C H A P T E R
24
Trichoderma Enzymes for Food Industries
Adinarayana Kunamneni*, Francisco J. Plou, Miguel Alcalde,
Antonio Ballesteros
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain
*Corresponding author email: adikunamneni@rediffmail.com
O U T L I N E
Introduction339
Fungus of Industrial Interest
Trichoderma Enzymes for Industries
Xylanases341
Cellulases341
Other Enzymes
INTRODUCTION
The application of microorganisms, such as bacteria,
yeasts and principally fungi, by the food industry has led
to a highly diversified food industry with relevant eco-
nomical assets. Fermentation, with special reference to
the production of alcoholic beverages, bioethanol, dairy
products, organic acids and drugs which also comprise
antibiotics, is the most important example of microbio-
logical processes.
The enzyme industry, as it is currently known, is the
result of a rapid development of biotechnology, ­especially
during the past four decades (Alcalde et al., 2006). Since
ancient times, enzymes found in nature have been used
in the production of food products such as cheese, beer,
wine and vinegar (Kirk et al., 2002).
Enzymes which decompose complex molecules into
smaller units, such as carbohydrates into sugars, are nat-
ural substances involved in all biochemical p ­ rocesses.
Due to the enzymes' specificities, each substrate has a
corresponding enzyme. Although plants, fungi, bacteria
Biotechnology and Biology of Trichoderma
http://dx.doi.org/10.1016/B978-0-444-59576-8.00024-2
β-Mannanase342
α-L-Arabinofuranosidase342
340
Food Industry 342
340
Perspectives for Biotechnological Production
of Enzymes by Trichoderma 343
342
and yeasts produce most enzymes, microbial sources are
more advantageous than their equivalents from animal
or vegetable sources. The advantages assets comprise
lower production costs, possibility of large-scale produc-
tion in industrial fermentors, wide range of physical and
chemical characteristics, possibility of genetic manipu-
lation, absence of effects brought about by seasonality,
rapid culture development and the use of nonburden-
some methods. The above characteristics make micro-
bial enzymes suitable biocatalysts for various industrial
applications (Hasan et al., 2006).
Therefore, the identification and the dissemination
of new microbial sources, mainly those which are non-
toxic to humans, are of high strategic interest. Besides
guaranteeing enzyme supply to different industrial
processes, the development of new enzymatic systems
which cannot be obtained from plants or animals is
made possible and important progress in the food
industry may be achieved. In this book chapter, we
have described the properties, production, and appli-
cations of Trichoderma enzymes in the food industry.
339
Copyright © 2014 Elsevier B.V. All rights reserved.
340 24. TRICHODERMA ENZYMES FOR FOOD INDUSTRIES

FUNGUS OF INDUSTRIAL INTEREST
Owing to progress in the knowledge of enzymes,
fungi acquired great importance in several industries
since they may improve various aspects of the final
product.
In fact, the fungi kingdom has approximately 20
known species of Trichoderma which produce enzymes.
They are isolated from soil, decomposing plants and air.
Trichoderma actually produces a great number of extra-
cellular enzymes, many of which are applied in biotech-
nology. Trichoderma reesei, Trichoderma viride, Trichoderma
atroviride, Trichoderma virens, Trichoderma harzianum,
Trichoderma lignorum, and Trichoderma longibrachiatum are
the best known.
The remarkable interest in T. reesei, a species of great
commercial interest with a highly promising future and
already widely applied in modern biotechnology, is due
to its several and diverse reactions (Lorito et al., 2010).
Moreover, T. reesei not only produces various enzymes
but it is one of the few species of the fungus kingdom
classified as GRAS (Generally Recognized as Safe) by
the Food and Drug Administration (FDA). The species
is used in the production of enzymes, its cell mass is
used as a component in animal feed and its fermenta-
tion ­produces organic acids and other compounds of
high economic value (Blumenthal, 2004; Schuster and
Schmoll, 2010).
TRICHODERMA ENZYMES FOR
INDUSTRIES
Trichoderma reesei produces a number of extracellar
enzymes (Tables 24.1–24.3). It produces at least four
endo-1,4-β-xylanases (XYN I, II, III and IV, EC 3.2.1.8,
Table 24.1), two β-xylosidases (EC 3.2.1.37), two endo-1,
4-β-D-glucan cellobiohydrolases (CBH I and II, EC

TABLE 24.1 Biochemical Properties of Purified T. reesei Xylanases

Xylanase MW* (kDa) pI Optimum pH Stability Specific Activity§ (IU/mg) pH Stability References

XYN I 19 5.5 4.0–4.5 24 h, 40 °C 70 2.5–4.5 Tenkanen et al. (1992),

Torronen et al. (1992)

XYN II 20 9.0 5.0–5.5 24 h, 45 °C 231 4.0–7.5 Tenkanen et al. (1992),

Torronen et al. (1992)

XYN III 32 9.1 6.0–6.5 24 h, 50 °C 258 5.0–8.0 Xu et al. (1998)

XYN IV 43 7.0 3.5–4.0 – – – Clarkson et al. (2001)

* MW, molecular weight in SDS-PAGE.

§ Specific activity was measured at 50 °C, pH 6.0, in 1% birch wood xylan solution by Xu et al. (1998). The specific activity of XYN I and II was reported to be 120 IU/mg and

810 IU/mg, respectively, at 60 °C by Lappalainen et al. (2000).

TABLE 24.2 Physical Properties of T. reesei Cellulases

Number of
Residues

Total Core Molecular Weight* (kDa) Isoelectric Point (pI) Position of the CBD Reference

CBH I (cel 7A) 497 430 59–68 3.5–4.2 C Reinikainen (1994)

CBH II (cel 6A) 447 367 50–58 5.1–6.3 N Reinikainen (1994)

EG I (cel 7B) 437 368 50–55 4.0–6.0 C Reinikainen (1994)

EG II (cel 5A) 397 327 48 5.5 N Reinikainen (1994)

EG III (cel 12A) 218 218 25 7.5 No Reinikainen (1994)

EG IV (cel 61A) 344 344 55 – C Karlsson et al. (2001)

EG V (cel 45A) 225 166 23 – C Reinikainen (1994)

BGL I (cel 3A) 713 713 75 8.7 No Reinikainen (1994)

BGL II (cel 1A) 700 700 114 4.8 – Foreman et al. (2003);
Viikari et al. (2001)

Abbreviations: CBH, cellobiohydrolase; EG, endoglucanase; BGL, β-D-glucosidases; CBD, cellulose binding domain; C, C-terminal; N, N-terminal.
* SDS-PAGE results.

F. INDUSTRIAL APPLICATIONS
 Cellulases
TABLE 24.3 Selected T. reesei Enzymes with Industrial Potential
Enzyme Function
β-Mannanase Degradation of mannan
α-L-Arabinofuranosidase Cleavage of side groups in xylan
α-Galactosidase Cleavage of side groups in xylan
Pectin methyl esterases De-esterification and gelling of pectins
Acetylxylan esterases Hydrolysis of acetyl side groups of xylan
Laccases Oxidation of wide variety of compounds
3.2.1.91), five endo-1,4-β-D-glucan-4-glucanohydrolases
(EG I, II, III, IV and V, EC 3.2.1.4, Table 24.1) and two β-D-
glucosidases (BGL I and II, EC 3.2.1.21) (Bailey et al.,
1993; Karlsson et al., 2001; Nogawa et al., 2001; Xu et al.,
1998; Zeilinger et al., 1996). Several other enzymes are
also produced by T. reesei: β-mannanase (EC 3.2.1.78),
β-mannosidase (EC 3.2.1.25), α-L-arabinofuranosidase
(EC 3.2.1.55), α-galactosidase (EC 3.2.1.22), acetylxylan
esterases (EC 3.1.1.72) and laccases (benzenediol:oxygen
oxidoreductases, EC 1.10.3.2) (Karlsson et al., 2001;
Roche et al., 1995; Shabalin et al., 2002).
XYLANASES
The use of xylanases for the production of xylooli-
gosaccharides is an interesting application of these
enzymes in the food industry. Many bacterial and fungal
species can produce a mixture of xylanase, β-xylosidase
and accessory side-group cleaving enzymes in order
to utilize xylan, a complex polymer which is the
major component of hemicellulose in the plant cell
wall. Xylan found in nature consists of a β-1,4-linked
xylopyranose backbone substituted with acetyl, arabi-
nosyl and glucuronosyl side chains (Kar et al., 2013).
Enzymatic hydrolysis of xylan to xylose is catalyzed
by endo-1,4-β-xylanase and β-xylosidase, the former
randomly hydrolyzing xylan to xylooligomers and the
latter producing xylose from xylooligomers. The side
chain groups are liberated by α-L-arabinofuranosidase,
α-D-glucuronidase, α-galactosidase and acetyl xylan
esterase (Subramaniyan and Prema, 2002). β-Xylosidase
shows high activity toward xylobiose but no activity
toward xylan (Bajpai, 1997). However, some xylanases
may also have an ability to hydrolyze xylooligomers
to xylose, especially in the cross-linked enzyme crystal
form (Finell et al., 2002).
The xylanase activity of T. reesei is composed of xyl-
anases I, II, III and IV, and xylan-digesting cellulases.
Xylanases I and II (pI 5.5 and 9, respectively) are approx-
imately 20 kDa proteins belonging to the family 11 of
glycosyl hydrolases (Lopez et al., 2011; Torronen and
Rouvinen, 1997). Xylanase III (pI 9.1, 32 kDa) is a family
F. INDUSTRIAL APPLICATIONS
341
Application References
Delignification of pulp Ohmiya et al. (1997)
Feed and baking Roche et al. (1995)
Digestion of guar gum; medicine Golubev et al. (2004)
Clarification of cider Haltmeier, et al (1983), Bhat, (2000)
Cooperation with xylanase Hakulinen et al. (2000)
Textile bleaching, biosensors, etc. Kiiskinen et al. (2004)
10 glycosyl hydrolase, first characterized from T. reesei
PC-3-7 (Xu et al., 1998). The pH optimum for xylanase
I is at pH 4.0–4.5, for xylanase II at pH 4.0–6.0 and for
xylanase III at pH 6.0–6.5. Of the total xylanase activity
in T. reesei PC-3-7 produced on a cellulose-based growth
medium, xylanase III accounted for over 25% (Xu et al.,
1998). Xylanase IV (pI 7.0, 43 kDa) was described in a
recent patent (Tenkanen et al., 2013). Its pH optimum is
at pH 3.5–4.0. The activity of xylanase IV increases effi-
ciently when it is combined with other xylanases. The
different properties of T. reesei xylanases are summarized
in Table 24.1.
The crystal structures of different family 11 xylanases
have been resolved, including the structures of T. reesei
xylanases I and II (Hakulinen et al., 2003; Lopez et al.,
2011). The protein structure is composed of two β-sheets
and a single α-helix forming a right hand-like structure.
Based on the structural information, a large number of
protein engineering studies have been performed with
family 11 xylanases utilizing site directed mutagenesis,
and also random mutagenesis techniques (Turunen
et al., 2004).
CELLULASES
Cellulose is degraded by three major classes of hydro-
lases (Table 24.3). Endoglucanases digest the amorphous
regions of cellulose, cellobiohydrolases cut the cellulose
to cellobiose from the free chain end and β-glucosidases
degrade small soluble oligosaccharides and cellobiose
to glucose. Efficient enzymatic degradation of insolu-
ble polysaccharides often requires a tight interaction
between the enzymes and their substrates. In the case
of cellulose degradation, many cellulases are known to
bind to crystalline and/or amorphous cellulose via cel-
lulose-binding domains (CBDs) which are distinct from
the catalytic domains (Ohmiya et al., 1997).
Cellulases are currently sold to the textile industry
for cotton softening and denim finishing and to deter-
gent markets for color care, cleaning and antiredeposi-
tion in washing powders (Cherry and Fidantsef, 2003).
Alkaline cellulase when it attacks cotton fiber relaxes
342 24. TRICHODERMA ENZYMES FOR FOOD INDUSTRIES
the rigidity of the fiber and releases the stains within the
interior of the fiber (Ohmiya et al., 1997). In the pulp and
paper industry, cellulases are used together with hemi-
cellulases to improve the drainage and runnability of the
paper machines and to enhance the deinking of recycled
fibers (Cao and Tan, 2002; Viikari et al., 1994).
Cellulases have replaced the use of volcanic lava stones
in the treatment of denim in order to achieve the so-called
“stone-washed” or abraded look appreciated by the con-
sumers. The stones caused considerable damage to the
machines and fibers, and nowadays the same effect can
be obtained by the use of cellulases (Ibrahim et al., 2011).
In the future, the cellulase market is expected to
increase dramatically if economical conversion of cel-
lulosic biomaterial to ethanol can be demonstrated. The
major barrier for this expansion is the current cost of cel-
lulases in biomass saccharification (Cherry and Fidant-
sef, 2003; Kataria and Ghosh, 2011).
OTHER ENZYMES
Besides xylanases and cellulases listed above (Tables
24.2 and 24.3), T. reesei is an efficient producer of many
other enzymes also, which are listed in Table 24.3.
β-Mannanase
Mannans and xylans are the main components of
wood besides cellulose and lignin. For the complete
hydrolysis of mannans the synergistic action of endo-
1,4-β-mannanases, β-mannosidases, β-glucosidases,
α-galactosidases and acetyl mannan esterases is
required. Endo-1,4-β-mannanase, which hydrolyzes
mannan yielding mannotriose and mannobiose, has
been reported to be produced by T. reesei (Stalbrand
et al., 1993). Mannases are also currently employed in
washing powders for removal of food-base stains.
α-L-Arabinofuranosidase
d-Xylose and l-arabinose are two most widespread
pentose sugars in biosphere. Arabinan, arabinoxylan
and some other arabinose-containing polysaccharides
release arabinose when hydrolyzed by T. reesei α-L-
arabinofuranosidase (Roche et al., 1995).
α-Galactosidase catalyses cleavage of terminal
α-galactose residues from α-O-galactosides includ-
ing galactose-containing oligosaccharides and
branched polysaccharides, such as galactomannans
and g­ alactoglucomannans. It may have an application
in digestion of guar gum, which contains about 40%
galactoses with α-1,6-linkages on a β-mannosyl back-
bone. α-galactosidase can be used in modification of
wood-derived materials because galactomannans and
F. INDUSTRIAL APPLICATIONS
galactoglucomannans are the main groups of hemicel-
luloses in softwoods. It may have an application also in
medicine for the treatment of Fabry disease (Golubev
et al., 2004; Shabalin et al., 2002; Zeilinger et al., 1993).
Pectinases are a group of enzymes (polygalacturo-
nase, pectin lyase, pectate lyase, and pectin esterase) that
break the glycosidic bonds of the long chains of galact-
uronic acid residues in pectic substances, which are the
structural polysaccharides of plant cells. The pectinases
have applications in fruit juice clarification and wine
production. A potential utilization of pectinases is treat-
ment of softwoods, which has been shown to improve
the efficiency of preservative treatment by rendering the
wood more permeable for chemical preservatives (De
Gregorio et al., 2002; Haltmeier et al., 1983).
Acetylxylan esterases represent a group of carbohy-
drate esterases with great potential in biotechnology
and carbohydrate chemistry. They deacetylate partially
­acetylated 4-O-methyl-D-glucuronoxylan, the main hard-
wood hemicellulose, or its fragments generated upon
the action of endo-1,4-β-xylanases (Hakulinen et al.,
2000). Other important enzymes, such as laccases and
proteases, are also secreted by T. reesei (Eneyskaya et al.,
1999; ­Kiiskinen et al., 2004). Laccases catalyze oxidation
of a wide variety of substrates, such as carbohydrates,
unsaturated fatty acids, phenols, and thiol-containing
proteins, are important components of various foods
and beverages. Their modification by laccase may lead
to new f­ unctionality, quality improvement, or cost reduc-
tion (Kirk et al., 2002; Minussi et al., 2002). Trichoderma
reesei protease digests the proteins in the medium under
acidic conditions (pH below 2.7). At higher pH, the pro-
teolytic reaction is limited. Glucose and cellobiose repress
the p­ roteolysis of cellobiohydrolase in a concentration-
dependent manner (Eneyskaya et al., 1999).
FOOD INDUSTRY
With their long history of safe industrial scale enzyme
production, Trichoderma spp. have been extensively
applied for production of food additives and related
products (Blumenthal, 2004; Nevalainen et al., 1994).
Currently, various Trichoderma enzymes are applied to
improve the brewing process (β-glucanases), as mac-
erating enzymes in fruit juice production (pectinases,
cellulases, hemicellulases), as feed additive in livestock
farming (xylanases) and for pet food. Cellulases are
mainly applied in baking, malting, and grain alcohol
production (Schuster and Schmoll, 2010).
However, not only enzymes but also metabolites of
Trichoderma spp. are used as additives. One of the first
products isolated from T. viride was a chemical with charac-
teristic coconut-like aroma, a 6-pentyl-α-pyrone with anti-
biotic properties, the production of which was constantly
 References
improved to reach concentrations of more than 7 g/l in
extractive fermentation cultures in T. atroviride nowadays
(Collins and Halim, 1972; Oda et al., 2009). An interesting
idea is the application of cell wall-degrading enzymes, for
example of T. harzianum, as food preservatives because of
their antifungal effect (Fuglsang et al., 1995), but so far this
suggestion has not found broad application. With a simi-
lar aim, T. ­harzianum mutanase can be used in toothpaste
to ­prevent accumulation of the polysaccharide mutan in
dental plaque (Wiater et al., 2005).
PERSPECTIVES FOR
BIOTECHNOLOGICAL PRODUCTION OF
ENZYMES BY TRICHODERMA
Biotechnology is an important tool for a more refined
search for microorganisms with commercial assets.
Microorganisms have existed on the planet Earth dur-
ing millions of years and are a source of biotechnological
possibilities due to their genetic plasticity and adapta-
tion. The isolation of new species from several and dif-
ferent habitats, such as saltwater and freshwater, soils,
hot springs, contaminated soils, caves and hostile envi-
ronments is required. Microorganisms adapted to these
conditions may have great biotechnological potential.
Methods such as the selection of mutants are simple
ways to obtain strains or strains with enzymatic possi-
bilities and these methods are widely used by research-
ers in academic pure science laboratories.
Trichoderma are potential producers of enzymes use-
ful for the food industry. Biotechnological tools are avail-
able for the selection and obtaining of strains and for
strains which increase enzymes' production on a large
scale. Progress and achievements in this area will bring
improvements in the food industry and, consequently, a
better health quality for mankind.
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