CA1325613C - Production of thermostable xylanase and cellulase - Google Patents

Abstract

ABSTRACT

The present invention provides for the use in the production of cellulolytic and xylanolytic enzymes, particularly xylanase and cellulase, of the microorganism Thermoascus aurantiacus 235E in a culture medium containing at least one of a cellulose or hemicellulose substrate whereby to produce thermostable enzymes, particularly cellulase and xylanase.

Description

t L32~13 The present invention relate3 to the production o~
xylanolytic and cellulolytic enzymes and particularly xyla-nase and cellulase. In particular the present invention relates to the production of thermally stable xylanase and cellulase enzymes, ~articularly xylanase enzymes, by cultur-ing a particular hemicellulolytic microorganism in a nutrient medium containing at least one cellulosic or hemicellulosic substrate.

Hemicelluloses constitute 20 to 35% by weight of wood and agricultural residues and serve as an abundant and inexpensive source of fermentable carbohydrates. E~ficient utilization of hemicellulose of biomass will enhance the economic competitiveness of bioconversion processes which must compete with petrochemical processess. Recently it has been demonstrated the technical fea-ability of using the extracellular xylanases produced by Trichoderma harzianum and Trichoderma reesei for the hydrolysis or combined , hydrolysis and fermentation of hemicelluloses. See for example Yu, E.X.C., Deschatelets, L. and Saddler, J.N., Applrt Microbiol. Biotechnol. 1984, 19, 365-372, Yu, E.K.C., Deschatelets, L. and Saddler, J.N., Biotechnol. Bioeng.
Symp. 1984, 14, 341-352 and Yu, E.K.C., Deschatelets, L., Tan, L.U.~. and Saddler, J.N., Biotechnol. Lett. 1985, 7, 425-430. Thus the applicants are presently developing a process for the bioconversion of wood sugars to fuels and chemicals. The process is an integration of the steam pre-treatment of wood residues, production of cellulase and xylanase enzymes, enzymatic hydrolysis of pre-treated wood cellulose and hemicelluloses with said enzymes to component sugaxs and subseque~t fermentation o the sugars to produce a variety of fuels and chemicals. At present enzymatic hy-drolysis step is carried out using non-thermostable cellu-, lases and xylanases obtained from the culture filtrates of Trichoderma harzianum F58 from the Forintek Canada Corp.culture collection. Although the enzymes produced by the ` `~ 132~6~3 .. .
aforesaid fungus have numerous desirable qualities, the enzymatic hydrolysis step is still a bottle~neck in the overall conversion scheme. This is in part due to the cost of producing the enzymes, the instability of the enzymes, particularly the xylanases, the low efficiency o~ the enzy-matic hydrolysis which must be carried out at temperaturesbelow 50C and the need to carry out hydrolysis under ster-ile conditions or in the presence of a~ undesirable preser-vative, such as sodium azide. The enzyme preparations from Trichoderma harzianum and Trlchoderma reesei suffer from lack of thermostability which has resulted in lower hydroly-sis efficiencies, high enzyme requirements and increased cost in carrying out hydrolysis under asceptic conditions.
It is anticipated that the use of thermostable xylanases in effecting the enzymatic hydrolysis at elevated temperatures over long periods of time would enhance the technical and economic feasabili~y of the hydrolysis process.

Xylanase production has been reported for many microorganisms including both fungi and bacteria. Most - notable examples of this group of xylanase producers are Trichoderma reesei which most researchers world wide con-sider as a source of standard enzyme and Trichoderma harzianum strain E58 from the Forintek Canada Corp. culture 25 collection. Although both fungi are prolific producers of extracellular xylanases, fungal grow~h and enzyme production can only be carried out at mesophilic temperature (28C).
Consequently the fermentation requires considerable cooling water during fungal growth and is easily subjected to bac-3~ terial contamination. The xylanase enzymes produced arealso thermally unst;able, losing over 90% of ~heir activities within half an hour incubation at 50C. As a result, enzymatic hydrolysis o hemicellulose (xylan) using these enzymes has to be carried out at a lower temperature about 35 37C to 45C. This in turn lowers the hydrolysis efficien-cies, necessitates asceptic conditions during hydrolysis, as ~32~3 , ~ ~
well as preventing prolonged enzyme use without replacement.

An ob~ect of the present invention there~ore i~ to provide a process for producing thermally stable cellulolytic and xylanolytic enzymes, particularly xylanase and cellulase enzymes which would be useful in the above hydrolysi~ process. The further object of the present invention is to provide a hemicellulolytic microorganism which if cultured in a nutrient culture medium .in a similar manner to the conventional.
hemicellulolytic microorganism to produce xylanase and cellulase, will produce thermally stable xylanase and cellulase enzymes.

It has now been found that fungus Thermoascus aurantiacus, particularly the strain ~her~o~sc~$ ~n~c~ 235E
whic~ i~ obtainable from the Forintek Culture Collect~on, Forintek Canada Corp., Ottawa, O~tario and is also available from ~merican Type Culture Collection (ATCC~ under ATCC, designation 20882, is highly suitable for producing such thermally stable cellulolytic and xylanolytic enzymes, particularly thermally stable cellulase and xylanase. Strain 235E is a recent Forintek reclassification of ~he same strain previously classified by it as C436.

Accardinging to the present invention therefore there is provided in a proces~ ~or the pro~uGtion of cellulolytic and xylanolytic enzymes which comprises culturing a hemicellulolykic microorganism in a nutrient culture medium containing at least one of a celllulose or hemicellulose substrate, the improvement wherein the microorgani~m is T~ oascus , auranti~u~, particularly strain 235E, to thus produce thermostable cellulolytic and xylanolytic enzymes.

The present invention also provides a biologically pura strain of the fungus ~h~moa~_~urantiacus 23SE, deposited in the Forintek cultllre collection, which is capable on culturing in a nutrient medium containing cellulosic and hemicellulosic sub~trate of producing thermally stabIe xylanase and oellulase enzymas.

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Thus, in accordance with the present invention, the fungus Thermoascus aurantiacus, particularly stxain 235E
from the Forintek culture collection, is grown in a culture medium solution containing cellulosic and/or hemicellulosic substrates such as Solka Floc, oat spelt xylan, steam-treated aspenwood and sawdust, at a temperature at 45C or above under culturing conditions. Suitable culture media includes Vogel's medium and Mandel's medium. Xylanase acti-vities are at their maximum levels in the culture medium after about S to 12 days whence the culture is filtered or centrifuged to obtain a culture filtrate. The culture il-trate containing the thermos~able xylanase enzyme may be used directly for the hydrolysis of hemicellulose substrates at elevated temperatures of 50C or above for a prolonged period of time to produce pentose (xylose) sugars. For uses requiring the hydrolysis of a mixture of hemicellulosic and cellulosic substrates the culture filtrate containing both the thermostable xylanase and cellulase enzymes may be used directly to carry out such hydrolysis at elevated tem-peratures of 50C or above for prolonged periods of time toprod~ce a mixture of pentose and hexose sugars.

In a particular embodiment of the present inven-tion, the thermostable xylanase and cellulase enzymes in the culture filtrate may be concentrated either by rotary eva-poration or by ultrafiltration. Thus, in particular the culture filtrate may be subjected to ultrafiltration through an ultrafiltration membrane having a low molecular weight cut-o~f point suitably between l,000 and 20,000 daltons to obtain a xylanase and a cellulase rich retentate. suitably the membrane has a low molecular weight cut-off point between 5,000 and 20,000, preferably between 5,000 and 15,000, more preferably between 5,000 and 12,000 and more desirably between $,000 and 10,000. Suitably the membrane is a non-cellulosic membrane such as a polysulfone membrane.
The retentate containing the concentrated thermostable . .~ , . . . . . . .~ , . .

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xylanases and cellulases may be used for the hydrolysis of hemicellulosic or combined hemicellulosic and cellulosic substrates at elevated temperature o~ 50C or above ~or prolonged periods of time to produce a mixture of pentose and hexose sugars for subsequent fermentation or deri-vatization. Further, to obtain pure thermostable xylanase the crude fungal filtrate or the retentate from the ultra-filtration may be i~cubated at elevated temperature of at least 60C to selectively inactivate the cellulase activi-ties in the mixture. Such pure thermostable xylanase can beused to selectively remove hemicellulose from mixed cellu-lose and hemicellulose substrates such as pulp.

The use of thermostable xylanase and cellulase enzymes obtained according to the present invention from Thermoascus aurantiacus 235E significantly enhances the .
technical and economic feasability of the hydrolysis set forth above. Being stable at higher temperatures of 50 to 70C the enzymes can be used at high reaction temperatures of 50C or above. This is highly desirable in that the rate of hydrolysis is increased drastically while the risk of contamination during hydrolysis is significantly reduced and i~ likely to be eliminated. Moreover the stability of the enzymes for prolonged periods of time at elevated tempera-tures enables the use of the enzymes for extended durationwithout replacement. This i5 also a very desirable property for enzymes to be used in commercial processes involving either enzyme recycling or a substrate fed batch process in continuous or semi-continuous hydrolysis systems. The ability to re-use the enzymes for prolonged periods of time also results in substantial reduction in the amount of enzymes required for the hydrolysis step thereby decreasing the cost o~ the enzyme production. Thus, the thermostable cellulases and xylanases produced in the process o the present invention have a substantial impact on the economic viability of the overall bioconversion process for woo.l , .

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residue realization.

The use of a cellulase free xylanase enzyme preparation to selectlvely remove hemicellulose ~rom pulp ln manufacturing high quality cellulose ~uch as rayon has already been e~fected as disclosed in our U.~. patent ~,725,5~4 issued February 16, 1988 using the fungus Trichoderma har~ianum E58 to produce the culture filtrate. The thermostable xylanase produced in the process o~ the present invention is also highly suitable in that the xylanase as present in the crude culture filtrates ~rom the culturing of Th~r~ç~ auranti3c~ 23~E is only contaminated by low cellulase ac~ivities (xylanase to carb~xymethyl cellulase activity ratio around 75 to 11. The preparation may be considsred to be essentially ~ree ~rom cellulase activlty based on relative thermostability o~ xylanase and cellulase activities.
The xylanase activity of ~he xylanase produced by the process of the present inYentiOn iS sta~le at 60C ~ith a half-life of 4 days whereas the corresponding half-life of the cellulase is 8 hours. Thus the cellulase activi~y o~ t.he enzyme preparation may be reduced to negligible levels while maintaining the xylanase ac~ivity and the thermostable cellulase ~ree xylanase enzyme may then be used in cleaning up cellulose pulp ~or industrial application.

In khe process of the present invention the fungus Thermoa~us ~ pus, particularly strain 235E, is grown at elevated temperature, 45~ or above and may be grown on a range of inexpensive commercial cellulosic substrates in~luding steam exploded wood with or without fractionation and sawdust without treatment. The Pungus so grown produces a full spectrum of other cellulolytic and xylanolytic enæyme~ and consequently the culture ~iltrates can ~erve as a source for both xylanase and cellulase enæymes for sub~equent application. Initial characterization of the xylanase enzyme~

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in the crude filtrate demonstrate that the enzyme activities are optimal at 75C, with a half-life at 70C and 60C o~ 90 minutes and 4 days, respe~tively. Le~s than a 10~ loss of enzyme activities occur when the enzymes are incubated at . S 50C for 12 weeks. The cellulase activities present in the same culture filtrates are also thermostable but to a much lower extent when compared with the xylanase activity. This property enables sel ctive inactivation. of the cellulase activities in avour of xylanase activities by simply incu-bating the enzyme preparations at eleva~ed temperatures.Thus, or example, thermostability of xylanase and carboxy-methyl cellulase activities at 60C dif$ers by a factor of 12 (half-life of xylanase and cellulase at 4 days and 8 hours, respectively). This coupled with the initial prolific production of xylanase relative to cellulase at over a 75 to 1 ratio results .in an essen~ially cellulase free thermostable xylanase for speciflc hydrolysis of hemi-cellulose (xylan). The thermostable xylanase enzyme or mixture of xylanase and cellulase enzyme may be used for the hydrolysis of biomass hemicellulose or combinëd hemicellu-lose and cellulose substrates, respectively. Both the xylanase and cellulase en2ymes in the culture fil~rates can be used directly or can be concentrated prior to appllca-tions as aforesaid by ultrafiltration through a membrane suitably with a molecular weight cut-off point at 10,000 daltons. Being thermostable, the xylanases or mixture of xylanase~ and cellulases in the same fraction can be used for the hydrolysis of hemicellulosic or combined hemicellu-losic and cellulosic substrates at elevated temperature, 50C or above, for prolonged duration. The component pentose and hexose ,sugars obtained from the hydrolysis of the hemicellulose or mixed hemicellulose and cellulose can then be used for the subsequent fermentation and deriva-tization to produce a variety of fuels and chemicals.
urantiacus strain 235E may be cul-. ' , -- ' .

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tured at elevated temperatures of 45C or above to reduce the risk of contamination and the cooling water requirement during fermentation. Enzyme production by Thermoascus aurantiacus 235E however, is superior to other fungi or bacteria in several important regards, namely 1. The organism is an extremely prolific producer of xylanase enzymes up to 576 IU of xylanase activities per ml of original culture fil~rate. 2. The sam~ culture filtrates also exhibit a full spectrum of other cellulolytic and :

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1325$1 3 xylanolytic enzymes, including exoglucanases, endoglucana-ses~ ~ -glucosidases, and ~-xylosidases; and 3. Inexpen-sive biomass residues, such as sawdust without treatment or steam-exploded wood, can be used as the sole substrate for S enzyme production. The extracellular nature of the enzyme also facilitates and drastically reduces the cost of enzyme harvesting in large scale commercial production schemes. In addition to the above benefits the xylanase enzymes also possess properties desirable in the actual hydrolysis of hemicelluloses. The enzymes in crude filtrates operated at an optimum temperature of 75C and are stable at elevated temperatures (half-life at 70C and 60C at 90 minutes and 4 days respectively). There is also no significant loss o xylanase activities at 50C after 12 weeks of incubation.
It is therefore feasible to carry out enzymatic hydrolysis at 50C or above temperatures at which the enzymes have high efficiencies of catalysis. The enzymes can also last longer - during use condi-tions without replacement, an ideal quality for en~yme recycling or substrate fed-batch in continuous hydrolysis processes. The use of higher temperatures for hydrolysis also eliminates or significantly reduces the risk of contamination during hydrolysis for extended periods in the absence of undesirable preservatives.

The thermostable xylanases present in the culture filtrate or concentrated in the retentates of the ultra-filtration process can be used to efficiently hydrolyse hemicellulosic substrates to fermentable sugars, predomin-antly pentoses. These thermostable cellulases present in the corresponding fractions can be used for the hydrolysis of cellulosic subst~ates to produce prèdominately glucose sugars for subsequent fermentation. The culture filtrates or their ultrafiltration retentates containing both xylanase and cellulase ac~ivities may also be used for the direct hy-drolysis of pretreated biomass residues containing both cel-lulose and hemicellulose substrates to produce a mixture of ~ g _ - i I i 132~3 ~..
fermentable hexose and pentose sugars. Alternatively the thermostable xylanases and cellulases in the retentates can be used in combined hydrolysis and fermentation of hemicel-lulose, cellulose or mixed cellulose and hemicellulose with a direct production of various fuels and chemicals. The pure thermostable xylanases may be used for the selective removal of contaminating hemicellulose (xylan) for high gr-ade celLulose pulp for manufacturing r~yon and cellophane and for reducing the amount o~ hemicellulose in aspen mech-anical pulp. The crude thermostable xylanases may also beused in the bioconversion process during ~he hydrolysis of hemicelluloses derived from wood or agricultural rèsidues to produce pentose ~xylose) sugars, for subsequent fermentation to fuels and chemicals such as ethanol, acetone, butanol and butanediol and for the derivatization to form xylitol, arti- -fical sweetners, furfural, etc. The thermostable xylanases can be used in processes for the manufacture of liquid cof-fee, the adjustment of wine characteristics, for the enhan-cement of astaxanthin extraction or the clarification of fruit juices and for the treatment of waste watex from the wheat starch industry. The thermostable cellulases may be used for improved hydrolysis of the cellulose components of wood and agriculture residues to produce a glucose syrup which can then be converted to liquid fuels and chemical feed-stock. The cellulases may also be used in the manu-facture of liquid coffee, clarification of fruit juices, treatment of was~e water and from the wheat starch industry and enhancing the recovery in the distillation step in the alcoholic beverage industry. Since a mixture of xylanases ~o and cellulases are generally required in several of the afo-resaid applicationslit is advantageous that the culture fil- ..
trates or the retentate fractions produced by the process of .

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~ t ~ i32S6~3 the present invention contained both active and thermostable xylanases and cellulases. Consequ~ntly the fractions may be used as the so].e enzyme source for the hydrolysis of mixed cellulose and hemicellulose substrates as well as for manufacturing coffee, clarification fru.it juices and treatment o~ waste water from food industry.

The present invention will be~further illustrated by way of the following Examples:

. pnoDucTIoN OF CEL~UL~LIT~C ~ND ~YLAN~LST~C ENZ~H85 1/ ~rganlom, edla, and c~lture conaitlon~
She thormophlllc ~ungu~, T. ~ur~ntl~cua ~traln235F ~ro~
Forlntok ~uleur~ tlon~ m~l~t~ln~d ~n ~it n~r ~ ) m~dlu~ ~a~ t 25-C- ~h~ ~tock ~ultur~a uor~
thun u~qd to lnoculato r~ah m~lt ~gar rl~nts ~nd Lncu~tsa ot 45~C ln ~ tur~-~ontrollod lncubator ~o~ i-9 dnys untll ~n~xlmal mycellal growth uao obto~n~d. ~he ~lnnt~ u~r~ then mal~t~ln~d at 2~-C. ~ 3p~re ~noculum ~ u~a to lnltlate ~outh ln Erlenmeyor flank~ tlS~ ~L medlum ln 5~D mL
rlaX~1 ualng ~arlouo ~ub~trat~a at 1~ t~ elther 0 ~ogel'a modlum tTablo 2) or ~n~lflod ~ndel~0 ~edlu~ tsabl~

3). ~h~ ~laoka uero ~nc~bDted at ~5-~ on ~ ahakln~
plat~rm ~ 10D rpm) for 7-1~ day~ untll ~axl~al xylan~q nctl~ltlee u~re obtulned; ~h~ cultureu ~rn ~lltered ~through a ~hatman glao~ flb~r fllter ana thc ~ rate~ ~ere?
u~ed ~or ~n~y~e 4s~ay~ ~nd chnr~ctar~atlon ~tudlen.

2/ Enzym~ pro~lles u11 r~n~ o~ ~11u101ytIG nnd ~y1ano1ytLo 2nr~met ~ctlvltle~ ~re det~t~d ln tha crud~ ungal ~u1ture fl1~r~t~o ~nb1e 4). Illghost xy1~nag~ act1vit~-~ obtaln~d Iro~ he~lc~l1ulo~ ~oat~p~1t xy1nn~ nnd c~11u10~501ka F1~) ~ern 575.9 ~mL and 365.~ IU~, r~epec~ 1y- ~h~
~ter-In~ol~b1e? reRlduz~ of steam-~p10d~d a~p~n~ood t24~-C~ ~0~ lch contnlnod prQdomln~nt1y ~e11uluas ~nd 1lgnln, aw ~ ra~duat ~Ith ~r u-thout ba11-mll11ng) ~lno d~mon~rn~a proml~ a~ pu~ntl~1 lna~p~n~l~e?
ren11et~c ~botralt~ for ~utur~ 1arg~ c~ oum~scl~1 rroductlon o~ th~ ~y1-n~e an~y~.

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B. INITIAL CHARACrrERIZA~ION 0~ ~ ~NASE ENZYMES:
- crude culture ~iltrates without further treatment were used instead of puxified enzyme 1/ Optimal xylanase activity was observed at pH 4.8-5.8 and 70-80C

2~ Xylanase activity was thermostabla wtth half-life (i.e., the time o~ incu~ation at a particular temperature during whicA 50% of the original enzyme activity is lost) at 70C and 60C being around 1 1~2 h and 4 d, respectively. Xylanase activity was stable at 50C ~or at least 12 weeks.

3/ The crude culture ~iltrate could hydrolyze oatspelt xylan at 70C, 60C, and 50C to release mixtures of xylotetraose, xylotriose, xylobiose ~predo~inant product), xylose, and arabinose (Table 5).

C. APPLICATIONS OF THERMOSTABLE XYLANASE ENZYMES:
1/ ~ydrolysis o~ hemicellulo~e by xylanase in fungal culture filtrate: Xylanase in cultrue filtrate can be used directly, or after being conaentrated either by rotary evaporation or by ultra~iltration, for the ef~ective hydrolysis of xylan (model hemicellulo~e subs~rate~ or of aspenwood hemiceIlulo~e present in the water-extract o~ ste~m-exploded wood ta realistic substrate obtained as a major ~yproduct in ~5 ~ioconversion processes to produce fuels and chemicals from wood or agricultural residues~ ~able 6~. Bas~d on the hemicellulose (xylan) content of ~he sub~trate, hydrolysis o~ hemicelluloses by the enzyme pr~paration can approach near completion within ~8 hours, even at high substrate concentration~ (10%, dry wei~ht~vol);

~/ Combined hydrolysis and fermentation of h~micelluloses for the direct production of fuels and chemicals:

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132~3 Xylanase enzyme preparation~ from 1/ can be used in conjunction with fermenatative organisms to simulataneously hy~rolyze hemicellulo~e to sugars and ferment the sugars released to a variety of fuels and chemicals. An example o~ this i~ the u~e of Klebisella ~LCU cni~ (8l~ki~LL~ o~toca ATCC 8724) with the enz~me preparation to produce 2,3-butanediol, ethanol, acetoin, and acetic aci~ from xylan and st~am-exploded aspenwood hemicellulose (Table 7).

- 12a -;r - , . ~, ~ . . , ~ ~32~3 3J Hydrolysis o~ mixed cellulose and hemicellulose substra~es by mixed xylana~e and cellulase enzyme preparations. The thermostable xylanase enzyme preparations obtained from 1/ al~o contain~
thermostable cellulase~ and can therefore be used for the hydrolysis of substrates containing both cellulose and hemicellulo6e carbohydrate~. For an exa~ple, 6team-exploded aspenwood containing around 54.0~ (w/w) c~llulose ~hexosan) and 13.4% ~w/w) hemicellulose ~pentosan) can be ef~iciently hydroly2ed ~o release a mixture o~ hexo~e and pentos~ sugar ~Table 8).

~/ Combined hydrolysis and fermentation o~ mixed cellulose and hemicellulose substrateæ by mixed xylanase and cellulase enzyme preparations in conjunction with fermenta~ivR microorganisms, ~utaned~oI and ethanol production can be achieved by inaubating the thermostable enz~me preparations obtained from 1/ and ~ E~S9~ml~ with steam~exploded aspenwood.

5/ Selective hydrolysis of hemicellulose in ~ub trates containing both cellulose and hemiaellulose by cellulase-free xylana~e enzyme preparations: Based on the dif~eren~ial thermostability of the xylanase and cellulose enzyme present in culture filtrates, enzyme preparation~ einriched w~th xylanase activities with neg~igible level of cellulose activitîes can be obtained. For an example, half-lifei of xylanase activities at 60C i5 4 day~ whilei that of c~llulase (carboxymethylcellulase) activities is only 8 h. This twelve ~old difference in thermostability, together with the initial xylanase ackivities in the culture ~iltrate~ being over 75 ~old higher than the aellulase activities, could result in a ~imple yet noYel method ~or obtaining essentially ceIlula~e-~ree thermostable xylanase enzy~e prepara~ions. Such ther~osta~e pure xyl~nase enzyme can be o~ great com~ercial potential, ...

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132~3 e . g ., in selecti~ely cleaning up contaminating amount of hemicellulo~a present in commercial cellulo~e pulp for the manufacture o~ high grade cellulose (~uch aE;
rayon or cellophane).

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' ~L 3 ~ 3 , Table 1. Composition oE malt agar (MA) medium Difco Dacto* Malt extract 20.0 g/L
DiEco Dacto* agar 20.0 g/L
Distilled Water 1000 mL

5 Table 2. Composition of Vogel's Medium Medium In~[Eedients Concentration ~/
Salts: Na3 Citrate.2H20 2.5 g KHaP04 5.0 g NH4NO3 2.0 g 10MgSO4 7H20 0.2 g CaC12 . 2H20 0.1 g Vitamins: Biotin 0.5 llg Myoinositol 2.0 mg Ca-panthothenate 0.2 mg Pyridoxine - HC1 0.2 mg Thymine - HC1 0.2 mg Trace Elements: Citric Acid.H20 5.0 mg ZnS04 . 7H20 5.0 mg Fe(NH4)2(SO4),-5H20 1.0 mg CuS04 . 5H20 0.25 mg MnS04 . 2H20 0.05 mg H3B03 0.05 mg Na2M0204 . 2H20 0.05 mg Bacto-Peptone* 1.0 g Tween 80* 2.0 mL
Carbon source 10.0 g Table 3. Composition of Mandel's Medium M um Ingredients Concentration (/L) Salts: (NH4)2 S~4 1.4 g KHaP04 2.0 g MgS04 7H20 0.15g Trace Elements: FeS04 . 71120 5.0 mg MnS04 . HaO 1.6 mg ZnS04 . 7HaO 1.4 mg CoCla 2.9 mg CaCl2 . 2H2 Urea 0.3 g Peptone 1.0 g Tween 80* 0.2 g Carbon source 10.0 g * Trade-Mark : 7i _, ..,~.

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Table ~. Production of extracellular cellulolytic and xylanolytic enzymes by T. aurantiacus strain 235E

Substrate _ Enzyme activities (IU/mL) (1~, w.v) Xylanase Endoglucanase Filter paper B-glucosidase A. Vogel's medium SolXa Floc 365.8 ( 7~ 10.1 ( 7) 0.16 ( 5) 0.74 (10) SEA-WI98.2 (10) 2.9 (10) 0.16 (10) 0~31 (10) Sawdust40.5 (10) 0.6 (10) 0.05 ( 4) 0.14 (10) Sawdust (Ball-174.4 (10~ 5.6 ( 7) 0.27 (10) 1.34 (10) milled) Xylan575.9 (10) 7.3 (10) 0.40 (10) 0.66 (10) SEA66.1 (10) SEA-WS6.2 (10) B. Modified Mandel's medium Solka Floc 94.6 (10)7.3 (10) 0.13 ( 4) 0.39 (10) SEA-WI 62.6 ( 7) 2.7 (10) o.o9 (10) 0.18 (10) Sawdust 61.6 (10)0.9 (10) 0-05 ( 4) 0.16 (10) Sawdust (Ball-milled) 80.7 (10) Xylan 283.3 ~10) SEA 39.1 (10) SEA-WS 3.0 (10) Notes:
1/ Values in parenthesis were the respective incubation time when maximum activities of the enzyme were detected in the culture filtrates.
2/ B-xylosidase activity was also assayed in cultures grown on solka floc or xylan in Vogel's medium to be around 0.07 IU/mL.
3/ Extracellular protein concentrations in filtrates of cultures grown on Solka Floc or xylan in Vogel's medium were 0.26 mg/mL and 0.90 mg/mL! respectively.

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Table 5~ Hydrolysi~ of xylan by culture ~iltrates of ~ura~iac~ 235E at various temperatures ~emp. Reducing 5uga~ ~y HPLC ~L) (C) sugars g/L Xylose Xylo- Xylo- Xylo- Arabine biose triose tetraose .,.... ~
~0 24.8 4.8 1~.5 6.2 9.2 1.2 60 2~0 4.5 10.4 5.0 8.~ 0.9 10 5018.5 1.6 6.0 4.4 7.~ ~0O
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~ydrolysi was carried out on oat-spelt xylan (5~, w/v~ u6ing arude culture filtrate of T. aurantia~u~ 235E (at xylanase activity cf 70 IU/mL). Incu~ation was carried ou~ at various temperaturQs with æhaking for 24 h.

~able 6. ~ydrolysis of Hemicellulose Substrate~ by culture ~iltrates of T.. aurantiacus ~35E concentrated by ultrafiltration.

Substrate R. S . ~ _Su~ y HPLC lg~)T - . . - .
& Conc. (g/L) Xylose Xylo- Xylo- Xylo- Arabine biose trio~:e tetraose ~- _ _. _ __ _ . . _ . __ _ . __ _ ~__ _ . , ~ _ . . : m~ . . _ _ . . _ _ _ 20g/L 8.7 4.7 6.7 0.0 0.5 1.1 25 50 g/L 24.1 12.7 12.2 0.3 0.0 2.4 100 g/L 48.1 33.8 22.3 1.7 7.1 7.0 Steam~Exploded Aspenwood Water-Soluble~:
20 g/L 5.3 4.9 6.1 0.1 0.0 1.1 50 g/L 10.2 11.2 14.3 0.9 0.0 3.6 100 g~L 24.1 23.8 23.9 1.7 2.0 4.8 .....
Hydrolysis wa~ carried out at 50C with shaking for 48 h, using culture filtrates of T. aur~ ~~ 235E concentrated by passage through a P~llicon membrane ~ith molecular wei~ht cut-off of 10 KD.

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. . , , ~

~ ~32~3 , Table 7. Butanediol and ethanol production by a CHF approach, using ~l~bslella ~Ç~oni~e and ~S.~l~En~ a culture filtra~e concentrated by ultrafiltration.

Xylan ~uta~çqiçl_lg11~L~ thanol ~L~
(g/L) Day 1 Day 2 Day 4 Day 1 Day 2 Day 4 .. . _ " _ 1.3 1.6 2.0 0.3 0.4 0.5 2.4 5.2 5.0 0.6 1.9 1.8 10 100 3.7 9.2 12~2 ~.0 3.2 ~.0 ,, _ _ . _ . ,, , ", ,, , , _ _ _ _ . _ _ _ Table 8. En~ymatic hydrolysis o~ combined cellulose and hemicellulose ~ubstrates by the thermostable enzyme preparations ~rom ~ 3n~i _ ~
SEA Reducing Sugars % ~Iydrolysis (based on conc. (g/L) released (g/L) c~r~ohydrate content of substrate) ~0 7.4 56.5 16.8 51.8 -- :~_~-. ~ _ _ ~.____ I _ ~ ......... ~.__.______ -- - r Hydrolysiæ was carried out at 50~ for 48 hours using culture filtrates o~ T. a~ranti~ç~s concentrated by ultrafiltration.
Substrate used wa~ aspenwood after steam-explosion (2~0C, 80 without extr~ction.

-.r . . , ~ . .- . . .

P 132~3 ~EL~Z

In the accompanying drawings of ~xample 2:

Figure 1. Xylanase production by .T~ aurantiacus 235E grown on various hemicellulosic and lignocellulosic ~ubstrates.
~, oat spelt xylan: ~, steam-exploded aspenwood, and , the water-soluble fraction ~he~icellulose) o~ steam-exploded a~p~nwood.

Figure 2. Temperature optimum of xylanase and B-xylosidase activitie~ assayed in citrate-phosphate buffer (pH 5.0) for 30 min. Activitîes obtained at 50C were taken as the 100~ reference levels for both enz~mes.
O, xylanase activity: ~, B-xylosidase activity.

Figure 3~ p~ optimum of xylanase and B-xylosida~e activities assayed in citrate-phosphate buffer at 50~C for 30 min.
Activities obtained at pH 4.~ an~ 5.0 were taken a~ the 100% reference levels ~or xylana~e and B-xylosidase, respec~iveIy. ~, xylana~e activity; ~, B-xylo~ida~
activity.

Figure 4O Thermostahility of xylanase activity at 70C.
~, filtrate of fungal culture grown on solka ~loc;
~, filtrate of fungal culture grown on xylan.

Figure 5. ~her~ostability of xylana~e activity at ~O~C.
0, filtrate of fungal culture grown on solka ~loc;
Q, filtrate of fungal culture grown on xylan.

~,~r' :
,.:, `, !. ;

. ~ ' ' .' ' ` : : ':

~ ~` 132~13 . .
MATERIALS AND METHODS
Microor~anisms:

All of the thermophilic fungi used were taken from the Forintek culture collection ~Table 9). Cultures were generally maintained on malt agar (MA) medium, containing 20 g of Difco Bacto Malt extract and ?O g of Difco Bacto agar per liter of distilled water. However,.strains B499 (un-classified), C416 (Mucor spp. ), C433 (Myricoccum al~omyces), C437 (Malbranchea pulchella), a~d C491 (Thielavia terres-tris) were maintained on yeast phosphate soluble star~h (UPSS~ agar medium, containing 4.Q g of Difco Bacto yeast extract; 1~0 g of potassium monophosphate; 0 5 g of magne sium sulfate heptahydrate; 15.0 g of Difco soluble starch;
and 20.0 g of Difco Bacto agar per liter of distilled water.
Fungal cultures to be used as inocula were grown on their respective maintenance media at 45C for 6-9 days to maxi-mize mycelial growth, and were then maintained at room temperature. A spore inoculum was then used to initiate growth in tubes and in shake ~lasks.
.
Media and culture conditions: ~

Initial screening of cellulolytic and xylanolytic fungi was carried out on solid medium supplemented with gelrite and various cellulosic and hemicellulosic substrates as detailed in the following section. Incubation was carried out at 45C in moisture-saturated plastic bags to minimize desiccation due to the high incubation tempera-ture. The tubes were analyzed weekly for the depth of clearing of the subgtrates~

Studies on the production of cellulase and xyla-nase enzymes in the culture filtrates were carried out in 150 ml of aqueous medium (in 500 ml Erlenmeyer flasks).
Unless otherwise specified, all fungi were grown in Vogel's ~ 19 -- '' - , .. . . ... . ..

325~

medium, Montenecourt, B.S. and Eveleigh, D.E. Appl. Environ.
Microbiol. 1977, 34, 777-784 containing 1~ (w/v) Solka Floc BW 300 tBrown and Co., N.H., U.S.A.) as the cellulose sub strate. The flasks were incubated at 45C on a shaking platform (100 rpm). Samplings were carried out at various times during the incubation period by asceptically with drawing a portion of the fungal cultures. Cultures were B then filtered through a Whatman ~lass ~iber filter and the Eiltrates were ~ssayed for the activities of the various hydrolytic enzymes.

PreIiminary studies on maximizing the production of xylanase and cellulase enz~mes by Thermoascus aurantiacus 235E were carried out in aqueous medium, using Vogel's or modified Mandel's medium, Mandel, M. Bioconversion of Cellu-lose Material to Ener~y~ ~ and Microbial Protein (Ghose, T.K., ed.), ITT, New Delhi, India, 1977 on various cellulosic substrates as detailed in the ~ollowing section.
Incubation conditions and sampliny procedures were carried out as earlier described. All studies were carried out in duplicate and repeated at least once.

Substrates:

All substrates used in the tube-clearing assay, except acid swollen cellulose, were all ball-milled for 3 weeks prior to use to facilitate analysis of tube clearing.
Unless otherwise specified, all substrates used in submerged aqueous medium were not ball-milled. Substrates used, other than the Solka Floc~already mentioned, included:
(1) Aspenwood saw ~ st (ASD) (2) Steam-exploded aspenwood (SEA). Aspenwood chips pretreated by steaming at 240C ~or 180 seconds in a ;
Masonite-type reactor and then explosively decompressed, Saddler, J.N., ~rownell, H.H., Clermont, L.P., and Levitin, N. Biotechnol Bioen~ 1982, 24, 1389-1402.
.
c~de~ ark . .

:

P~ ~ ~32~3 (3) Steam-exploded aspenwood, water-insolubles (SEA-WI).
Aspenwood chips treated as in (2) except that the resulting materials were further extracted with water at room~tempera-ture for 2 hours at a concentration of 5% (dry weight per volume).

(4) Steam-exploded aspenwood, water-solubles (SEA-WS). The water extracts obtained in (3) which were concentrated to dry powder by rotary evaporation u~der pigh vacuum.

(5) Aspenwood xylan prepared by alkali treatment of extractive-free aspenwood sawdust by the modified methods of Jones et al, Jones, J.K.N., Purves, C.B. and Timell, T~E.
Can~ J. Chem. 1961, 39, 1059-1~66 and Koshijima et al, Roshijima, T., Timell, T.E. and zimbo, M. J. Polymer Sci.
1965, 11, 265-270.
(6~ Oat-spelt xylan purchased from Siqma Chem. Co. (St.
Louis, Mo., U.S.A.) (7) Acid swollen cellulose (ASC) (8) Cellulose- ~hatman No.l filter paper (CW1) Analytical me~hods:

Total reducing sugars were estimated colori-metrically using dinitrosalicylic acid reagent; Miller, G.L.
Anal Chem. l9S9, 31, 426-428.
Xylanase (Endo-1,4-~ -D-xylanase) activity was determined by incubating 1 ml of an appropriately diluted enzyme preparation (crude fungal culture filtrate) with 10 mg of oat-spelt xylan in 1 ml of 0.1 M citrate phosphate buffer (pH 5.0) at 50C for 30 minutes. The reaction was terminated by the addition of 3 ml of 3,S-dinitrosalicylic acid reagent. One unit of xylanase activity was defined as 1 ~mole of xylose equivalents released per minute. Filter paper activity was determined by the method of Mandel et al (Mandel, M. Andreotti, R. and Roche, C., ~iotechnol Bioeng.
Symp~ 6 1976, 21-34. Endogluconase (carboxymethycellulase~

'` 132~
and B-glucosidase (salicinase) activities were assayed under conditions previously established, Saddler, J.N., Hogan, C.M., Chan, M.K.-~ and Louis-Seize, G. Can. J Mic~b.L~. 1982, 28, 1311-1319. Xylobiase ~B-xylosida~e) activity was determined u~ing p-nitrophenyl xylopyranoside r~agent, Dekker, R.F.H.
Bi~tecbnol. Bi~eng. 1983, 25, 1127-1146. Soluble protein was detexmined by the method of Lowry ~_~l, Lowry, O.H., Rosebrough, N.J~, Farr, A.L. and Randall, R.J. J. Biol. Chem. 1951, 193, 265-275 a~ modified by Tan e~ ~l, Tan, L.U.L., Chan, M.K.-H and Saddler, J.N. f~ e~SI,_k~t5. 1984, 6, 199-204 using bovine serum albumin as ~tandard.

.o~,~i.ma~iQ~con~ $

Studie~ on pH optimizations were performed in 0.05 M citrate phosphate buffer adjusted to initial pH values of 3.5 to 7.5.
~he ~eaction mixtures, containing oat-spelt xylan (0.5%, w/v) and an appropriately di~uted enzyme preparation (crude culture filtrate), were incubated at 50C for 30 min. Temperature optimization studies were similarly carried out in citrate phosphate buffer at pH 5.0 and assayed for reducing su~ars released after incubation at various temperature for 30 min.

;~

. , ~ , . . :: ..

.

L 3 2 ~
Tl~e ~,Qs,t,.~bili~y Studies:

Thermostability of the en~yme activities of the crude ~ungal culture filtrates were determined by incubating the filtrates at 70C, 60C, and 50DC ~or variou~ durations ranging ~rom hours to weeks. ~he trea~ed flltrates were then assayed for the respective enzyme activities by incubating an appropriately diluted aliquot o~ the treated sample with the assay substrate in citrate pho~phate ~uffer at pH 5.0 and 50C ~or 30 min., as de~cribed in the previous section.

10 S~E.L

Solka Floc tB.W. 300 F.C.) was ob~ained from Brown and Co., Berlin, ~H. Carboxymethylcellulose (medium viscosity, D.P.
1100), æalicin, oat-spelt xylan, and p-nitrophenyl xylopyr~noside were purchased from Sigma Chem~ Co. (St. Louis, Missouri). 3,5-Dinitrosalicylic acid was purchased from Eastman Kodak Co.
(Rochester, NY). All other chemicals ware obta~ne~ ~rom Fisher Chem~ ~o. (Ottawa, Canada) and o~ reagent grade.

~ ~3 --~r;

13 2 ~ ~ L 3 , F~E$ULTS AND I:)ISCU~;~OI~I

Initially twenty-one thermophilic fungal strains in Forintek culture collection were screened for ~ylanase and cellula~e enzyme production in solid mediu~ using the tube-clearing method as proposed by ~ansey. Based on the results obtained from earlier studies on xylanase and cellulase production by mesophilic ~ungi, aspenwood xylan wa~ selected as the substrate in the present study for detecting the production and secretion of extracellular fungal xylanase enzymes, while acid swollen cellulose and aspenwood sawdust were chosed as substrates to screen for the cellulose enzymes~ Whatman ~ilter paper, and steam exploded aspenwood cellulose (i.e., the water-insoluble fraction of steam-exploded a~penwood) were all found to be unsatisfactory as routine assay substrates since they generally 1~ did not result in clear zones of hydrolysis (data not shown).

Table lO summarizes the results obtained from the solid medium tube clearinq assay for both the xylanase and cellulase production by the thermophilic fungi. All fungal strains, with the possible exceptions of strains B499, C416 ( u~or sp. ), and C491 (T:~içl~ erres~ri~3, showed visible growth on all the substrates used. ~hese three strains, as well as strain B519~
also failed to exhibit any clearing on mos~ or all of the substrates tested. Two other strains, Phanero~h~t~
ahr~so~p~riu~ A387 and ~ 9 5 ~ e~ C4~3, also showed low overall cl~aring abilities on all the substrates used. All the remaining strains were found to be ~trongly active in clearing aspenwood xylan, with nine of the strains resulting in complete æubstrate clearing within 2-3 weeks~ Only two strains, Malbran~hea ~ul~ell~ C437 and ~y~ kL_LEluginosa C466, which ~0 showed good ~ubstrate clearing when grown on xylan, failed to exhibit any apparent clearing when grown on all cellolosic sub~trates~ Substra~e clearing by the thermophilic strains grown on aspenwood sawdu~t and acid-swollen cellulo~e generally showed good correlation, with ~ost ~ungal strains exhibiting stronger clearing activities when grown on aspenwood sawdust. Th~re were, .... .
,, ~ ,~ .

.. , - ~ :

--" ' 132~
however, several exceptions to this trend, particularly with one strain~ QL-y~L~Lo~ C433, which showed complete clearing of acid swollen cellulose and yet poor clearing of aspenwood sawdust. Among all the thermophilic fungal strains, Sporgt~Lchum thermQp~ C419, P~an~roç~e~ çhrysospQrium A435, ~Em~ç~
aurant~acus C412 and 235E appeared to be the best strains in the overall clearing of both the cellulosic and hemicellulosic substrates.

~o ensure thak the tube clearing asæay i8 a true reflection of th~ abilities of the organisms ~o produce extra~ellular hydrolytic enzymes, the fungi were also grown in liquid aul~ures and analyzed for their enzyme pro~uction and secretion in culture filtrates (Table 11). General correlation~
between the tube assay ''~;, , .

. .

` ` ~ 132~ 3 for estimating the cellulolytic and xylanolytic potential o~ a particular fungal strain and the actual enzyme production in liquid cultures were obtained. Several strains which showed poor clearing in tubes, such as B4ss~ C491 ~Thielavia t~rr~E~), and C416 (Mucor sp.) ~Table 10), also exhibited low cellulase or xylanase enzyme activities in their culture filtrates ~Table 11).
Other strains, such as ~her~oascus aurantiacus C412 and 235E
showed good clearing of the substrates in the tube assay (Table 10) and also good enzyme production in liquid medium (Table ll).
There were, however, several notable exceptions to the general correIation of the tube assay and enzyme production in liquid medium. Phane~ocha~e chrys~s~Qrium A387, which sho~ed no apparent clearing on xylan and little clearing on cellulosic substrates in solid medium (Table 10), was found to produce high levels of endoglucanase and xylanase activity when grown on Solka Floc in liquid medium (Table 11). On the other hand, P~
Çh~Y9~lQ~i9~ A435, which demonstrated near complete clearing on all ~ubstrates when grown in solid medium (Table 10), was found to produce only very low levels of both cellulase and xylanase ~nzyme6 in liquid medium (Table 11). This, however, could be becau~e the medium and culture conditions for the fungi used in the screening studles have not yet been established, thereby preventing optimal production of the desired enzymes. It is apparent, however, that both the tube clearing assay and the mr .
, - ~ 132~ 3 \
liquid culture studies are use~ul in screening work, and that the choices of one approach over the other will lik~ly depend on the nu~ber of cultures to be studied.

~ h~rm~s~ auran~iacus 235E was demonstrated to be the ~est producer of extracellular xylanase in liquid culture (Table 11).
Culture filtrates of the fungus were also found to exhibit high cellulase activities, with particularly high B-glucosidasa activity. A preliminary study was th~re~ore ini~iated to examine enzyme production by the fungus when grown in common fungal fermentation medium on various cellulosic substrates of commercial potential (such as steam-exploded aspenwood and aspenwood sawdu~t) (Table 12). In general, Vogel's mediu~
appeared to be more favourable for fungal enzyme production than Mandel's medium (Tables 11 and 12). However, the poten~ial of using the simpler and less expensive Mandel's medium in place of Vogel's medium was still demonstrated by tha high levels of xylanase and cellulose activiti~s detected in the filtrates of culture~ grown in Mandel~s medi1l~. Solka Floc appeared to be the most effective substrate for enæyme production, but high levels of extracellular xylanase ~with accompanying cellulase activities were also detected in filtrates o~ cultures grown on stea~
exploded aspenwood cellulose [i.e., water-insoluble residues of stea~-exploded aspenwood) and sawdust without any ,:~

~ 132~3 treatment. The economic potential o~ u~ing these letter substrates should be further explored, particularly w~th respect to sawdust. Being inexp~nsive and abudnant, th~y should be more attractive substrates for large scale enæyme production. The present study also ~howed that sawdust after ball-milling became an excellent substrate for xylanase and cellulase enzyme production (Table 12). The levels o~ filter paper activity and B-glucosidase activity in filtrates o~ cultures grown on ball-milled sawdust aatually surpassed those obtained with Solka Floc (~ables 11 and 12). Although it is unlikely that ball-milling would be a realistic pretreatment method due to its high energy - ~' 1'~

- . :: :
- , :, , ~ ' ~ ~' P ~32~3 1 ~
. .
cost, it is conceivable that other pretreatment methods could be tested to more fully exploit sawdust as a substrate for enzyme production. The present level of 61. 6 IU/ml of xylanase activity produced from completely untreated sawdust might already constitute an economical method of enzyme production. The decision on whether to pretreat sawdust to enhance enzyme production might rest on the compromise of enzyme yields versus the additional pretreatment costs.

The production oE extracellular xylanase(s) from hemicellulose-containing substrates was also studied (FigO
1). Vogel's medium was again shown to be superior to Mandel's medium for enzyme production. A xylanase activity of 66.1 IU~ml was obtained in fil~rates of cultures grown in Vogel's medium on unextracted steam-exploded aspenwood, demonstrating that the crude lignocellulosic residue could serve as an effective substrate for enzyme production as well. The hemicellulose-rich wa~er-soluble fractions of steam-exploded aspenwood, on the other hand, resulted in poor fungal growth and enzyme production. Similar findings had earlier been reported with enzyme production from Trichoderma species and was attributed to the presen~e of water-soluble inhibitors associated wi~h the fraction, Saddler, J.N. and Mes-Hartree, M. Biotechno~. Adv. 1984, .2, 161-181. Oat-spelt xylan was found to be the best substrate for xylanase production~ Up to 575.9 IU/ml of xylanase ac~ivity was detected in ~he filtrates of cultures grown in xylan in Vogel' 5 medium (Fig. 1), surpassing the xylanase production in cultures grown on Solka ~loc (Table ll, for strain 235E). Maximum enzyme production occurred at day 10, after w~ich the enzyme ac~ivities in the culture filtrates started to decline ~data not shownJ. At the time when maximum xylanase activity was detected in the culture filtrate, the corresponding ~ -xylosidase, endogluc-ana~e, filter paper, ~ -glucosidase, and protein levels in the filtrates were 1.63 IU/ml, 8.70 IU/ml, 0.30 IU/ml, 0.87 ~ .

132~3 ` ~!

IU/ml, and 1.33 mg/ml, respectively. To our knowledge, the level of xylanase activity (both in terms of IU/ml and IU/mg, using standard assay techniques) detected in the crude culture filtrates of T. aurantiacus surpassed all other microbial sys~ems reported in the literature.
Moreover, being thermophilic, the fungus could be grown at elevated temperature (45C or above) which should reduce the risk of microbial contamination and the cooling requirements for ~he fermentation process.
Preliminary characterization of the xylanase activity in the crude culture filtrate.of T. aurantiacus was then carried out. Both the xylanase and ~ -xylosidase activities in the filtrates exhibited temperature optima at 75C (Fig. 2) and p~ optima around 5.0 (Fig. 3). The xylanase activity in the crude filtrate was also demons-trated to be thermally stable. The half~lives of the enzyme at 70C and 60C were around l.S hours and 4 days, respec-tively (Fig 4 and 5). The patterns were essentially similar to xylanase activity in filtrates o cultures grown on a cellulosic substrate (Solka Floc) or on a hemicellulosic substrate (oat-spelt xylan). The half-life of the enzymes at 50C have no~ yet been determihed, since over 90~ of the activity remained in the filtrates after incubation at 50C
~S for 12 weeks. The corresponding ~-xylosidase activity also appeared to be thermostable, though not to the same extent as the xylanase activity (half-life at 70C and 60C at around 36 minutes and 2.4 hours, respectively).

The obser~ed thermostability of the xylanase in the crude culture filtrates of T. aurantiacus was unusual when compared to the xylanases reported in the literature.
Although detailed information on the thermostability of th~
reported xylanases was generally lacking, xylanases produced by a wide range of mesophilic fungi and bacteria usually showed optimal enzyme activity at around 50C Woodward, J.

, ~ ~32~3 To~ic~ in Enzy~ ~nd Ferment~ti~n Bl~t~ y 1984, 8 9-30;
Dekker, R.F.H. and Richards, G.N. Adv. Carbohy~, Çhem. ~l~chem., 1976, 32, 277 352). Recent inter~st in thermostable enzymes have led to discovery of several t~ermophilic or thermotolerant fungi and bacteria capable of producing thermostable xylanases. These include ~albranc~ea pulchella, an alkalophilic thermophilic Bacillus, Therm~mo~Q~ sp., Cl~stridium~ e~rari~m, Sa~ch~romQ~s~ora virldis, Ther~onosp~ra ~uaca, T. cu~vata, T.
çh~Y~g~n~, ~ela~Q~e~ albomyces, Thie~avi~ ~e~E~@~
~ , ~ , and Ceratocy~t~s par~Q~. Direct comparisons of enzyme activities and thermostability among various organisms were not always possible due to variations in the methodologies used.
Nevertheless, based on published reports, it would appear that the xylanase6 oP ~ aY~a~iaEi~ were among the most thermostable xylanases produced. This ~inding, in combination with the excsedingly high levels o~ xylanase activities obtained in crude cultur~ ~iltrates, strongly indlcate the potential of th~ enzyme in future commercial applications.

CONCLUSIONS

Screenin~ of thermophilic cellulolytic and xylanolytic fungi in Forintek's culture collection successfully identified ~.
DlLaçu~ 235E as an extremely prolific producer o~
extracellular xylanase enæymes. The same culture ~iltrate also exhibited a full spectrum of other cellulolytic and xylanolytic enzy~e activities. Enzyme production could be achieved at elevated temperatures even in cultures grown on inexpensive biomass residues, such as untreated sawdust and steam-exploded aspenwood. The xylanase enzyme demonstrated unusually high , .. .
~; . . ~ :

_~ ~ 132~ 3 - `~
~ .

th~ tæblllty nnd c~uld ha~ t~ntlal ln f-aturn l~rg~
~loma~ hydrolyDls ~r ~ombln~d hyd~oly~ nd ~orr~ntatlon -Rr gc ea~
. , , . . . - . ' ~bl~ 3. Llst of thnrm-Jph~llc ~un~ orlntek ~tltur~
~:v~lectlsn 0 tudled .

~
strnln Fungu~ ~ Cl~ Tlc~tlon A3~7 Ph~n~rclchs~t~ hryno~pc~r~um Bn~ldlomyc~
J~435 Ph~n~ haete ~ ~n/lldlomyc~te . _ ~49g Uncl~ loa Unol~a~nl~lod ~ 5 ~ ~ Un c l a ~ l a d . ' Un c l a ~ d E15 19 Ur~cln~sl~led vrtcla~lsloa C34 1 Uncla~ led Uncla~lEled C37~ p~ t-lchum ~ Deut~ss~yceto C375 Uncla~fl~d . , Un~:lansl~l ~412 ~hermolil3c~ r an~la cu ~ orayce'c~
c4 16 ~lu~:er ~p . Ppycomyc~ te C4 19 ~ thern~ hll~ D~ut~cmyeete ~4 24 Vncla~ td Uncla~ led c433 Myrlcoccum lb~mycc~ D~utero~yc~t~
;~35E The~moa~cl3s au~asltlacu~ A~c~nycet~
. . ~
C437 llalbr:~nchea ~ulchella Deutq~omycl~t~

-- 3~ --~f~` ~ i32~3 Table 9 ~cont'd) C~363 Taloramyce~ e~er~onll Dout~r~ycot~
C464 Thielav1a ter~a~rl~ ~acomy~te C465 Alle~cheria terreE~tr~.s Ascc~my~t~
C4 66 llumlct~ la,~?~l~no~a ~eu~r~myc~te C467 ~lamlcc~ gr1~o~ Deu'e~rc~mycet~
C491 _ hlelavla terrestrl~ A~ myeQt~

S~ble 10 8~r-enlng o~ th~rm~phllle l'ungl on ~ol~a ~o~lL~Im x~ l a n 2~ n d ~ l a 2 ~ t l ~ l t l ~

. ~ . . .
tung~l D~pth o~ rlny ~ono t~ ln ~ u~ ~ubs~r~t~
~t~Rln ~ponvot~d xyll~n 7~pl~ O~ ~wdugt l~eld-0-l0110n c~llulo~Q
. . _ , J~3~ ~ t1) 3 ~4~
A43526 14) 4.D t2J 4D i3J
J499ID 11) , V
DS~ 2~ t4~ ) g ~4 JSl9 ~ t!) ~11 t C341 34~3J 13 54) 13 1~1 30C372 ~1D12) 16 ~4~ 13 l4) C375 .~ilD 53J 12 tA) 4 ~31 C412 4~t3~ ~ 40 ~1 - 10 14) ~:4 1 6 ~ ) 4~ 4 ~
. .
' C41~ .qo1~2~ 4~ ~2~ l6 ~4) C4~ 0 t~ ) lo ~
PJJ3 4~ ~2~ ) 4~ t2) - 33 ~ :

P ~325~3 Table 10 (cont'd) 235E 40 (3~ 40 (3) 10 (3) C437 40 (3) 0 (1~ o (1) C463 0 (1) 3 (3) 4 (3) ~464 3~ (3) lO (4) 1~ (4) C465 2~ (4) 13 (~) 13 (4) C466 29 (4) o ~1) 0 tl) C467 40 (3) 13 (4) 4 (3) C491 0 ~1) o ~1) o (1) 1 0 ~
* Cultures were grown in solid medium on various substrates and incubated at 45C for up to 4 weeks. Depth of clearing zone at 40 mm represents complete tube clearing. Values in paranthesi~ indicate the time of incubation ~in we~ks~ when maximum tube clearing was observed.

Table 11 Production of xylanolytic and cellulolytic ~nzymss by thermophilic fungi in liquid culture*

Fungal ~ n~y~ 3ç~ivities ~U~
strain Xylanase Endoglucanase Filter Paper B-gluc~sidase ..... . _ . . , .. . ~ .. ~
A38787.3 ( 7) 12.20 0.47 0.18 A4351.6 (10~ 0.27 0.02 0.03 B4990.1 ( 3) 0.06 0.02 0.03 B50812.4 (10) 2.79 0.~9 0.05 B519 n.d.** n.d. n.d n,d.
C34120.3 (10~ ~.84 ~ .09 C37218.5 ( 5~ 3.40 0.16 0.15 - 3~ -~r ~32~613 ,...................................................................... . .
Table 11 (Cont'd) C3753.3 (10) 2.20 0.0~ 0.23 C412143.1 (10)7.30 0.22 0.47 C4160.8 (10) 0.14 0.04 0.06 C41g14.0 ( 7)6.56 0.18 0.3 C42411.0 (lO)5.25 0.17 0.12 C43363.7 ( 7)8.50 0.67 0.~0 235E365.8 ( 7)10.10 0.16 0.74 C437125.3 (lO)7.78 0.29 0.04 C4~36.1 (lO) 1.4~ 0.04 0.06 C46415.5 ( 5)~.77 0.17 0.21 C4655.8 ( 7~ 3.37 0.16 O.V9 C466n.d. n.d. n.d. n.d.
C4675.9 (10) ~.38 0.44 0.07 C4910.1 ( 3) 0.04 0.02 0.02 ... . ..

* Cultures were grown on Solka Floc (1%, w~v~ in liquld Vogel's mediu~ and incu~ated at 45C with shaking for up to 10 days.

** Not determined. Strains B519 and C466 did not show any visible growth under the present culture conditions.

*** Enzyme activities listed were the maximum levels obtained.
Values in parenthesis indicate the culture age at the time that maximum xylanase activity was detected in culture filtrates. `

.~ ,.

` 132~ 3 Table 12 Production of xylanolytic and cellulolytic enzymes by Th~rmo~s.c~s~a~3~Ltiaçus 235E grown in various cellulosic suhtrates in liquid medium.

Subs~rate Enzyme A~iviti~ (IU/mL ! --(1%, w/v) Xylanase ~ndoglucanase Filter Paper B-glucosidase . Vogel's medium:

SEA--WI* 98~2 (10)** 2~87 0.16 0.31 Sawdust 40.5 (10) 0.63 0.05 0.14 Ball-milled Sawdust 174.4 (10) 5.62 0.27 1034 B. Mandel's medium:

Solka Floc 94.6 (10) 7034 0.13 0-39 ~EA-WI 62.6 ( 7) 2.70 0.09 0u18 Sawdust 61.6 (10) 0,91 0.05 0.16 Ball-milled Sawdust 80.7 (10) n.d.*** n.d. n.d * Water-in~oluble residue of steam-exploded aspenwood.

** Enzyme activitie~ listed were the maximum levels obtained.
Values in parenthesis indicate the culture age at the time that maximum xylanase activity was detected in culture filtrates.

*** Not determined.

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Claims (14)

1. A process for the production of a thermally stable xylanase enzyme preparation having no significant cellulase enzyme activity, and suitable for the selective hydrolysis of hemicellulose in mixed cellulose and hemicellulose substrates, which process comprises culturing the 235E strain Thermoascus aurantiacus microorganism in a nutrient culture medium containing at least one cellulosic or hemicellulosic substrate;
separating from the culture medium a culture filtrate containing a major proportion of xylanase enzymes and a minor proportion of cellulase enzymes; and incubating the culture filtrate at a temperature of at least about 60°C for a time sufficient to selectively inactivate the cellulase enzymes and recovering a culture filtrate having xylanase enzyme activity, but no significant cellulase enzyme activity.

2. The process of claim 1, wherein the culturing is effected at a temperature of at least 45°C.

3. The process of claim 2, wherein the culture medium contains cellulose pulp, oat-spelt xylan, steam treated aspenwood or sawdust as the substrate.

4. The process of claim 3, wherein the culture medium is Vogel's medium or Mandel's medium.

5. The process of claim 2, wherein the culturing is effected for a period of 5 to 12 days.

6. The process of claim 1, wherein the separation of the culture filtrate is effected by filtration or centrifugation.

7. The process of claim 1, wherein prior to incubating the culture filtrate at a temperature of at least about 60°C, said culture filtrate is concentrated by rotary evaporation.

8. The process of claim 1, wherein prior to incubating the culture filtrate at a temperature of at least about 60°C, said culture filtrate is concentrated by ultrafiltration through an ultrafiltration membrane having a low molecular weight cutoff point between 1,000 and 20,000 daltons to obtain a xylanase rich and cellulase rich retentate; and wherein the xylanase retentate is then incubated at a temperature of at least 60°C to selectively inactivate the cellulase enzymes.

9. The process of claim 8, wherein the membrane has a low molecular weight cut-off point between 5,000 and 20,000.

10. The process of claim 8, wherein the membrane has a low molecular weight cut-off point between 5,000 and 15,000.

11. The process of claim 8, wherein the membrane has a low molecular cut-off point between 5,000 and 12,000.

12. The process of claim 8, wherein the membrane has a low molecular cut-off point between 5,000 and 10,000.

13. The process of claim 8, wherein the membrane is a non-cellulosic membrane.

14. The process of claim 8, wherein the membrane is a polysufone membrane.