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DOI: 10.4025/actascitechnol.v32i1.7648

Acta Scientiarum. Technology Maring, v. 32, n. 1, p. 37-42, 2010

Production of xylooligosaccharides from enzymatic hydrolysis ofProduction of xylooligosaccharides from enzymatic hydrolysis ofProduction of xylooligosaccharides from enzymatic hydrolysis ofProduction of xylooligosaccharides from enzymatic hydrolysis of

xylan by whitexylan by whitexylan by whitexylan by white----rot fungirot fungirot fungirot fungiPleurotusPleurotusPleurotusPleurotus

Cristiano Ragagnin de Menezes*, sis Serrano Silva, rika Cristina Pavarina, AndriaFonseca de Faria, Elisngela Franciscon and Lucia Regina Durrant

Departamento de Cincia de Alimentos, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas,Cx. Postal 6121, 13083-862, Campinas, So Paulo, Brazil. *Author for correspondence. E-mail: [email protected]

ABSTRACT. Hemicellulose consists of non-cellulosic polysaccharides, with xylans andmannans as their main examples. In nature, xylan can be first degraded to

xylooligosaccharides and finally to xylose by certain microorganisms. White-rot fungibasidiomycetesPleurotus sp. BCCB068 andPleurotus tailandiawere used to degrade oat-spelts

xylan under submerged fermentation for a period of 40 days. The study obtained activitiesof endo-1,4--xylanase and -xylosidase and determination of xylan products bydegradation. The fungi reached significant levels of xylan degradation by Pleurotus sp.

BCCB068 (75.1%) and P. tailandia (73.4%), following formations of xylooligosaccharidesand sugar monomers. These Pleurotus strains proved to be a feasible alternative forbiotechnological processes related to degradation of hemicellulose sources.Key words: biodegradation,Pleurotus, xylan, hemicellulose, xylooligosaccharides.

RESUMO. Produo de xilooligossacardeos pela hidrlise enzimtica de xylana

por fungos Pleurotus. A hemicelulose um polissacardeo no-celulsico, tendo comoexemplos principais as xilanas e mananas. Na natureza, as xilanas podem ser degradadas pormicrorganismos, primeiramente a xilooligossacardeos e finalmente a xilose. Fungosbasidiomicetos Pleurotus sp. BCCB068 e Pleurotus tailandia foram utilizados para degradar

xilana de aveia em fermentao submersa durante o perodo de 40 dias. Foram obtidas asatividades de endo-1,4--xilanase e -xilosidase e a determinao dos produtos dedegradao da xilana. Os fungos atingiram nveis significativos de degradao da xilana por

Pleurotussp.

BCCB068 (75.1%) and

P. tailandia (73.4%), seguido da formao dexilooligossacardeos e monmeros de acar. Essas cepas dePleurotusdemonstraram ser uma

alternativa vivel para os processos biotecnolgicos relacionados degradao de fontes dehemicelulose.Palavras-chave: biodegradao,Pleurotus, xilana, hemicelulose, xilooligossacardeos.

InInInIntroductiontroductiontroductiontroduction

Hemicellulose is one of the most abundantpolysaccharides in nature present in agriculturalbiomass waste, and cannot be easily converted tosimple monomeric sugars due to its recalcitrantnature. Xylan, the major component of

hemicellulose in plant cell walls, is the second mostabundant polysaccharide after cellulose (TAN et al.,2008). Xylan polymer consists of a main chain of-1,4- linked D-xylose residues or some substitutesincluding L- arabinose, D-glucose, D-galactose, D-mannose, D-glucuronic acid, 4-O-methylglucuronic acid D-galacturonic acid residues, as wellas in less proportion the L-rhamnose, L-fucose and

various O-methylated neutral sugars (SUN et al.,2000). The complete degradation of this complexstructure depends on different enzymes acting insynergism endo--1,4-xylanases hydrolyze -1,4-

bonds between D-xylose residues in the main chainproducing xylooligosaccharides (XOS), and -D-

xylosidases convert xylo-oligosaccharides to xylosemonomers (DEN HANN; VAN ZYL, 2003). Someother specific enzymes such as -L-arabnofuranosidase, -glucuronidase, as well asseveral esterases have also a cooperative function

into the complete degradation of xylan(TENKANEN et al., 1996).

Xylan is degraded by bacteria and fungi throughthe production of a full complement of enzymes.The genera Trichoderma, Aspergillus, Fusarium and

Pichia are considered great producers of xylanases(CHRISTAKOPOULOS et al., 1996; ADSUL etal., 2005), and basidiomycetes usually secret largeamount of enzymes to degrade lignocellulosicmaterials: white-rot fungus Phanerochaete

chrysosporiumproduces high level of -glucuronidase

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(CASTANARES et al., 1995),andCoriolus versicolor,acomplex xylanolytic system (EL-NASSER et al., 1997).

Xylanase is also produced by Cuninghamellasubvermispora when growing on plant cell-wallpolysaccharides or on wood chips (SETHURAMANet al., 1998; SOUZA-CRUZ et al., 2004) and currentinvestigation suggests that xylanase is a key enzyme forhemicellulose degradation. Pleurotus ostreatus, Pleurotus

sajor-caju and Trametes versicolor (REDDY et al., 2003;VALASKOV; BALDRIAN, 2006) are also producersof lignocellulolytic enzymes. Multiple endoxylanaseisoenzymes are produced by microorganism and mayreflect the need to produce xylanases with strict andrelaxed specificities, capable of acting on differentsubstrates (MILAGRES et al., 2005).

Xylanases are used in the management of agro-industrial wastes to degrade partially xylan to a

renewable biomass, generating less environmentalimpact, and in the paper industry as pulp bleaching(COLLINS et al., 2002; TECHAPUN et al., 2002)to reduce the amount of chlorine in the process.

Agricultural wastes can also be hydrolyzed infermentable sugars and ethanol. Several bacteria,fungi and yeast are capable of fermenting xylose toethanol (INGRAM et al., 1999; LEE et al., 1986).Oligosaccharides produced by the action of

xylanases are used as functional food additives oralternative sweeteners with beneficial properties(PELLERIN et al., 1991; PULS et al., 1998).

The white-rot fungi basidiomycetes have beenstudied nowadays as xylanase sources, once itsextracellular system is produced to act in a widerange of lignocellulosic materials, and because theyare considered edible, these mushrooms is highlynutritional and safe (BUSWELL; CHANG, 1994)being source of important metabolites of interest tothe pharmaceutical, cosmetic and food industries(JONG; DONOVICK, 1989; QINNGHE et al.,2004).

In this present study, white-rot fungiPleurotus sp.BCCB068 and Pleurotus tailandia were evaluatedregarding the degradation of the xylan matrix with

production of xylanases using submergedfermentation. The ability of these fungi in degrading

xylan, the principal component of hemicellulose inagro-industrial wastes, suggests further research tooptimize xylanolytic enzymes, produce XOS anddecrease these residues into the nature.

Material andMaterial andMaterial andMaterial and mmmmethodsethodsethodsethods

MicroorganismsMicroorganismsMicroorganismsMicroorganisms

The white-rot fungi Pleurotus sp. BCCB068 andPleurotus tailandia belong to the Culture Collection of

the Systematic and Microbial Physiology Laboratory(Faculty of Food Engineering State University ofCampinas, So Paulo State, Brazil) and were cultivatedin PDA (Potato Dextrose Agar - Difco) at 30C forseven days up to complete superficial growth into Petridishes, using three portions of small disks with 6 mm

in diameter, when one disk is used per 10ml of liquidmedium as inoculum.

Experimental conditionsExperimental conditionsExperimental conditionsExperimental conditions

The submerged fermentation was carried out in125 ml Erlenmeyer flasks containing 50 ml of sterilebasal medium (pH 5.5) plus 1% xylan. Basalmedium presented the following composition perLiter: 1.4 g (NH4)2SO4, 2.0 g KH2PO4, 0.1 g urea,0.3 g MgSO4.7H2O, 0.3 g CaCl2, 5.0 mgFeSO4.7H2O, 1.56 mg MnSO4.H2O, 2.0 mg CoCl2,1.4 mg ZnSO4.7H2O. Flasks were inoculated as

described above and incubated at 30C for 40 daysunder aerobic static condition. Triplicate of sampleswere collected each 10 days of cultivation, and pHwere measured. Extracellular fluids obtained byrefrigerated centrifugation at 33,450 x g for 15minutes were stored at -20 C for enzymaticanalyses and determination of xylan degradation.

Enzymatic activitiesEnzymatic activitiesEnzymatic activitiesEnzymatic activities

Endo-1,4--xylanase (EC 3.2.1.8) were assayedby measuring of reducing sugar released fromsubstrate (MILLER, 1959). The reaction mixturecontaining 1 mL of culture supernatant (enzymatic

extract), 1 mL of 1% oat-spelts xylan (Sigma) in 0.05M acetate buffer pH 5.0, and 2 mL 3,5-dinitrosalicilic acid (DNS) was incubated at 50C for30 minutes, and the enzyme-substrate system havebeen shacked periodically to keep xylan insuspension. Tubes containing the reactions wereread in spectrophotometer (Bausch and Lomb) at550 nm. The values were expressed in U mL-1,

when one unit is mol of xylose produced per ml ofmedium per minute.

-Xylosidase activity was routinely determinedby measuring the amount of p-nitrophenol released

from PNPX. The assay was carried out at 50 C in50 mM sodium phosphate buffer (pH 6.5), with 50L of 10 mM PNPX (Sigma) plus an appropriatelydiluted enzyme solution, in a total volume of 0.5mL. The p-nitrophenol released was measured bymonitoring the increase in the absorbance at 410 nmfollowing 10 min. of incubation. The reaction wasstopped by the addition of 1.0 mL of 1.0 M sodiumcarbonate. One unit of b-xylosidase activity wasdefined as the amount of enzyme required to release1 mol of p-nitrophenol min.-1mL-1(IEMBO et al.,2005).

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Production of xylooligosaccharides from enzymatic hydrolysis of xylan 39


High performance liquid chromatographyHigh performance liquid chromatographyHigh performance liquid chromatographyHigh performance liquid chromatography

The degradation of xylan was analyzed in aShimadzu chromatographer LC-6A using an ionexchange Supelcogel C-610H column (Supelco). A

volume of 20 L was previously filtered into PVDF0.22 m membrane (Millipore) and injected, using0.1% phosphoric acid in ultrapure water as mobilephase, a flow rate of 0.5 mL min. -1 and refractiveindex detector RID 10A (Shimadzu), specific forcarbohydrates and organic acid analyses.

Results andResults andResults andResults and ddddiscussioniscussioniscussioniscussion

ResultsResultsResultsResults

Xylanase activity was present practically in thewhole period of cultivation, and the greatestactivities corresponding to 20th, 30th and 40th days

were reached by Pleurotus sp. BCCB068: 0.64, 0.58,

and 0.60 U mL-1, respectively, as shown in Figure 1.P. tailandia presented higher activity of xylanase on20thday (0.25 U mL-1); however, very low values ofthis enzyme have been shown during this period(Figure 2).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

5 10 15 20 25 30 40

U.m

l-1

0

1

2

3

4

5

6

7

days

pH

xilanase

-xylosidase

pH

UmL-1

Figure 1.Activities of xylanase, -xylosidase and pH produced byPleurotus sp. BCCB068 during 40 days under submergedfermentation using xylan as carbon source. Data are the mean oftriplicate and standard deviation is indicated in the error bars.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

5 10 15 20 25 30 40

U.m

l-1

0

1

2

3

4

5

6

7

days

pH

xilanase

-xylosidase

pH

UmL-1

Figura 2.Activities of xylanase, -xylosidase and pH produced byP. tailandiaduring 40 days under submerged fermentation usingxylan as carbon source. Data are the mean of triplicate andstandard deviation is indicated in the error bars.

In general, the fungi were capable of hydrolyzingxylan and forming some sugar monomers (xylose,arabinose, fructose, mannose, fructose, and othersnon-identified), and oligosaccharides as indicatedin Tables 1 and 2. Pleurotus sp. BCCB068 shownconsiderable degradation of xylan on the 5 th day

(70.1%), followed by production of monomers andoligosaccharides. A peak of xylan hydrolysis wasdetected on the 20thday (75.1%), together with thebest production of xylanase activity. Sugarmonomers produced previously have indicatedanother decrease in concentration from the 15 thdayon due to a possible absorption of thesecompounds to be part of the fungal metabolism.

Arabinose and xylose have been identified asproducts of xylan degradation by Pleurotus sp.BCCB068 comparing these peaks with previousanalysis of carbohydrate standards (data not show).

In 10 days of cultivation there was a drop of xylosepeak and absence of arabinose, however, thesemonomers have increased on the 15thday throughbreakdown of XOS that were previously formed.From this period on a steady consumption of thesecompounds have been verified, moreover, newother compounds were formed up to 30 days ofcultivation.

Table 1.Degradation of xylan, yield of xylooligosaccharides andsugar monomers by Pleurotus sp. BCCB068 under submergedfermentation.

Days of

cultivation

% Xylan

degradationXylooligosaccharidesa

Sugar

monomers

Non-identifiedintermediatemetabolitesb

5 70.1 + Arabinosec,Xylosed

+

10 55.0 +++ Xylosed +++15 42.8 ++ Arabinosec,

Xylosed++

20 75.1 + NDe +25 72.7 + NDe +30 66.9 + NDe ++40 67.2 + NDe ++a, bYield expressed in intensity +/++/+++ by comparison of peak areas in thechromatograms; c, dsugar monomers identified by comparing retention times in thepreviously performed carbohydrates standard chromatogram; enot detected.

Table 2.Degradation of xylan, yield of xylooligosaccharides andsugar monomers byP. tailandiaunder submerged fermentation.

Days ofcultivation

% Xylandegradation

Xylooligosaccharidesa Sugarmonomers

Non-identifiedintermediatemetabolitesb

5 NDg NDg Arabinosec,Maltosed

++

10 65.0 +++ NDg +15 70.2 ++ Arabinosec,

Xylosee++

20 73.6 + Arabinosec,Xylosee,Mannosef

+

25 56.2 ++ Mannosef ++30 58.4 + Mannosef +++40 61.2 + Mannosef +++a, bYield expressed in intensity +/++/+++ by comparison of peak areas in thechromatograms; c, d, e, fsugar monomers identified by comparing retention times in thepreviously performed carbohydrates standard chromatogram; gnot detected.

pH

pH

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DiscussionDiscussionDiscussionDiscussion

The highest activities of xylanase were verify byGarzillo (GARZILLO et al., 1994) following 16 daysof cultivation of P. ostreatus; nevertheless, studiesregarding the effect of growth of P. ostreatus 3004and the digestibility of corncob producing

cellulolytic enzymes under submergedfermentation, have verified low activities of

xylanases (SERMANNI et al., 1994). Corncobcontains high level of xylan (33%) and was used asinduction substrate to the production of xylanases(QINNGHE et al., 2004), as well as organic andinorganic nitrogen compounds including peptone inoptimized conditions (KALL et al., 1995),promoting an increase in xylanase production by

P. ostreatus. In our results, xylan was used as thesingle carbon source, and due to the absence of anyco-substrate, there was low activity expressed. There

was no use of such nitrogen sources, once our aimwas not to optimize xylanase production, but utilizethese enzymes as parameters to xylan degradation

with no enrichment source. -xylosidase enzymewas detected since the 5thday, verifying the presenceof monomers through the degradation of XOS,maintaining its activity during the whole period ofcultivation with some variation in values (0.01-0.09U mL-1), reaching a best activity on the 40 thday, asshown Figure 1.

Analyzing pH values, Pleurotus sp. BCCB068showed an oscillation from 5.03 to 6.66, and

P. tailandia,from 4.42 to 5.99. Studies conducted byQinnghe (QINNGHE et al., 2004) havedemonstrated that P. ostreatus performed higheractivity of xylanase in pH 5.4, confirming greatmycelial growth of the same fungus in pH 5.0(FURLAN et al., 1997). In this present work, thesupernatant of P. tailandia revealed the highest levelof xylanase activity (0.25 U mL-1) in pH 5.41 withpresence of xylan degradation (20thday, Figure 1). Inthe same period,Pleurotus sp. BCCB068 showed thebest degradation of xylan and xylanase activity (0.64U mL-1) and pH 6.66. Burla and co-workers

(BURLA et al., 1992) demonstrated that higherproduction of biomass to P. ostreatus was in pH 6.0,using basal medium supplemented by 0.2%sunflower oil and wheat straw, and a study by Bajpai(1997) has reported that from pH 5.0 though 6.5,

xylanases are generally produced by fungi.The activity of xylanase toP. tailandiawas slightly

less than described previously by Pleurotus sp. CCB068, showing very low level of enzyme on the 5thday (no xylan degradation detected), and following25, 30 and 40 days of incubation, when there was nolonger an increase in the degradation rate.

Nevertheless, the greatest activity of xylanase(0.25 U mL-1) was observed on the 20th day,accompanied by higher levels of degradation of

xylan by this fungus (Figure 2).P. tailandia did notshow activities of -xylosidase, except on the 10thday(0.01 UI mL-1; Figure 2). Iembo (IEMBO et al.,

2005) performed experiments with thermophilousfungus Humicola grisea in optimized conditions andfound considerable value (12.98 UI mL-1) whenusing oat-spelts xylan as carbon source. The fungusStreptomyces sp.CH7 growing in xylan substrate alsoreached great levels of -xylosidase: 26.3 and6.6 U mL-1of protein, according to Pinphanichakarnand colleagues (PINPHANICHAKARN et al.,2004). Both studies carried out by these authorsshowed the stability of enzyme between pHs 6 and 9.

Regarding the results obtained by Pleurotustailandia, only on the 5thday was hydrolysis of xylan

not observed; however, a significant degradation ofthis matrix was detected on the remaining days.Higher degradations of xylan were reached from the10th day on (Table 2). Moreover, XOS wereproduced early on the 10thday, as well as a gradualdisappearance of these compounds up to the 25 thday. In this period, a new production ofoligosaccharides and sugar monomers was detected,

with a drop in these productions on the 40 thday, dueto its consumption by the fungal metabolism. Sugarmonomers were produced through the breakdownof XOS, and it is likely some metabolites producedin the xylan degradation presented the sameretention time of xylan into the chromatogram,causing an overlaying of peaks, indicating a falseraise of xylan peak.

According to the chromatographic profilespresented by fungi, there was a concomitantdegradation of xylan and XOS, as well asassimilation of sugars and other products, suggestingthe action of an enzymatic system containing

xylanases and other hydrolytic and oxidativeenzymes produced to degrade this complex carbonsource. Studies conducted by Cotta and Zeltwanger(COTTA; ZELTWANGER, 1995) have shown the

use of xylan as substrate by B. fibrisolvens reaching75% of degradation with production of soluble acid-alcohols, and these products have been utilized by S.ruminantum to produce XOS. The degradation of oatspealt xylan and subsequently XOS by B. fibrisolvensgenerated a mixture of low-molecular-weighintermediate metabolites containing severalcompounds with different levels of polymerization(e.g. xylose, arabinose and uronic acid), according toHespell and Cotta (HESPELL; COTTA, 1995).Products from acid hydrolysis of xylan formed a

variety of mixture of XOS when these products

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Production of xylooligosaccharides from enzymatic hydrolysis of xylan 41


were depending on several xylanolytic enzymespresent into medium, and the degradation of XOSproduced by xylanases was better metabolized thanby acid hydrolysis procedure (COTTA;ZELTWANGER, 1995).

ConclusionConclusionConclusionConclusion

Despite the low production of endo-1,4--xylanase in this present work, a considerabledegradation of xylan indicated the action of otherspecific and unspecific enzymes not studied in this

work, responsible for xylan hydrolysis.In conclusion, Pleurotus sp. BCCB068 and

P. tailandiawere capable of degrading xylan in up to75%, producing XOS and several sugar monomers.These results have shown a promising applicabilityin residues with high concentration ofhemicellulose, which when delignified are viable

matrices for the production of oligosaccharides.Therefore, complementary studies may be done tooptimize degradation processes and enzymaticproduction, once these fungi have shown a greatpotential for future biotechnological processes.

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

The authors thank the Foundation for ResearchSupport of So Paulo State (FAPESP, Brazil) for thefinancial support for this research.

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