~ = 1.5 was chosen in order to obtain greater precision for the estimates of the quadratic effects. This value for the axial distance o~does not fulfill the conditions for orthogonality for all coefficients. Four center points were used to estimate the experimental error. A commercially available program STATGRAPHICS (Statistical Graphics Corp.. USA) was used for the contour plotting.
Results and discussion
Effect +~ carbon sources T h e effect of c a r b o n s o u r c e s o n the e n z y m e yield was tested by the use of the basal m e d i u m with a d d i t i o n of 28.5 g I ~ yeast extract. T h e c o n c e n t r a t i o n of each C source was 25 g 1 ~. Table I s h o w s the e n z y m e titers o b t a i n e d from different C s o u r c e s after 5 d a y s of cultivation. Significant d i f f e r e n c e s in the rate of x y l a n a s e
Table 1 Effect of carbon sources on xylanase production
Carbon source
Enzyme activity assays. Endo-1.4-,0-D-xylanase was assayed according to Bailey et a/.: by incubating the diluted enzyme solution at a pH of 6.5 and a temperature of 50°C for 5 rain using a substrate solution of 1.0% (w/v) birchwood xylan (Roth, Karlsruhe, FRG). Reducing sugars were assayed by adding 3 ml of DNS (2-hydroxy-3,5 dinitrobenzoic acid) reagent, boiling for 5 rain, cooling, and measuring the absorbance at 5411nm. s Mannanase was determined in an analogous manner using a solution of 0.5% (w/v) carob seed galactomannan as substrate. /3-Xylosidase was determined using a modified method according to Lachke et al. ~ Onehalf milliliter 11.05 M citrate buffer of adjusted pH, 0.25 ml diluted sample, and 0.25 ml of a solution of 3 mg mlp-nitrophenyl-/3-n-xylopyranoside(NPX) were incubated at 50°C. After 10 rain the reaction was stopped by addition of
678
E n z y m e M i c r o b . T e c h n o l . , 1993, vol. 15, A u g u s t
Alfalfa Birch pulp (bleached) Corn cobs (powdered) Corn cobs (coarse) Glucose Jute fiber (steamed) Poplar wood chips Rice husk Soya oil Oat spelts Starch (soluble) Wheat straw (steamed) Xylan (beechwood) Xylose
Xylanase activity (nkat m1-1) 545 1,900 7,952 26,700 8 1,600 338 1,630 25 1,020 28 3,870 17,300 4,270
Xylanase from Thermomyces lanuginosus: H. Purkarthofer et al. production occurred by variation of the C sources. Xylan or a xylan-containing C source is necessary for a satisfactory production of xylanase by T. lanuginosus, not only because it is the main C source, but probably also because its hydrolysis products act as inducers. Xylosc also had an inducing effect on the formation of xylanolytic enzymes. With media that did not contain inducers, xylanase liters of only about 25 nkat ml-~ were obtained. From these data it seems to be clear that a low level of xylanase formation is constitutive. Culture filtrates were found to bc frec of filter paper activity. The S D S - P A G E pattern of the excreted enzymes from cultivation with several different carbon sources is shown in Figure 1. When corn cobs and xylan were used as C sources, xylanasc was the main component in the culture filtrate, as can be judged from the good correlation between xylanasc activity and intensity of the major band. Xylanasc occurs as a single band with a molecular wcight of 26.0 kD, a value that slightly differs from those molecular weights of 22.5 and 21.5 kD previously determined by S D S - P A G E using different isolates of T. lanttginosus, a'''14 Taking into consideration the low costs of corn cobs, which are a cheap agricultural waste material, further steps of the optimization process were carried out using this C source. In the following experiments the particle size of corn cobs was found to have a strong influence on xylanase production. A particle size of 2-7 mm was optimal. These data indicate that a slower solubilization of reducing sugars is beneficial for xylanase production. Steam treatment of the corn cobs to possibly attain a better accessibility of the substrate and hot water extraction for a reduction of the initial concentration of soluble sugars in the medium did not affect enzyme production.
.... . .
.
.
.
. . .
. . . . . .
.
-~,o-...:~
. . .
Table 2 Effect of organic nitrogen sources on xylanase production during utilization of xylan and corn cobs (2-7 ram) as C source Xylanase activity (nkat ml 1) N source
Corn cobs
Xylan
24190 22000 19370 18750 18090 17820 17770 16540 16400 13620 1600
16670 10990 15100 12650 13300 11300 16940 19670 10270 11150 2450
Yeast extract Casein peptone Meat extract Pha rmamedia Cornsteep powder Cottonseed Fish peptone Fish peptone a Meat peptone Gelitaflex Soya meal a 30% yeast blend
Effect o f nitrogen sources The effect of various organic nitrogen compounds on the formation of xylanase by T. lanuginosus was tested by the use of the basal medium together with corn cobs or xylan (25.0 g 1-~) and the organic N source. The concentrations of the N sources used were based on their total nitrogen content as estimated by the Kjeidahl method. The concentration of organic nitrogen was fixed at 3.0 g N 1- ~. The xylanase activities after 5 days of cultivation using different N sources are shown in
Table 2. All N sources promoted growth of the fungus. In all but one medium containing corn cobs, the pH value after 5 days of incubation (pH 7.5-8.4) was higher than in those containing xylan as C source (pH 6.0-6.5), which seems to be more favorable for xylanase production. With soya meal as N source, the final pH value was 5.5 (corn cobs) and 6.0 (xylan). With all N sources except for fish peptone, e n z y m e production using corn cobs was higher than with xylan. The protein pattern of the culture filtrates did not change by varying the N source. Yeast extract was chosen as the best N source for further optimization.
Optimization o1" the m e d i u m
- - -
1
-
2
.
.
.
3
.
.
.
.
.
.
.
.
.
.
4
. .
-.
5
.
v
6
7
.
8
.
•
9
10
Figure 1 SDS-PAGE of culture filtrates of T. lanuginosus grown on different carbon sources. Lane 1, MW standards; lane 2, wheat bran; lane 3, alfalfa; lane 4, starch; lane 5, oat spelts; lane 6, wheat straw; lane 7, corn cobs (coarse); lane 8, soya oil; lane 9, xylan; lane 10, MW standards
Using two-level factorial designs, we found that addition of calcium, iron, and magnesium ions as well as several trace elements (Zn, Mn, Co, Cu, Ni, B) had no influence on xylanase production when tap water was used for media preparation (data not shown). Therefore, these supplements as well as ammonium sulfate were omitted in the following experiments. An increase in the concentrations of C and N sources up to a level of 30 to 35 g 1- ~led to a prolonged production phase. The highest activities of xylanase at these concentrations were reached after 7 days of cultivation. Realizing that the C and N sources were by far the most important factors for xylanase production, the Enzyme Microb. Technol., 1993, vol. 15, August
679
Papers
optimal concentrations o f media components were determined using a central composite design with the two variables C and N source. The media consisted of 5.0 g l - ~ KH2PO ~ and different concentrations of corn cobs and yeast extract. The levels of the two variable factors are presented in Table 3. The results were evaluated by the response surface statistical method similar to that used by McDaniel et al. ~ Some modifications had to be made taking into account that the design was not completely orthogonal. A regression analysis was run to fit a second-order model to the experimental data. It yielded the least squares estimated coefficients: b,: 36390; b~: - 9 3 2 : b,: - 4 9 2 : b ~
/
: I
X\XI
\
/ ....
-2372:
.........
\ 1
\
b_,2" - 1574; b 1 2 : - 7 8 8 X
The model is an adequate approximation to the data at the 99% probability level. The coefficients b~ and bl2 were found not to be significantly different from zero at a significance level of c~ = 0.10. Nevertheless. these coefficients were not dismissed as unnecessary in the regression equation, since there was no reason for making the hypothesis b, = 0 and b,2 = 0, respectively, and they were the best estimates for this effect. The optimum was determined at x, = - 0 . 1 8 and x, = - 0 . I0, which correlates to 31.2 g I ~ corn cobs and 30.2 g I ~ yeast extract. The contour plot (Fi~,,ure 2) shows a rather broad plateau region with the highest xylanase activity, in which the activities changed relatively little with changes in nutrient levels. In order to determine the accuracy of the optimization procedure, shake-flask experiments with nutrient media combinations representing the estimated optimum were repeatedly conducted yielding 36,160 -'- 1,880 nkat ml (n = 6), which is one of the highest xylanase activities ever reported. The addition of 0. I% polypropyleneglycol (PPG) as an antifoaming agent did not influence the rate of xylanase production, whereas soya oil in the same concentration reduced the xylanase yield by 25%. The final pH of the media to which soya oil was added was 6.1, as compared to a pH value of 7.9 in the media without soya oil.
Time course o f e n z y m e production in s u b m e r g e d culture The time course of the production of various lignocellulolytic enzymes which could play a major role in bleaching of pulp was determined in shake flasks using the optimized medium. For all detected enzyme activi-
Table3
Range of values for the central composite design
- 1.5 21.3 19.0
- 1.0 25.0 23.0
0 32.5 31.0
- 1.0 40.0 39.0
- 1.5 43.8 43.0
Concentrations of corn cobs and yeast extract are given in g I
680
Enzyme
Microb.
Technol.,
/
-
/ •
.
z
i
F i g u r e 2 Contour plot of the equation calculated to fit the data of the central composite design. Concentrations of corn cobs and yeast extract are s h o w n in Table 3. Xylanase activity is given in/2kat m1-1
ties (/3-xylosidase,/3-glucosidase, acetyl esterase, and o~-L-arabinofuranosidase), the pH optima were determined. These data are shown in Fi~,,ure 3. The pH dependency of acetyl esterase activity suggests that it consists of at least two different enzymes with different pH optima. The culture filtrate did not show any filter paper or CMCase activity. Mannanase and [3-mannosidase activities were also not detectable. As can be seen in Table 4, the excretion of the enzymes ~-xylosidase, /3-glucosidase, tx-arabinofuranosidase, and acetyl esterase was very low as compared to that of xylanase. The optimum of/.3-xylosidase and o~-arabinofuranosidase activities was reached at the sixth day of cultivation, whereas/3-glucosidase and acetyl esterase activity increased with pronounced autolysis of the fungus. Since the pH optima of the other enzymes differ from the pH optimum of 6.5 found for xylanase, the actual ratio of xylanase to the activity of these other enzymes is even greater, as indicated in Table 4 when the culture filtrate is used for bleaching purposes at this pH value.
Solid-state cultivation
Levels Variables Corn cobs Yeast extract
j
1 9 9 3 , v o l . 15, A u g u s t
The effect of nitrogen concentration on the production of xylanase by T. lanuginosus in solid-state cultivation was studied using media with an initial water content of 70% (w/w) at a temperature of 50°C, which has proved to be optimal for production. These values were
Xylanase from Thermomyces lanuginosus: H. Purkarthofer et al. 120
140(300
----a~
100
JB-Glucosidase
=
~-Xylosidase
8o
120000
'
~m 100000
-
80000 -"
•~
•
60
60000 -
~ 40
40000 -'
211
20000 "
~
•
0 i
----o~
J
i
i
ct-Arabinofuranosidase
•
i
200
100
i
Acetylesterase
1.75 % 21% 5.7% 7.0%
300
Hours Of cultivation
Figure 4 Time course of xylanase production in solid-state cultures by T. lanuginosus at different yeast extract concentrations
80
20
3
i
i
i
!
i
4
5
6
7
8
9
pH
Figure 3 Effect of pH on the activity of acetyl esterase, B-glucosidase,/3-xylosidase, and (~-arabinosidase of T. lanuginosus
somewhat higher than the values of 45°C and 65% moisture content found by Kitprcevanavich et al.~6 Yeast extract was added in the amounts of 1.4, 1.75, 2. I, 2.4, 2.9, 4.4, 5.7, 7.0, 8.3, and 9.6% (w/w), respectively. Strong growth of the fungus occurred on the surface of the corn cob particles, with an average particle size of 5 mm after a lag of I day, except in the case of the two media containing the highest amounts of yeast extract. The concentration of the nitrogen source had a strong influence on the production of xylanase. Surprisingly, low amounts of nitrogen lead to a higher e n z y m e production. The 1.75% yeast extract was found to be optimal. After 9 days of cultivation, an activity of 125,000 nkat ml-~ culture centrifugate (336,700 nkat g ~ dry solid) was determined. Selected time courses of xylanase production with media containing low amounts of yeast extract arc prcsented in Figure 4. The final xylanase titers after 179 h of cultivation using the media not shown in Figure 4 were 105,000 nkat ml - t (1.4%), 95,600 nkat ml i (2.9%), 85,600 nkat ml - i
Table 4 Time course of various lignocellulolytic enzymes using the optimized medium in shake-flask experiments Activity (nkat m1-1) Enzyme
3 days
Xylanase 5380 /3-Xylosidase 1.8 /3-Glu cosidase 1.3 a-Arabinosidase 0.1 Acetyl esterase --
4 days
5 days
6 days
7 days
11880 20630 32170 36150 4.1 6.5 7.5 5.2 1.2 1.6 2.1 3.0 0.1 0.8 1.0 0.3 0.6 0.9 1.3 1.5
(4,4%), 59,300 nkat m l ~ (7.0%), 45,000 nkat ml -~ (8,3%), and 11,300 nkat ml ~(9.6%), respectively. Agitating the solid-state culture continuously at low speed (15 rev rain -~) in a rolling bottle (20 1) reduced the production of xylanase drastically to a value of 14% in comparison to that in static culture. This could be explained by shear stress sensitivity of the fungal mycelium. The molecular weight of the xylanase was identical to that produced in submerged culture. The xylanase yield based on the nitrogen source which was the most expensive medium compound in solid-state cultivation (43, I00 nkat mg t N) was superior to that in submerged culture ( 11,300 nkat mg t N). Furthermore the productivity was much higher (solid state: 553 nkat ml ~ h j" submerged: 215 nkat ml ~h ~). Although these data are promising, a scaleup of this solid-state process seems to be difficult due to the generally known problems of heat transfer, inhomogeneity of media, and aeration. These problems are exacerbated by the shear sensitivity of T. lanuginosus. The thermophilic nature of the fungus represents an advantage with regard to heat transfer within the medium. The culture filtrates of T. lanuginosus did not show any filter paper or CMCase activities, which is of tremendous importance for pulp bleaching, since treatment of pulp with xylanase preparations containing cellulases has resulted in a reduction of the polymerization degree of the cellulose fibers and a drop in product quality. J7 Therefore, it is possible to use these filtrates for pulp bleaching without previous purification, which dramatically lowers the costs of application. The lack oflignocellulolytic enzymes other than xylanase makes this strain very promising, not only for bleaching purposes, but also for processes where a relatively pure xylanase without expensive purification procedure would be needed.
Acknowledgements The skillful technical assistance of Ms. Sabine Mauler is gratefully acknowledged. This work was financially supported by the V O E S T A L P I N E lndustrieanlagenbau GesmbH, Linz, Austria.
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2 3
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5 6 7
8 9
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Senior, J. S., Mayers, P. R. and Saddler, J, N. Production and purification of xylanases. In Plant (.'ell Wall Polymers. Bio~,enesis and Biodek, radation (l,cwis, N. G. and Paice, M. G., eds.) ACS Symposium Series 399, American Chemical Society. Washington, I)C, 1989, pp. 641-654 Poutanen, K., Ratio6, M., Puls. J. and Viikari, L. Evaluation of different microbial xylanolytic systems. J. Biotectmol. 1987, 6, 49-60 Paice, M. G., Bernier. R., Jr. and Jurasek. I,. Viscosityenhancing bleaching of hardwood kraft pulp with xylanase from a cloned gene. Biotechnol. Bioeng. 1988, 32, 235-239 Wizani, W., Esterbauer. H., Steiner, W. and Gomes, J. Xylanase manufacture with Thermomyces. Ear. Pat. Appl. 1991, 10 Sinner, M., Ditzelmtiller, G., Wizani, W., Steiner, W. and Esterbauer. H. Papier. 1991, 45, 403-410 Biely, P. Microbial xylanolytic enzymes. 7)'end,~ Biotechnol. 1985, II, 286-290 Bailey, M. J., Biely, P. and Poutanen, K. lnterlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 1992, 23, 257-270 Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959, 31,426-428 I,achke, A. H.. Deshpande. M. V. and Srinivasan. M. C. Extra-
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cellular/3q~-xylosidase of Sclerotium rolfsii. Enzyme Microb. lechnol. 1985, 7, 445-448 IUPAC. Measurement ofcellulase activities. Pure Appl. Chem. 1987, 59, 257-268 Poutanen. K. and Sundberg, M. An acetyl esterase of Trichoderma reesei and its role in the hydrolysis of acetyl xylans. Appl. Microbiol. Biotechnol. 1988, 2,8, 419-424 Lacmmli, U. K. Nuture 1970. 227, 680-685 Anand. L.. Krishnamurthy. S. and Vithayathil, P. J. Purification and properties of xylanasc from the thcrmophilic fungus. tltmlic~la lanu,l,,ino.~a (Griffon and Maublanc) Bunce. Arch. Biochem. Biophys. 1990, 276, 546-553 Kitpreechavanich, V.. Hayashi, M. and Nagai, S. Purification and properties of cndo-l,4-/:t-xylanase from Humicola lanuk, ino.~a. J. Ferment. Technol. 1984, 62, 415-420 McDaniel, L. E., Bailey. E. G., Erhiraj, S. and Andrews, H. P. Application of response surface optimization technique to polyene macrolide fermentation studies in shake flask. Det,. Ind. Mi('rohiol. 1976, 17, 91-94 Kitprcechavanich, V. Production of xylan degrading enzymes by thermophilic fimgi, A,~pergillus .fimfigatus and Humicola lanu~,,ino.sa. J. Ferment. Tec'hm~/. 1984, 62, 63-69 Senior, D. J., Mayers. P. R.. Miller, D.. Sutcliffe, R.. Tan. 1,. and Saddler. J. N. Selective solubilization of xylan in pulp using a purified xylanase from Trichoderma harzianum. Biotechnol. l.ett. 1988, 10, 907-912