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Review

Enzymology of the thermophilic ascomycetous fungusThermoascus aurantiacus

Michel BRIENZO, Valdeir ARANTES, Adriane M. F. MILAGRES*

Department of Biotechnology, Escola de Engenharia de Lorena University of Sao Paulo – USP Lorena, Estrada Municipal do Campinho,

s/n� – CP 116, 12602-810 Lorena/SP, Brazil

a r t i c l e i n f o

Article history:

Received 28 February 2008

Received in revised form

6 April 2009

Accepted 21 April 2009

Keywords:

Amylases

Cellulases

Enzyme application

Pectinases

Thermoascus aurantiacus

Xylanases

* Corresponding author. Tel.: þ55 12315950E-mail addresses: michel@debiq.eel.us

(A. M.F. Milagres).1749-4613/$ – see front matter ª 2009 The Bdoi:10.1016/j.fbr.2009.04.001

a b s t r a c t

Different strains of the thermophilic ascomycetous fungus Thermoascus aurantiacus have

been reported in the literature to produce high levels of a variety of industrial interest

enzymes (i.e. amylases, cellulases, pectinases and xylanases), which have been shown to

be remarkably stable over a wide range of temperatures and appear to have tremendous

commercial potential. Most studies on enzyme production by T. aurantiacus are carried

out in chemically defined liquid medium, under conditions suitable for induction of

a particular enzyme. A few studies have investigated the production of some enzymes

by T. aurantiacus by solid-state fermentation, using lignocellulosic materials. The present

review focuses on the enzymes produced by T. aurantiacus, their main kinetic parameters,

and the effect of different culture conditions on production and enzyme activity. It also

provides a view of the possible applications of T. aurantiacus enzymes, considering that

this thermophilic fungus could comprise a potential source of thermostable enzymes.

ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

1. Introduction 47.5 �C and 60 �C, respectively (Deploey, 1995), which were

Thermoascus aurantiacus is a thermophilic fungus that belongs

to the ascomycetous class, first isolated from selfheating hay

by Hugo Miehe in 1907 as reported by Cooney and Emerson

(1964). T. aurantiacus has a bright orange color, elliptical asco-

spores, and no asexual stage (Maheshwari et al., 2000), although

in the past the description given by Cooney and Emerson (1964)

depicted it as having an asexual stage. So far, about 19 T. auran-

tiacus strains have been classified (Table 1).

Optimum and maximum temperatures for T. aurantiacus

ascospore germination have been reported to be about

00; fax: þ55 1231533165.p.br (M. Brienzo), ara

ritish Mycological Society

higher than those reported for hyphal growth (40–45 �C and

55 �C, respectively) (Cooney and Emerson, 1964). The

minimum temperature for ascospore germination (32.5 �C–

35 �C) (Deploey, 1995), is also higher than the minimum for

hyphal growth (20 �C–25 �C) (Cooney and Emerson, 1964). In

addition, the percentage of germination has been shown to

be slightly affected by culture age (Deploey, 1995).

Noack (1920) studied the behavior of T. aurantiacus sub-

jected to anaerobiosis and found that the withdrawal of

oxygen severely affected its respiration and growth. He also

investigated the effects of subminimal temperatures and

ntes@debiq.eel.usp.br (V. Arantes), adriane@debiq.eel.usp.br

. Published by Elsevier Ltd. All rights reserved.

Table 1 – Classified strains of T. aurantiacus

Strain References

T. aurantiacus 179-5 Martins et al., 2002

T. aurantiacus 26904 Feldman et al., 1988

T. aurantiacus 34115 Feldman et al, 1988

T. aurantiacus A-131 Kawamori et al, 1987

T. aurantiacus ATCC 204492 Milagres et al, 2004

T. aurantiacus BJT 190 Alam et al., 1994

T. aurantiacus BT 2079 Gomes et al., 2007

T. aurantiacus CBMAI-756 Martins et al., 2007

T. aurantiacus IF0 9862 Wang et al., 1998

T. aurantiacus IFO 31693 Ko et al., 2005b

T. aurantiacus IFO 9748 Hong et al., 2003a

T. aurantiacus II S96 Katapodis and Christakopoulos, 2004

T. aurantiacus IMI 173038 Katapodis and Christakopoulos, 2004

T. aurantiacus IMI 216529 Parry et al., 2001

T. aurantiacus Miehe Ortner et al., 2006

T. aurantiacus NBRC 31693 Ko et al., 2005a

T. aurantiacus SL 16 W Kongbuntad et al., 2006

T. aurantiacus TUB F43 Nampoothiri et al., 2004

T. aurantiacus var. levisporus Upahyay et al., 1984

Hydrolases of Thermoascus aurantiacus 121

rewarming and observed that when an actively growing

culture of T. aurantiacus was cooled just a little lower than

the minimum temperature for ascospore germination, its

respiration after 2 d declined only minimally, but those

cultures kept at 21 �C for 24 h stopped respiring. When the

culture medium was rewarmed to 46 �C, practically no respi-

ration was observed, showing the low resistance to lower

temperature. However, until more information is available,

it should not necessarily be assumed that mycelial cultures

of thermophilic fungi cannot be stored under refrigeration or

at subminimal temperatures without loss of viability.

T. aurantiacus has been shown to grow efficiently on ligno-

cellulosic biomass from agro-industrial and forest wastes

(Da Silva et al., 2005; Kalogeris et al., 2003; Milagres et al.,

2004; Santos et al., 2003; Yu et al., 1987). The good growth adap-

tation on such materials of high abundance and low cost is an

attractive and promising alternative for production of

enzymes.

The following sections describe the main physico-chemi-

cal characteristics of hydrolytic enzymes produced by T. aur-

antiacus. The considerable biotechnological potential of most

of these enzymes is also briefly discussed.

Cellulases

Cellulose, the major constituent of plant cell walls, consists

of an unbranched polymer of cellobiose, and is one of the

most abundant organic compounds in the biosphere. Cellu-

lose crystallinity and the large amount of hydrogen bonds

are two essential factors affecting the biodegradation of

native cellulose; a group of enzymes acting in a synergistic

manner is required to facilitate complete cleavage of the

cellulose bonds to form glucose. Cellulose hydrolyzing

enzymes (cellulases) are divided into three major groups:

endo-1,4-b-D-glucanases (EC 3.2.1.4), exo-1,4-b-D-glucanases

(EC 3.2.1.91) (or cellobiohydrolase) and b-glucosidases (EC

3.2.1.21), all three attacking b-1,4-glycosidic bonds.

T. aurantiacus can produce all cellulolytic enzymes required

for complete degradation of cellulose to glucose. However, as

reported for some other well known cellulase-producing

microorganisms such as Trichoderma reesei, Termonospora fusca

and Aspergillus niger endo-1,4-b-D-glucanase is the main cellu-

lase activity produced by T. aurantiacus (Himmel et al., 1999).

Efforts to increase the performance of cellulase mixtures for

the hydrolysis of pretreated biomass have sometimes focused

on the selection of enzymes with high thermal stability to

enable operations at higher temperatures, which implies

possibilities for prolonged storage increased and reduce risks

of contamination. A summary of some relevant published

data for the properties of cellulases from different strains of

T. aurantiacus is given in Table 2.

b-glucosidaseb-glucosidase production by T. aurantiacus is highly depen-

dent on the culture conditions. Thus, larchwood xylan, blot-

ting paper, bagasse, pectin (Khandke et al., 1989a), enhance

the production of b-glucosidase. It was suggested that in

T. aurantiacus more than one type of b-glucosidase exists

and have important role in the formation of endo and exo-

glucanase inducers from cellulose (Feldman et al., 1988; De

Palma-Fernandez et al., 2002; Tong et al., 1980). In general,

its b-glucosidases show high stability, half lives at 70 �C of

23.5 h (Gomes et al., 2000), optimum temperature and pH

between 65 �C and 80 �C, and 4.5 �C and 6 �C, respectively

(Hong et al., 2006; Parry et al., 2001). Leite et al. (2007) observed

a first-order kinetic for thermo inactivation of b-glucosidase

from T. aurantiacus 179-5 when exposed to temperatures

higher than 80 �C.

Kalogeris et al. (2003) identified b-glucosidases from T. aur-

antiacus IMI 216529 having a pI of 3.9. An extracellular b-gluco-

sidase purified from T. aurantiacus IMI 216529 has a molecular

weight of 120 kDa (based on gel permeation) hydrolyzed disac-

charides with b,1-1, b,1-6 and b,1-2 glucosidic bonds. The pNP-

cellooligosaccharides were used to demonstrate that the

enzyme cleaves one glucose unit at a turn from the non-

reducing end (Parry et al., 2001). A high degree of N-terminal

amino acid sequence identity between T. aurantiacus b-gluco-

sidase and other b-glucosidases of family 3 suggested that the

enzyme could be a member of glycoside hydrolase family 3

(Parry et al., 2001).

The synergism in a mixed culture of T. aurantiacus and

A. niger on oat straw semi-solid fermentation resulted in

high levels of b-glucosidase activity (500 IU/g), there were

2.5-fold higher than the activity observed when A. niger was

grown in monoculture (Stoilova et al., 2005).

A b-glucosidase gene (bgl2) of T. aurantiacus IFO 9748 was

isolated and expressed in Pichia pastoris KM71H. The recombi-

nant enzyme retained 50 % of its activity in the presence of

1.39 M of glucose, while the wild enzyme was inhibited at

low glucose concentrations.

Endo-1,4-b-D-glucanaseEndoglucanases cleave randomly b-glycosidic bonds in b-1,4-

glucan chains to produce free chain ends. Different T. auran-

tiacus strains have been shown to produce endoglucanases

with pIs (around 3.5–3.7) (Kalogeris et al., 2003; Parry et al.,

2002). In general, endoglucanases from T. aurantiacus display

Table 2 – Characteristics of cellulases produced by different T. aurantiacus strains

Enzymes Strain Activity Stability Optimumtemperature

(�C)/pH

Optimum medium References

Endoglucanase IMI 216529 50 �C/48 h 70–80/4.4 Wheat bran Parry et al., 2002

Miehe 60 U/mL 60 �C/1 h 75/5–5.5 Corncob – green grass Da Silva et al., 2005

179-5 758 U/mL Sugarcane and orange

bagasse

Martins et al., 2003

BT 2079 1130 nkat/mL 70 �C/t1/2¼ 98 h 75/4 Solka floc Gomes et al., 2000

IMI 216529 1572 U 80 �C/42 min Wheat straw Kalogeris et al., 2003

N/M 1,71 U/mL 70 �C/8 h 65/5 Corn cob Khandke et al., 1989a

A-131 70 U/mL 60 �C/24 h Alkali treated bagasse Kawamori et al., 1987

Celobiohydrolase N/M 1,71 U/mL 70 �C/low stability 65/5 Corn cob Khandke et al., 1989a

N/M 50 �C/8 h 65/4.1 Maheshwari et al., 2000

Miehe 46,1 U/g Sugarcane-orange

bagasse

Martins et al., 2002

IMI 216529 70 �C/48 h 80/4.5 Wheat bran Parry et al., 2001

Cellobiohydrolase

recombinant

IFO 9748 70 �C/2 h 70/6 Avicel Hong et al., 2003a

b-glucosidase N/M 8.2 UI/g Wheat bran Stoilova et al., 2005

BT 2079 94 nkat 70 �C/t1/2¼ 23.5 h 75/5 Solka floc Gomes et al., 2000

IMI 216529 50 �C/2 h 80/4.5 Wheat bran Parry et al., 2001

b-glucosidase

recombinant

IFO 9748 65 �C/pH 5 65/6 Xylan - avicel Hong et al., 2006

FPAa N/M 5.9 UI/g Wheat bran Stoilova et al., 2005

a-D-Glucuronidases N/M 2.2 U/mg 70 �C/t1/2¼ 40 min 65/4.5 – Khandke et al., 1989b

N/M – not mentioned.

a FPA – Filter paper activity.

122 M. Brienzo et al.

high stability at 70 �C half live of 98 h, (Gomes et al., 2000),

optimum temperature and pH between 65 �C and 80 �C and

4.0 �C and 5.5 �C, respectively (Da Silva et al., 2005; Gomes

et al., 2000; Khandke et al., 1989a; Parry et al., 2002).

Parry et al. (2002) described an endoglucanase with

a molecular weight of 34 kDa (based on SDS-PAGE) and a pI

of 3.7. This enzyme contained high levels of Asp (15.4 %) fol-

lowed by Gly, Ala, Glu and Ser (8 %–10 %). The levels of Cys,

His and Arg were in the range of 0.4 %–2.2 %. Complete 3-D

structural analysis and substrate specificity allowed the

enzyme to be classified in a new subfamily of glycoside

hydrolases in family 5.

The structure of T. aurantiacus endoglucanase was deter-

mined by multiple isomorphous replacements, which showed

a catalytic module with compact eight-fold b/a barrel architec-

ture (Leggio and Larsen, 2002). In comparison with family 5,

two Gly, one catalytic Glu and six other residues were

conserved. The catalytic residues were identified as Glu 133

(acid-base) and Glu 240 (nucleophile) (Leggio and Larsen,

2002). This enzyme had been classified in subtype A6 of the

endoglucanase family in previous work done by Leggio et al.

(1997).

Constitutive levels of endoglucanase (1.6–4.0 U/mL) were

detected in the culture filtrates of T. aurantiacus A-131 (Kawa-

mori et al., 1987). However, enzyme activity was induced by

amorphous polysaccharides with b-1,4 linkages, such as

bagasse treated in alkali medium, reaching 70 U/mL (Kawa-

mori et al., 1987). Kalogeris et al. (2003) reported high levels

of endoglucanase activity for T. aurantiacus IMI 216529 on

solid-state fermentation using lignocellulosic materials,

specially on wheat straw supplemented with (NH4)2SO4 (1 %

w/v), (NH4)2HPO4 and corn steep liquor.

Exo-1,4-b-D-glucanaseExoglucanases have a strong preference for acting at chain

ends, thus degrade the molecule by removing cellobiose units

from the free chain. The molecular weight of exoglucanases

from T. aurantiacus have been reported to be in the range of

32–58 kDa (Khandke et al., 1989a; Maheshwari et al., 2000;

Tong et al., 1980), optimum pH between 4.1 and 5.0, optimum

temperature at about 65 �C, and stable for 8 h at 50 �C

(Khandke et al., 1989a; Tong et al., 1980).

In general, exoglucanase has been reported to be produced

in lower levels by T. aurantiacus, in comparison with other

enzymes of the cellulolytic complex (Hong et al., 2003a;

Khandke et al., 1989a), a common pattern in cellulase-

producing microorganism. However, high exoglucanase

activity (758 U/mL) was detected in cultures of T. aurantiacus

179-5 when cultivated in semi-solid media containing sugar-

cane/orange bagasse (Martins et al., 2003).

An exoglucanase gene named cbh1 was isolated from

T. aurantiacus IFO 9748. The amino acid sequence was shown

to be very similar to the sequence of family 7 of glycoside

hydrolase. This gene was expressed in Saccharomyces cerevi-

siae, a fungus that does not produce cellulases. The recombi-

nant enzyme was produced by S. cerevisiae and characterized

as exoglucanase. The protein structure was shown to be

highly glycosylated, and stable after incubation at 65 �C for

1 h, and in the pH range 3–9 (80 % of the initial activity)

(Hong et al., 2003b). In a similar way, Benko et al. (2007) cloned

Hydrolases of Thermoascus aurantiacus 123

an exoglucanase from T. aurantiacus and expressed it in T. ree-

sei Rut-C30 using the cbhl promoter.

Applications of thermostable cellulasesThe significant industrial importance of cellulases was prob-

ably peaked during the 1990’s, mainly within the textile, deter-

gent and cellulosic pulp industries. In the current industrial

processes, cellulolytic enzymes are employed in: (i) clarifica-

tion of juices and wines; (ii) detergents causing color bright-

ening and softening; (iii) pretreatment of biomass to

improve nutritional quality of forage; and (iv) pretreatment

of industrial wastes (Bhat, 2000; Haki and Rakshit, 2003).

For instance, the biopolishing process of cotton in the

textile industry requires cellulases stable at high tempera-

ture (Ando et al., 2002). During this process, the cellulases

act on small fiber ends that protrude from the fabric surface,

where the mechanical action removes these fibers and

polishes the fabrics (Sreenath et al., 1996). Cellulases can

improve drainage of recycled pulps by hydrolyzing the amor-

phous hydrophilic cellulose, which is the main constituent of

the fines formed during refining (Oksanen et al., 2000). Endo-

glucanase treatment can also decrease viscosity and increase

the alkaline solubility of dissolving pulp (Rahkamo et al.,

1998).

Nowadays, the conversion of lignocellulosic into ferment-

able sugar is the main focus for biofuel production. In this

matter, the lignocellulosic conversion has clear advantages

when thermostable cellulases are employed, as well as low

activity losses occur during processing, even at elevated

temperature often used in raw material pre-treatments.

Some studies have shown that for high conversion efficiency,

a long reaction time is required (Sassner et al., 2008; Zhu et al.,

2008; Borjesson et al., 2007; Ohgren et al., 2007), so, considering

that most studies were performed near 50 �C, the use of ther-

mostable enzymes, such as T. aurantiacus cellulases would

guarantee that enzymes would still remain stable until the

end of the process, improving significantly the hydrolysis

process. In general, thermostable enzymes have enjoyed

much attention because of their advantages in usage under

industrial conditions since mass transfer is facilitated at

higher temperatures due to decreased fluid viscosity; contam-

ination risks are reduced and enzymes can be purified and

stored at room temperature without inactivation (Krahe

et al., 1996). However, due to low specific growth, rates large

scale commercial cultivation of thermophiles for enzyme

production remains an economical challenge (Santos et al.

2003).

Hemicellulases

Hemicelluloses are the second most abundant polysaccha-

rides present in the cell walls of herbaceous and woody plants.

Xylans of many plant materials are heteropolysaccharides

with homopolymeric backbone chains of 1,4-linked b-D-xylo-

pyranose units. Besides xilose, xylans may contain arabinose,

glucuronic acid or its 4-0-methyl ether, and acetic, ferulic, and

p-coumaric acids. The frequency and composition of

branches are dependent on the source of xylanare the most

abundant hemicellulose-type polysaccharides constituent in

plants (Ward and Moo-Young, 1989).

Synergistic effects among hemicellulases have been docu-

mented in many works (Coughlan and Hazlewood, 1993;

Biely et al., 1986; Tenkanen et al., 1992). The key factors

that influence xylan hydrolysis are chain length and degree

of substitution (Li et al., 2000). The main enzymes involved

in hemicellulose degradation are endo-b-1,4-xylanase (EC

3.2.1.8) and b-xylosidase (EC 3.2.1.37) (acting in the main

chain), and enzymes that act on the substituted groups,

called accessory enzymes, such as ferulic acid esterase (EC

3.1.1.73), p-coumaric acid esterase (EC 3.1.1.-), acetylxylan

esterase (EC 3.1.1.6), a-arabinofuranosidase (EC 3.2.1.55) and

a-glucuronidase (EC 3,2,1,131) (Beg et al., 2001; Polizeli et al.,

2005).

T. aurantiacus can produce most of the hemicellulolytic

enzymes, however, endoxylanase is the main enzyme

detected in its culture. Many well known hemicellulase-

producing microorganisms, such as A. niger (Kang et al.,

2004; Yuan et al., 2005) and T. reesei (Tenkanen et al., 1992)

possess complete xylan-degrading enzyme systems. Some

characteristics of T. aurantiacus hemicellulases are described

in the following sections, and additional detailed properties

of hemicellulases from various strains of T. aurantiacus are

presented in Table 3.

Endo-b-1,4-xylanaseEndo-b-1,4-xylanases cleave the internal xylan chain and

release short xylooligosaccharides (Shallom and Shoham,

2003). T. aurantiacus endoxylanases are in the range of

33–39.7 kDa (Kalogeris et al., 2001; Viswamitra et al., 1993),

and have been shown to be highly active and thermo-

stable. T. aurantiacus C 436 produces endoxylanases that

are stable at 50 �C for 12 weeks, retaining 90 % of the initial

activity (Yu et al., 1987). Alam et al. (1994) determined that

the strain BJT 190 retains 80 % of initial activity after incu-

bation for 15 d at 55 �C.

Xylanases from different strains of T. aurantiacus have been

shown to be optimally active at about 70–75 �C and around pH

5 (Alam et al., 1994; Gomes et al., 2000; Yu et al., 1987). More-

over, Da Silva et al. (2005) reported that xylanase from T. auran-

tiacus CBMAI-756 isolated in Brazil was stable in a wide range

of pH (3–9) at room temperature.

Only two isozymes active on xylan, one with a pI 6.8 and

the other with a pI of 5.5, have been reported to be produced

by T. aurantiacus IMI 216529 (Kalogeris et al., 2001). This may

imply that in general T. aurantiacus does not produce many

xylanases isozymes, thus differ from other filamentous fungi

that have demonstrated a great capability for secreting a wide

range of xylanases.

Solid-state fermentation of T. aurantiacus has been shown

to produce maximum xylanase activities in a short period of

time (Kalogeris et al., 1998; Martins et al., 2007; Santos et al.,

2003; Souza et al., 1999). T. aurantiacus ATCC 204492 cultivated

on sugarcane bagasse displayed xylanase production depen-

dent on the aeration (Santos et al., 2003). Other variables

such as nitrogen and carbon sources have also been reported

to affect xylanase production by T. aurantiacus, as observed for

T. aurantiacus Miehe 216529 (Kalogeris et al., 1998). For T. auran-

tiacus ATCC 204492, the use of sugarcane bagasse with 80 %

initial moisture content was proven ideal for the xylanase

activity, whereas inoculum level and cultivation time were

Table 3 – Characteristics of hemicellulases produced by different T. aurantiacus strains

Enzymes Strain Activity Stability Optimumtemperature

(�C)/pH

Optimum medium References

Endoxylanase N/M 45.5 UI/g Wheat bran Stoilova et al., 2005

Miehe 107 U/mL 60 �C/1 h 75/5 Corncob – green grass Da Silva et al., 2005

ATCC 204492 1597 UI/g SCBa Milagres et al., 2004

N/M 500 U/g SCBaþ rice bran Santos et al., 2003

IMI 216529 6193 U/g t1/241 min/80 �C 70/4.5 Wheat straw Kalogeris et al., 1998

N/M 705.7 U/g 55 �C/15 d 70/5 Wheat bran Alam et al., 1994

C 436 575.9 U/mL 50 �C/84 d 75/5 Oat spelt xylan Yu et al., 1987

179-5 1288.8 U/g Sugarcane-orange

bagasse

Martins et al., 2003

b-xylosidase BT 2079 5 nkat/mL 75/5 Solka floc Gomes et al., 2000

ATCC 204492 27 UI/g SCBa Milagres et al., 2004

BT 2079 3479 nkat/mL Solka floc Gomes et al., 2000

C 436 1.63 U/mL t1/236 min/70 �C 75/5 Oat spelt xylan Yu et al., 1987

b-mannanase BT 2079 Low level Solka floc Gomes et al., 2000

b-manosidase N/M 10 nkat/mL 50 �C/5 h 76/2.5–3 Bean gum Gomes et al., 2007

b-galactosidase BT 2079 Low level Solka floc Gomes et al., 2000

a-galactosidase BT 2079 Low level Solka floc Gomes et al., 2000

a-arabinofuranosidase N/M 1083.5 nkat/g 50 �C/48 h 70/4 Sugar beet Roche et al., 1994

a-glucuronidase N/M 2.2 U/mg protein t1/240 min/70 �C 65/4.5 Khandke et al., 1989b

N/M – not mentioned.

a SCB – Sugarcane baggase.

124 M. Brienzo et al.

of lower significant effect on the production of enzyme

activity (Souza et al., 1999).

Commonly, endoxylanases are classified by substrate

specificity in family 10 or 11 based on hydrophobic cluster

analysis and amino acid sequence homologies. Enzymes

from family 10, in contrast to endoxylanase of family 11,

exhibit low substrate specificity and is capable of attacking

the xylosidic linkage next to the branch and towards the

non-reducing end. Kalogeris et al. (2001) concluded based on

the catalytic properties that the major endoxylanase purified

of T. aurantiacus belongs to glycosyl hydrolases of family 10.

Vardakou et al. (2003) revealed that xylanase from T. aurantia-

cus hydrolyzed two times more efficiently insoluble water-

unextractable arabinoxylans than the family 11 enzyme

showing better capability of cleaving glycosidic linkages in

the xylan main chain closer to the substituents. In spite of

higher molecular mass, xylanses of family 10 have smaller

substrate binding sites than xilanases of family 11 (Biely

et al., 1997).

b-xylosidaseThere are only a few reports dealing with b-xylosidase from

T. aurantiacus. It appears that b-xylosidase is produced at

lower levels by this fungus compared to some other hemicel-

lulolytic enzymes, such as endoxylanases. A study on the

ability of fourteen substrates, such as beech and birch wood

xylan and methyl b-D-xylopyranoside, for the production of

hemicellulases by T. aurantiacus indicated that only low levels

of constitutive b-xylosidase were produced by T. aurantiacus,

even in the presence of cited inducers (Gomes et al., 1994).

On the other hand, in the presence of wheat straw, a b-xylosi-

dase with an acidic pI (3.5) was induced (40 UI/g) in cultures of

the T. aurantiacus IMI 216529 (Kalogeris et al., 1998; Kalogeris

et al., 1999).

a-L-arabinofuranosidasea-L-arabinofuranosidases hydrolyze non-reducing arabinofur-

anose residues in short oligosaccharides and branched

arabinoxylans substituted at positions 2 and 3 on b-D-xylopyr-

anosyl (De Vries et al., 2000).

Low levels of exo-a-L-arabinofuranosidase were detected in

cultures of T. aurantiacus from solid-state fermentation of

leached sugar beet pulp based medium (Gomes et al., 2000).

Moreover, Roche et al. (1994) reported that the optimal

medium conditions for maximum arabinofuranosidase

production were sugar beet pulp at 77.8 % humidity supple-

mented with mineral solution and yeast extract (1.2 %) at pH

4.5. This arabinofuranosidase showed optimum temperature

and pH at 70 �C and 4 �C, respectively, and was stable for

48 h at 50 �C.

a-D-glucuronidasea-D-glucuronidases cleave the a-1,2-glycosidic bond of the

4-O-methyl-D-glucuronic acid side chain of xylans (Puls et al.,

1987).

A purified a-D-glucuronidase from T. aurantiacus with a single

polypeptide chain and molecular weight of 118 kDa (based on

SDS-PAGE), cleaved the a,1-2 linkage between 4-O-methyl-a-D-

glucuronic acid and the xylose residue in xylan and several glu-

curono-xylooligosaccharides. The enzyme was stable at 70 �C

with a half live of 40 min, optimum temperature and pH at

65 �C and 4.5 �C, respectively (Khandke et al., 1989b).

b-1,4-MannanaseMannan is composed of b-1,4-linked mannopyranose and glu-

copryranose units, which in softwood is present as acetylated

galactoglucomanans. b-1,4-mannanase (EC 3.2.1.78) cleaves

randomly within the 1,4-b-mannan (McCleary and Mathen-

son, 1986).

Hydrolases of Thermoascus aurantiacus 125

Araujo and Ward (1990) investigated the effect of carbon

and nitrogen sources on mannanase production by eight ther-

mophilic fungi. Mannose has displayed the greatest inducer

effect on mannose production by T. aurantiacus, and sodium

nitrate was the best nitrogen source. Gomes et al. (2007) eval-

uated different carbon and nitrogen sources and found that

the best sources for mannanase production (10 nktal/mL) by

T. aurantiacus were locust bean gum and soymeal. According

to Gomes and Steiner (2000) the enzyme was much more ther-

mostable than other mannanases from some other strains of

T. aurantiacus. In another work Gomes et al., 2000 also reported

simultaneous production of mannanase under optimized

conditions for xylanase and endoglucanase production by

T. aurantiacus BT 2079. Mannanase production was about 232

and 75-fold lower than xylanase and endoglucanase, respec-

tively, and similar to the level of a-galactosidase.

b-mannosidaseb-mannosidase (EC 3.2.1.25) catalyzes the hydrolysis of

mannose units from the non-reducing end of mannosides.

b-mannosidase is commonly produced at low levels by

various microorganisms and a similar pattern has also been

described for T. aurantiacus BT 2079 (Guy et al., 1997). Gomes

et al., 2007 detected a b-mannosidase in cultures of T. aurantia-

cus of 99.9 kDa (UREA-PAGE electrophoresis), pI 4.8, and stable

at 50 �C for 5 h. Among different inducers tested locust bean

gum (15 g/L), induced the highest b-mannosidase activity. In

addition, the enzyme also displayed transglycosylation

activity by transferring the mannosyl group from p-nitro-

phenyl-b-D-mannopyranoside to methyl-a-D-mannopyrano-

side for the synthesis of disaccharide.

Ortner et al. (2006) studied the characteristics of regioselec-

tivity and acceptor tolerance in transglycosylation reaction by

culture filtrates from T. aurantiacus, and observed that the

structure of the acceptor caused difference in the anomeric

configuration bond when b-mannopyranosyl fluoride was

employed as the donor.

Applications of thermostable hemicellulasesThe pool of hemicellulases, especially endoxylanases, b-

xylosidases, and the accessory enzymes (b-fructofuranosi-

dase, a-L-arabinosidase, b-mannosidase, etc) can be used

for the saccharification of the hemicellulosic fraction of

lignocellulosic materials for sugar production, which can

then be converted to chemicals and fuels in a similar

manner to that of cellulases in the conversion of cellulosic

materials. For instance, Cardona and Sanchez (2007) appli-

cation have shown that ethanol can be produced from

xylose in a simultaneous process, in which xylose is initially

isomerized and then fermented.

During the production of cellulosic pulp, lignin is removed

by a multistep bleaching process, in which the addition of

xylanases has proved to be an interesting and efficient alter-

native to improve pulp bleaching, besides collaborating with

environment preservation (Wong and Saddler, 1992, 1993; Vii-

kari et al., 1993). For example, in kraft processes, the wood

chips are cooked at a high temperature and alkaline pH. These

processes cause xylan dissolution which precipitates on the

surface of cellulose microfibrils, acting like a barrier to the

chemical removal of lignin. Alternatively, xylanase can

efficiently remove xylan chains exposing lignin to the action

of bleaching reagents. For this purpose, the most appropriate

xylanases would be thermostable and alkaline active xyla-

nases (Battan et al., 2007; Jacques et al., 2000). In this viewpoint,

T. aurantiacus xylanases appears to fulfill these requirements,

and so, would seem to have great potential for biobleaching of

cellulosic pulps.

Besides xylanases, hemicellulases are comprised of

several other enzymes whose potential have not been fully

explored. Arabinofuranosidase can be used as auxiliary in

the conversion of hemicellulose substrate to ferment

sugars. In the some way, it can be used in improvement

of animal feedstock digestibility, hydrolysis of grape mono-

terpenyl a-arabinofuranosylglucosides to increase aroma

during wine making, and clarification and thinning of juices

(Saha, 2000).

Amylases

Plants as cereal grains (e.g. wheat, rice, corn, sorghums, oats,

barley) store glucose as the polysaccharide starch. Amylases

can be divided into two classes, endoamylases (EC 3.2.1.1)

and exoamylases (EC 3.2.1.3). Enzymes belonging to the

second group, the exoamylases, either exclusively cleave

a,1-4-glycosidic bonds such as b-amylase (E.C.3.2.1.2) or cleave

a,1-6-glycosidic bonds like amyloglucosidase or glucoamylase

(EC 3.2.1.3) and a-glucosidase (EC 3.2.1.20) (Fogarty and kelly,

1990).

Only limited information is found in literature about

amylases of T. aurantiacus. It was reported that small quanti-

ties of amylase was produced in extracellular growth media

(Adams, 1992). However, some characteristics of T. aurantiacus

amylases are described below and some others are given in

Table 4.

Endo amylase and ExoamylaseEndo amylase (or a-amylase) hydrolyzes a-1,4 bond randomly

in amylose and amylopectin releasing maltooligosaccharides,

while exoamylases hydrolyzes the penultimate a-1,4 bond

starting from non-reducing end releasing maltose. (Guzman-

Maldonado and Peredes-lopez, 1995).

Ohno et al. (1998) used starch as carbon source for amylase

production by T. aurantiacus IFO 31693 and detected the

production of both exo (75 kDa) and endoamylase (56 kDa).

The authors observed different stability behavior for both

enzymes. The endo was more stable (60 �C/3 h) and showed

better activity at higher temperature (70 �C) than the exoamy-

lase (65 �C).

A mixed culture of T. aurantiacus and A. niger on oat straw

semi-solid fermentation resulted in higher levels of a-amylase

activity if compared to the activity detected in monoculture

(Stoilova et al., 2005).

a-glucosidasea-glucosidases are exoenzymes that hydrolyze the glucosidic

bond a-1,4 and/or a-1,6 in short oligosaccharides. They are

divided into three types: type I hydrolyzes heterogeneous

substrates (e.g. sucrose); type II hydrolyzes mainly maltose

and isomaltose and shows low activity on heterogeneous

substrates; and type III has the same specificity as type II,

Table 4 – Properties of pectinases and amylases produced by T. aurantiacus

Enzyme Strain Activity/Stability Temp opt (�C) pH opt Optimum medium References

Pectin Ko et al., 2005b

Polygalacturonase 179-5 43 U/g. Stable in the acidic

to neutral pH range and at

60 �C for 1 h

65 10.5–11.0 Wheat bran or orange

bagasse

Martins et al., 2002

Pectin lyase 179-5 40180 U/g. Stable at acidic

pH and at 60 �C for 5 h

65 5.0 sugarcane bagasseþorange bagasse

Martins et al., 2002

Pectin esterase IFO 31693 0.156 U/mL Pectin Ko et al., 2005b

Endoamylase IFO 31693 60 �C for 3 h 70 4.5 Starch Ohno et al., 1998

Exoamylase IFO 31693 65 5.0 Starch Ohno et al., 1998

126 M. Brienzo et al.

but also hydrolyzes polysaccharides such as amylose (Frand-

sen and Svensson, 1998).

Carvalho et al. (2006) detected a-glucosidase in cultures of

T. aurantiacus 179-5 grown in solid-state fermentation using

wheat bran as substrate. The enzyme showed specific activity

for oligomers of maltose, however starch, dextrins (2–7 G) and

a-PNPG was very slowly hydrolyzed, suggesting that the

enzyme is a a-glucosidase type II. It also showed to be very

thermostable, with half-life of 15 min at 80 �C, and stable in

a wide range of pH (3–10).

Applications of thermostable amylasesProcessed starch is mainly used for glucose, maltose, and

oligosaccharide production, but a number of other products/

intermediates can also be produced via cyclodextrins (Turner

et al., 2007).

Starch processing is usually performed in a two-step

hydrolysis process of liquefaction and saccharification.

During liquefaction starch is gelatinized by thermal treatment

requiring high temperature. A thermostable a-amylase is

added before the heat treatment. Consequently, a highly ther-

mostable enzyme is required to be active during the whole

procedure. T. aurantiacus has an extracellular a-amylase

enzyme that shows promising characteristics for applications

in the starch industry due to its high thermostability in the

absence of metal ions and is active even at pH 4.0 (Ohno

et al., 1998). The saccharification of the remaining oligosaccha-

rides to produce a hydrolyzate syrup with more than 95 %

glucose is done by using exo-acting glucoamylase and pullula-

nase that efficiently hydrolyzes a,1-6-glycosidic bonds (Maarel

et al., 2002).

Pectinases

Pectins are the third main structural polysaccharide group of

plant cell walls. It is abundant in sugar beet pulp and fruit,

e.g. in citrus fruit and apple, where it can form up to half of

the polymeric content of the cell wall (Brummell, 2006). It is

composed mainly of linearly connected b-1,4-D-galacturonic

acid units and their methyl esters, interrupted in places by

1,2-linked L-rhamnose units (Willats et al., 2001).

For complete hydrolytic degradation of this component,

a set of enzymes, including endo-polygalacturonases (EC

3.2.1.15), exo-polygalacturonases (EC 3.2.1.67), lyases (EC

4.2.2.10) and pectin esterases (EC 3.1.1.11) (Gummadi and

Panda, 2003) are required.

The characteristics of some of the pectinases produced by

T. aurantiacus are described below and some others are shown

in Table 4.

PolygalacturonasesPolygalacturonases (PGases) catalyze the hydrolytic cleavage

of the polygalacturonic acid chain through introduction of

water across the oxygen bridge. They are the most extensively

studied enzyme among the family of pectinolytic enzymes.

The PGases involved in the hydrolysis of pectinaceous

substances are endo-PGase and exo-PGase. Endo-PGases are

widely distributed among fungi, bacteria and many yeasts,

whereas exo-PGases occur less frequently (Jayani et al., 2005).

It is generally agreed that the optimum medium for the

enhanced production of extracellular pectinase is that con-

taining pectic materials as an inducer (Martins et al., 2002).

T. aurantiacus 179-5 grown in solid-state fermentation

using wheat bran or orange bagasse produced the maximum

activity (43 U/g), whereas a decrease of PGase production

was observed when mixed to sugarcane bagasse. Exo-PGase

had optimum activity at pH 5.0, and was stable at pH values

of 7.0–8.0 and maintained 85 % of its activity at pH 4.0–6.5.

The optimum temperature was 65 �C, and at 70 �C, 88 % of

maximum activity was retained. According to the authors,

exo-PGase production by T. aurantiacus 179-5 was high

compared to those reported for pectinolytic microorganisms,

such as A. niger, A. foedidus, Penicillum italicum and Penicillium

frequentans (Martins et al., 2002).

Among forty thermophilic fungi screened for the produc-

tion of polygalacturonases, most of them did not show detect-

able activity, however, one isolate of T. aurantiacus produced

pectinases in medium containing 5 % (w/v) pectin, which

was markedly enhanced when citrus peel was used instead

of purified pectin. Polygalacturonase from T. aurantiacus was

one of the most stable, showing achieve at 65 �C with a half-

life of 20 min at 55 �C (Maheshwari et al., 2000).

Recently, an unusual polygalacturonase was isolated from

5-day culture filtrates of T. aurantiacus CBMAI-756 and purified

by gel filtration and ion-exchange chromatography (Martins

et al., 2007). Only one form of PG (30 kDa) was detected in

culture medium, which exhibited both endo and exo-PG activ-

ities. The enzyme was maximally active at pH 5.5 and 60 �C–

65 �C. The enzyme was 100 % stable at 50 �C for 1 h and

showed a half-life of 10 min at 60 �C. PG was also shown to

be stable at pH 5.0–5.5 and maintained 33 % of initial activity

at pH 9.0. Metal ions, such as Znþ2, Mnþ2, and Hgþ2, inhibited

Hydrolases of Thermoascus aurantiacus 127

50, 75 and 100 % of enzyme activity, respectively. According to

the authors, the purified enzyme showed to be less thermo-

stable than unpurified enzyme, suggesting the fungi may

produce some thermostabilizer factor.

Pectin lyasesPectin lyases (PL) produce a trans-eliminative split of the

pectic polymer, breaking the glycosidic linkages at C-4 and

simultaneously eliminating H from C-5, producing a D 4:5

unsaturated products (Jayani et al., 2005).

Martins et al. (2002) reported the growth of T. aurantiacus

179-5 and PL production in solid-state fermentation using

wheat bran, orange bagasse and mixtures of these materials

with sugarcane bagasse, and found that the medium con-

taining pectin, in their case orange bagasse, afforded higher

PL production than those with wheat bran. However, the

highest yields of PL activity were obtained by the addition

of sugarcane bagasse to orange bagasse. Maximal PL

activity (40.18 U/g) was obtained in a medium composed of

10 % (w/v) sugarcane and 90 % (w/v) orange bagasse. PL

exhibited maximal activity at pH 10.5–11.0 and 65 �C, retain-

ing 88 % of maximum activity at 70 �C. The authors also

observed that PL production by T. aurantiacus 179-5 was

high compared to other pectinolytic microorganisms, such

as A. niger, A. foedidus, P. italicum and P. frequentans (Martins

et al., 2002).

Applications of thermostable pectinasesPectinases have been applied in fruit juice clarification, juice

extraction, manufacture of pectin free starch, refinement of

vegetable fibers, degumming of natural fibers, waste-water

treatment, curing of coffee, cocoa and tobacco and as an

analytical tool in the assessment of plant products (Turner

et al., 2007). High temperatures are commonly employed in

such processes for different proposes. In most of these

processes the material submitted to heating needs to be

cooled down before enzymatic treatment. Thus, the use of

thermostable pectinases, such as the pectinases produced

by T. aurantiacus, could eliminate the cooling down step,

decreasing the final process costs. In other applications, it

can be more convenient to use thermostable enzymes, partic-

ularly when using substrates (which can also be other natu-

rally occurring glycoside-containing molecules with similar

linkages as in pectin) that are poorly soluble at ambient

temperatures, such as naringin and rutin, present in fruits

(Birgisson et al., 2004).

2. Concluding remarks

T. aurantiacus produces a large pool of enzymes of industrial

interest, i.e. amylases, cellulases, xylanases, etc., which have

been shown to be remarkably stable over a wide range of

temperature and pH. Many studies with T. aurantiacus have

been carried out on lignocellulosic materials, which were

shown to induce or increase production of several enzymes.

The mechanism involved is poorly understood, although it is

well established that the species secrete different types of

hydrolytic enzymes. The annotated gene set of T. aurantiacus,

transcript patterns, and comparative analyses with other

thermophilic fungi will advance our understanding of these

complex mechanisms involved in lignocellulose conversions.

Enzymes of T. aurantiacus are potentially applicable in

a wide range of industrial process mainly due to their extraor-

dinary operational stability at high temperatures and dena-

turant tolerance. Although such enzymes are described for

their ability in the chemical, food, pharmaceutical, paper,

textile and other industries, they are however, still today,

often limited for applications in small scale and testing of

such applications are under investigations (Gomes et al.,

2000; Milagres et al., 2003).

Recently, increasing attention has been devoted to the

utilization of biomass, especially lignocellulosic materials in

various industrial fields, as a possible alternative to fossil

resources because of their abundance, low cost and renew-

ability. The use of T. aurantiacus in biorefinery and for enzyme

production, especially lignocellulolytic enzymes, have gained

a great deal of interest as T. aurantiacus has good growth adap-

tation on lignocellulosic materials such as those originated

from agro-industrial and forest waste, and its enzymes ther-

mostability has tremendous advantages in biorefinery when

compared to the temperatures and pressures required for

chemicals conversion.

The study of T. aurantiacus has added greatly to our under-

standing of thermostable enzyme production and biochem-

ical features. It can be seen as a fungus of great importance

to bio-industrial processes due to its high thermal stability

and its enzymatic systems. However, much more work is

required to fully explore its biotechnological potential, espe-

cially in regard to the molecular and genetic fields.

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