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Enzyme Technology and Mycoremediation by

White Rot Fungi

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M. P. Singh Sanjay K Vishwakarma

University of Allahabad, Allahabad - 211002, India Veer Bahadur Singh Purvanchal University
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In: Recent Trends in Biotechnology: Volume 2 ISBN 978-1-61761-797-3
Editors: M.P. Singh, Anju Agrawal, Bechan Sharma © 2010 Nova Science Publishers, Inc.

Chapter 8

Enzyme Technology and

Mycoremediation by White Rot Fungi

M.P. Singh*, V. K. Pandey, A. K. Srivastava, and S. K. Vishwakarma

Department of Biotechnology, V.B.S. Purvanchal University,
Jaunpur (UP)-222001, India

Abstract
White rot fungi has attracted global attention because of the potential use of its
versatile enzymes as tools of agriculture, medicines, industries, and bioremediation. The
extracellular lignin modifying enzymes of the fungi have very low substrate specificity
which makes them suitable for degradation of many toxic, hazardous compounds like
polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB),
trinitrotoluene (TNT) etc. having structural similarities with lignin. Among the white rot
fungi the four genera which have shown tremendous potential for bioremediation are
Phanerochaete, Trametes, Bjerkandra and Pleurotus. Out of these four genera focus has
shifted towards Pleurotus which possess lignin degrading enzymes- laccases,
peroxidases, aryl alcohol oxidases, glyoxal oxidases and pyranase oxidases with
extraordinary oxidation potential. Besides, the fungus also produces appreciable quantity
of cellulases and xylanases. Cellulases have potential application in the textile and
detergent industries and in the hydrolysis of cellulose biomass for the production of
ethanol as renewable liquid fuel. Xylanases can be utilized in bioconversion of
lignocelluloses to sugars, clarifying fruit juices, increasing the nutritional value of silage
and fodder and in textile industry for improvement of fiber quality.

*
Corresponding Author, E-mail: mpsingh.16@gmail.com
 158 M.P. Singh, V. K. Pandey, A. K. Srivastava and S.K. Vishwakarma

Introduction
Enzyme technology can be referred to as the technology associated with the application
of enzymes as the tools of agriculture, medicine, industries and bioremediation. Enzymes are
key substances of our life. They play vital role in our activities as they participate and
catalyzed the various metabolic reactions in the cell. With the exception of a small group of
catalytic RNA molecule, all enzymes are proteins, and are highly effective catalyst,
commonly enhancing reaction rates by a factor of 105 to 1017. Their catalytic activity depends
on the integrity of their native protein conformation [8]. Human beings have been using
microbial enzymes to make bread, cheese, beer, wine etc. since long. The application of these
enzymes extended from food industries to pharmaceutical industries, leather industries,
detergent industries, paper and pulp industries and in bioremediation.
Mycoremediation are fungal mediated bioremediation which uses the extracellular
enzymes to reduce or remove toxic wastes from the environment. Fungi can not only be used
to breakdown harmful pollutants in the environment but they can also act as filters for the
environment which can be used in buffer zones around streams to filter the run- off from
farms, highways and suburban zones.
White rot fungi is a physiological group of fungi which grow naturally on lignocellulosic
substrate and metabolize the lignin portion of the complex leaving a white rot residues. The
potentialities of white rot fungi gained attention in early 1990s due to their capability to
degrade recalcitrant compounds and resistance to toxic level of pollutant in comparison to
bacteria [1]. The model organism used in this study was Phanerochaete chrysosporium [3, 9],
although some non-white rot fungi like Aspergillus niger, Penicillium janthienellum and
Cunninghamella elegans were also tested [18,4,7]. Pleurotus has gained importance in recent
times because of its powerful enzymatic machinery and capability to grow vigorously on
fresh non fermented lignocelluloses [13,14]. Pleurotus are popularly called oyster mushroom
because of the characteristic nature of their fruiting bodies; with an extrinsic stalk attaches to
the piles, which opens up like an oyster shell during morphogenesis. They grow naturally in
temperate or tropical forests on dead and decaying wooden logs or sometimes on outer bark
of living trees. Pleurotus (Fr.) kummer belongs to the family Lentinaceae of the order
Poriales, subclass Holobasidiomycetidae, class Basidiomycetes and the division
Basidiomycota [5]. Most of the known species of the genus Pleurotus are edible. White rot
fungi in general and Pleurotus in particular have powerful enzymatic machinery which have
potential application in food, textile, detergent, paper and pulp industries and bioremediation
of recalcitrant, toxic and mutagenic substances.

Enzyme Technology
Enzyme technology is best described as the technology associated with the application of
enzymes as the tools of industries, agriculture and medicine [2]. With a few exceptions all
enzymes are the most remarkable and highly specialized proteins with extraordinary catalytic
power often much more than the synthetic or inorganic catalysts. Enzymes differ from other
catalysts by being very specific. Like other proteins the molecular weight varies from about
12,000 to more than one million. Some enzymes require no chemical groups for activity other
 Enzyme Technology and Mycoremediation by White Rot Fungi 159

than amino acid residues. Other require an additional chemical components called cofactor
either one or more inorganic ions such as Fe2+, Mg2+, Mn2+ , Zn2+ or a complex organic or
metalloorganic molecule called a coenzyme [8]. Some enzymes require both a coenzyme and
one or more metal ions for activity. A coenzyme or metal ion which is very tightly or even
covalently bound to the enzyme portion is called prosthetic group. A complete catalytic active
enzyme together with its bound coenzyme and/ or metal ions is called holoenzyme. The
protein part of such enzyme is called the apoenzyme or apoprotein [8]. Enzymes generally
enhance reaction rate by factor of 105 to 1017 and reaction takes place at the active site of
enzymes where substrates gets bound and is chemically transformed into products. The
reaction can be written as:

E+S ES EP E+P

Where E, S and P are Enzymes, substrate and product; ES and EP are enzyme with
substrate and enzyme with product, respectively. ES and EP are transient complexes of the
enzyme.
Today enzyme has wide range of applications as illustrated in table. Majority of enzymes
used in biotechnological applications are microbial origin and derived mainly from fungi and
bacteria i.e. Aspergillus, Bacillus, Rhizopus, streptomyces, Pleurotus etc. some enzymes are
also derived from plants and animals. Modern biotechnology in general and genetic
engineering techniques in particular has brought revolution in enzyme technology and has
improved quality and production efficiency of enzymes in the industries. Companies can
tailor enzymes specifically to meet customer demands for products with derived properties.
Enzymes can be encapsulated, entrapped or immobilized without loss of their activity and
thereby they can be reused and recycled. In food industries enzymes are used in the
production of bread, cheese, beer, wine, fruit juices and other beverages, desired flavours and
aromas etc. They are heavily used in pharmaceutical industries in making diagnostic kits and
also in drug manufacturing. Enzyme can be used to detect and measure amount of glucose in
blood. The amount of glucose in the blood and urine is a crucial indicator in the diagnosis of
diabetes; this is when there is a deficiency of insulin resulting in high glucose levels in the
blood. It is detected using the enzyme glucose oxidase which is impregnated onto a strip of
paper, and a biosensor. This instrument uses glucose oxidase as its biological system. The
enzyme catalyses the reaction between the glucose and oxygen to form gluconic acid. The
biosensor then uses the amount of gluconic acid produced to indicate the quantity of glucose
and oxygen there was in the blood, this is indicated by a colour change [2].
Leather industries use a wide range of chemicals which include lime, sodium sulphide,
salt, solvent etc. during leather processing leading to dumping of hazardous wastes in
environment. This severely pollutes the water bodies and badly affects aquatic flora and
fauna. To overcome these problems microbial enzymes – protease, lipase and cellulases can
be use in soaking, dehairing, baiting and degreasing operation of leather processing. Enzymes
are extensively used in the detergent industries as ingredients in powder and liquid detergents,
stain removers/laundry pre-spotters, automatic dishwashing detergents and
industrial/institutional cleaning products to improve the cleaning efficiency. Detergent
enzymes account for about 30% of the total worldwide enzyme production and represent one
of the largest and most successful applications of modern industrial biotechnology [6]. A
 160 M.P. Singh, V. K. Pandey, A. K. Srivastava and S.K. Vishwakarma

small amount of enzyme added to a laundry detergent can give a big cleaning benefit to the
consumer since proteases break down protein based stains, lipolases break down lipid or fat
based stains and amylases break down starches and other carbohydrate based stains. In pulp
and paper industries xylanase, cellulase and laccase are used in prebleaching, bleaching and
deinking. Cellulases are used in textile industries in textile biopolishing. The enzyme removes
pills from fabric, restoring its appearance and conditioning it for resistant for further pilling.
Other applications of cellulases are deinking and dewatering paper, processing of fruit juices
and other beverages, baking and alcohol production [20]. After proteases and amylases,
cellulases are the third most important industrial enzymes in dollar value [21]. Hence, various
applications which employ microbial enzymes are not only cleaner and safer but also efficient
in energy and resource conservation.

Table 1. Application of enzymes

ENZYMES APPLICATIONS
Protease Leather soaking baiting, dehairing, tendering
Chill proofing of bear
Generating flavors and aromas
Digestive acids
Cheese manufacturing
Detergents
Biomedical applications
Amylase Production of glucose from starch
Brewing
Digestive acids
Cellulases Biofinishing agent in textile industry
Detergent
Animal forage and feed to increase digestibility
Improvement of beatability, drainage and solubility of pulp
Deinking and dewatering paper
Fruit juice and beverage processing
Baking
Ethanol production
Pectinases Clarification of juice and wine
Fermentation of coffee bean
Glucose isomerase Manufacture of high fructose syrup and high sweetness
Glucose oxidase Analysis of blood glucose levels
Hemicellulases Baking
Brewing
Bioconversion of lignocelluloses to sugars
Production xylitol and ethanol
Clarifying fruit juices
Increasing the nutritional value of silage and fodder
Laccases Pulp bleaching and improvement in pulp brightness
Effluent detoxification
 Enzyme Technology and Mycoremediation by White Rot Fungi 161

Dye decolourization
Removal of phenolics from wines
Transformation of antibiotics and steroids
Washing powder components

Mycoremediation
Mycoremediation is term that is applied to the use of fungi to clear the environment of
contaminating substances. In another words it refers to the use of fungi in reduction/removal
of toxic wastes from the environment. All of the three types of fungi-saprophytic, parasitic
and mycorrhizal species participate in bioremediation process. Saprophytic fungi secrete
extracellular enzymes to degrade biological materials as well as recalcitrant substances.
Parasitic fungi and mycorrhizal fungi destroy bacteria and many other pathogens. The
enzymes of fungi are used as tools for detoxification of pesticides residues, dye’s effluents,
polycyclic aromatic compounds and many other hazardous wastes. In recent years, the
possibility of using white rot fungi for bioremediation strategies have initiated several
research efforts in academic, industrial and government institution [11]. The interest in this
area arises from the ability of white rot fungi to degrade an extremely wide range of
environmental pollutants using the extracellular lignin degrading machinery produced by
organisms. The non-specific nature and extraordinary oxidation potential of the lignin-
modifying enzymes enable these organisms to also degrade toxins and contaminants
including many pesticides, polyaromatic hydrocarbons, polychlorinated biphenyls and other
halogenated aromatics (including dioxins), trinitrotoluene (TNT) and other nitro aromatic
explosives and arrange of other toxic pollutants such as cyanides, azide, carbon tetrachloride
and pentachlorophenol [19]. Hence, the use of fungi in remediation is gaining momentum.
The role of fungi in dye decolourization is increasingly realized. Manufacture and use of
dye is a multibillion dollar industry which releases huge amount of toxic effluent in the
environment. They adversely affect water resources, soil fertility, aquatic organism and
ecosystem integrity [10]. The synthetic dyes are generally toxic, mutagenic and resistant to
biodegradation. Many processes like chemical, physico-chemical and biological have been
employed for dye decolourization. Biological method of removal involves the use of
microorganisms, bacteria and fungi to turn these pollutants into non-toxic harmless
substances [15]. Most studies on dye decolourization have been focused on bacteria.
Constrains associated with bacterial systems are that they are unable to degrade these dyes
efficiently. The reductive products of dye degradation by bacteria are generally aromatic
amines that are potentially hazardous to living organisms. White rot fungi were found to have
ability to decolourize the azo dyes effectively. The ability of white rot fungi to degrade dyes
has directly been correlated to its ability to degrade lignin by secreting extracellular enzymes
[16,17]. The organism degrades the dye molecules with the lignin degrading machinery [11].
Singh et al., studied nine species of Pleurotus for azo dye decolorization. They found four
species i.e. P. flabellatus, P. sajor-caju, P. ostreatus and P. fossulatus are very effective in
decolourization of azo dyes [15].
In vitro results overwhelmingly show white rot fungi very efficient in degrading highly
toxic and complex organopollutants. This may not be true in field application under in vivo
 162 M.P. Singh, V. K. Pandey, A. K. Srivastava and S.K. Vishwakarma

condition. However, rapid growth in the base of knowledge on bioremediation capabilities of

white rot fungi under in vitro condition will facilitate to utilize this pool of information in the
field application effectively and efficiently after overcoming the constrains.
Fungi have also shown to accumulate radionuclide metal and even rare earth elements.
They are potentially useful in detecting the presence of deleterious compounds.

Conclusion
We cannot think of life without enzymes. They are key substances of life. They are
synthesized in living cells and can function within and outside cell. Many of the enzymes
continue to function under in vitro condition in isolation when separated from living cells.
This unique ability of enzymes to catalyse chemical reaction and perform specific chemical
transformation in isolation has led to extensive use of enzymes in various industries
collectively known as enzyme technology. White rot fungi can be highly promising for the
sustainable treatment of coloured effluents and other toxic wastes. Environmental
compatibility and lower costs white rot fungi are inducement that permits bioremediation
technology to compete more effectively for site clean up consideration.

References
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[2] Chaplin, M. F. and Bucke, Christopher. (1990). Enzyme technology. Cambridge
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[3] Cripps, C., Bumpus, J.A. and Aust, S.D. (1990). Biodegradation of azo and heterocyclic
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