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Bioresource Technology

89 (2003) 1734

Review Paper

Developments in industrially important thermostable enzymes: a

G.D. Haki, S.K. Rakshit *
Bioprocess Technology Program, Asian Institute of Technology (AIT), P.O. Box 4, Klong Luang, Pathumthani 12120, Thailand
Received 18 November 2002; received in revised form 3 January 2003; accepted 13 January 2003

Cellular components of thermophilic organisms (enzymes, proteins and nucleic acids) are also thermostable. Apart from high
temperature they are also known to withstand denaturants of extremly acidic and alkaline conditions. Thermostable enzymes are highly
specific and thus have considerable potential for many industrial applications. The use of such enzymes in maximising reactions
accomplished in the food and paper industry, detergents, drugs, toxic wastes removal and drilling for oil is being studied extensively. The
enzymes can be produced from the thermophiles through either optimised fermentation of the microorganisms or cloning of fast-growing
mesophiles by recombinant DNA technology. In this review, the source microorganisms and properties of thermostable starch hydrolysing
amylases, xylanases, cellulases, chitinases, proteases, lipases and DNA polymerases are discussed. The industrial needs for such specific
thermostable enzyme and improvements required to maximize their application in the future are also suggested.
2003 Elsevier Science Ltd. All rights reserved.
Keywords: Thermostable enzymes; Thermophilic microorganisms

1. Introduction

1994; Bharat and Hoondal, 1998; Bauer et al., 1999;

Kohilu et al., 2001).
While the most widely used thermostable enzymes are
The role of enzymes in many processes has been known
the amylases in the starch industry (Poonam and Dalel,
for a long time. Their existence was associated with the
1995; Crab and Mitchinson, 1997; Emmanuel et al., 2000;
history of ancient Greece where they were using enzymes
Sarikaya et al., 2000), a number of other applications are in
from microorganisms in baking, brewing, alcohol
various stages of development. In the food related industry,
production, cheese making etc. With better knowledge and
they have been used in the synthesis of amino acids (Satosi
purification of enzymes the number of applications has
et al., 2001). In the petroleum, chemical and pulp and paper
increased manyfold, and with the availability of
industries, for example, thermostable enzymes have been
thermostable enzymes a number of new possibilities for
used for the elimination of sulphur containing pollutants
industrial processes have emerged. Thermostable enzymes,
through the biodegradation of compounds like
which have been isolated mainly from thermophilic
dibenzothiophene (Bahrami et al., 2001), in the production
organisms, have found a number of commercial
of 1,3-propanediol from glycerol and in replacing polluting
applications because of their overall inherent stability
chemical reagents causing toxic products (Peter et al.,
(Demirijan et al., 2001). Advances in this area have been
2001). Currently, a number of publications have
possible with the isolation of a large number of beneficial
extensively discussed developments in this area.
thermophilic microorganisms from different exotic
Adaptation of extremophiles to hot environments (Danson
ecological zones of the earth and the subsequent extraction
et al., 1992; Stetter, 1999), production of heat-stable
of useful enzymes from them (Burrows, 1973; Antranikian
enzymes from thermophiles and hyperthermophiles (Knor,
et al., 1987; Groboillot,
1987; Jakob, 1989; Huber and Stetter, 1998; Niehaus et al.,
1999), structure and function relationships of thermozymes (heat*
tolerant enzymes) (Zeikus et al., 1998a,b)
Corresponding author. Tel.: +66-2-524-6115; fax: +66-2-524-6111.
E-mail address: (S.K. Rakshit).

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734


0960-8524/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved.

and biotechnological and industrial applications of

thermostable enzymes (Franks, 1993; Lasa and Berenguer,
1993; Leuschner and Antranikan, 1995; Cowan, 1996;
Holst et al., 1997; Hough and Danson, 1999; Eichler, 2001)
are among the topics that have been studied.
In the present review, an attempt is made to document
the research activities conducted in the area and indicate
the source microorganisms of some important thermostable
enzymes. The need for thermostable catalysts, the optimum
conditions for an efficient catalytic activity of the enzymes
and current industrial applications are presented. Besides
discussing the reason for the thermostable character of the
enzymes, possible improvements and a number of expected
developments are also suggested in the article.

2. Geothermal sites as sources of extremophiles

In situ temperatures between 80 and 115 C were found
to be conducive biotopes for a number of
hyperthermophiles (Huber and Stetter, 1998). Some
examples are mentioned in Table 1. Extremophiles are also
adapted to live at low temperatures in the cold polar
regions, at high pressure in the deep sea and at a very low
and high pH values (pH 03 or 1012), or at a very high
(530%) salt concentration (Herbert and Sharp, 1992).
In the last decade, a number of hyperthermophilic
archaea, the least understood domain of life (Woese et al.,
1990; Rotschild and Manicinelli, 2001) have been isolated
and are able to grow around the boiling point of water
(Niehaus et al., 1999). The organisms with the highest
growth temperatures (103110 C) are members of the
genera Pyrobaculum, Pyrodictium, Pyrococcus

Table 1

and Methanopyrus. Within the bacteria, Thermotoga

maritima and Aquifex pyrophilus exhibit the highest
growth temperatures of 90 and 95 C respectively (Herbert
and Sharp, 1992). These properties imply extremely
important industrial and biotechnological implications due
to the fact that enzymes from such microorganisms can be
employed for use in harsh industrial conditions where their
specific catalytic activity is retained.

3. Resistance of thermophiles to high temperatures and

Microorganisms, like all living things, adapt to the
condition in which they have to live and survive.
Thermophiles are reported to contain proteins which are
thermostable and resist denaturation and proteolysis
(Kumar and Nussinov, 2001). Specialized proteins known
as chaperonins are produced by these organisms, which
help, after their denaturation to refold the proteins to their
native form and restore their functions (Everly and Alberto,
2000). The cell membrane of thermophiles is made up of
saturated fatty acids. The fatty acid provides a hydrophobic
environment for the cell and keeps the cell rigid enough to
live at elevated temperatures (Herbert and Sharp, 1992).
The archae, which compose most of the hyperthermophiles,
have lipids linked with ether on the cell wall. This layer is
much more heat resistant than a membrane formed of fatty
acids (De Rosa et al., 1994).
The DNA of thermophiles contains a reverse DNA
gyrase which produces positive super coils in the DNA
(Lopez, 1999). This raises the melting point of the DNA
(the temperature at which the strands of the double helix
separate) to at least as high as the organ-

Examples of sites serving as sources of microorganisms which can provide thermo tolerant enzymes




Hot spring
Hot spring
Deep sea hydrothermal vent
Marine solfatare
Decomposed plant samples from
a lake
Hot spring
Compost of fermenting citrus peels,
coffee and tea extract residues

Thermus sp.
Bacillus sp. WN.11
Staphilothermus marinus
Thermococcus litoralis
Clostridium absonum CFR-702

Cellulase free xylanase

Shaw et al. (1995)

Mamo and Gessese (1999)
Canganella et al. (1994)
Brown and Kelly (1993)
Swaroopa and Krishna (2000)

Bacillus thermoleovocans ID-1

Bacillus strain MH-1


Dong-Woo et al. (1999)

Kenji et al. (1998)

Korean salt fermented anchovy
Deep sea hydrothermal vent
Sediments of hot springs
Garbage dump
Compost treated with artichoke juice

Bacillus stearothermophilus CH-4

Bacillus sp. KYJ963
Pyrococcus abyssi
Bacillus sp. 3183
Bacillus circulans
Bacillus sp.

Alkaline phosphatase
a-Amylase-like pullulanase

Kenji et al. (1994)

Young et al. (2001)
Sebastien et al. (2001)
Badal et al. (1989)
Ashita et al. (2000)
Jean-Jacques et al. (1987)

Table 2
Bioconversion reactions and applications of thermostable enzymes

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734


Temperature range (C)



a-Amylase (bacterial)


Starch!dextrose syrups

a-Amylase (fungal)

4565, 105a

Starch!dextrose syrups
Starch!dextrose syrups
Craft pulp!xylan+lignin

Starch hydrolysis, brewing, baking,

Production of maltose
Production of glucose syrups
Pulp and paper industry
Food, cosmetics, pharmaceuticals,


4555, 95c

Chitin!N-acetyl glucosamine
N-acetyl glucosamine!
glucosamine (deacetylation) Chitin!
chitosan (deacetylase)



Protein!amino acids and peptides



Fat removal, hydrolysis,

interesterification, alcholysis, aminolysis

DNA polymerase
Xylanase from Thermotoga sp.
Within this range enzyme activity was high. cCellulases

DNA amplification

from Thermotoga sp.

isms maximum temperature for growth. Thermophiles also

tolerate high temperature by using increased interactions
that non-thermotolerant organisms use, namely,
electrostatic, disulphide bridge and hydrophobic
interactions (Kumar and Nussinov, 2001).
Thermostable enzymes are stable and active at
temperatures which are even higher than the optimum
temperatures for the growth of the microorganisms (Saboto
et al., 1999). These authors indicated that relatively few
studies have been carried out in this regard and the only
available information is that thermophilic enzymes are
more rigid proteins than their mesophilic counterparts. A
clearer understanding of this capacity should be possible
with new methodologies that clearly indicate changes in
protein structure.
The effect of structural fluctuations of a-amylases of
mesophilic and thermophilic sources were studied and
reported by Fitter et al. (2001). While determining the
conformational entropy of both enzymes, the folded state
showed a higher structural flexibility for the thermophilic
protein than the mesophilic homologue. It has, thus, been
assumed that a mechanism characterized by entropic
stabilization could be responsible for the higher
thermostability of the thermophilic enzyme.



General advantages of enzymes from thermophiles

Thermostable enzymes are gaining wide industrial and

biotechnological interest due to the fact that their enzymes
are better suited for harsh industrial processes (Leuschner
and Antranikan, 1995; Fredrich and Antrakian, 1996; Diane

Cellulose hydrolysis, polymer

degradation in detergents
Baking, brewing, detergents, leather
Dairy, oleo chemical, detergent, pulp,
pharmaceuticals, cosmetics and
leather industry
Genetic engineering/PCR

et al., 1997; Zeikus et al., 1998a,b). Some biocatalytic

conversions and industrial applications of thermostable
enzymes are presented in Table 2.
One extremely valuable advantage of conducting
biotechnological processes at elevated temperatures is
reducing the risk of contamination by common
mesophiles. Allowing a higher operation temperature has
also a significant influence on the bioavailability and
solubility of organic compounds and thereby provides
efficient bioremediation (Becker, 1997). Other values of
elevated process temperatures include higher reaction
rates due to a decrease in viscosity and an increase in
diffusion coefficient of substrates and higher process
yield due to increased solubility of substrates and
products and favorable equilibrium displacement in
endothermic reactions (Mozhaev, 1993; Krahe et al.,
1996; Kumar and Swati, 2001). Such enzymes can also
be used as models for the understanding of
thermostability and thermo-activity, which is useful for
protein engineering. The following sections describe
specific thermostable enzymes, their sources,
characteristics and application requirements of high
temperature resistance.


Amylolytic enzymes

The starch industry is one of the largest users of

enzymes for the hydrolysis and modification of this
useful raw material. The starch polymer, like other such
polymers, requires a combination of enzymes for its
complete hydrolysis. These include a-amylases,
glucoamylases or b-amylases and isoamylases or
pullulanases (Poonam and Dalel, 1995). The enzymes are
classified into endo-acting and exo-acting enzymes. aamylase is an endo-acting enzyme and hydrolyses


G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

linkages in a random fashion and leads to the formation

of linear and branched oligosaccharides, while the rest
are exo-acting enzymes and attack the substrate from the
monosaccharides. The starch hydrolytic enzymes
comprise 30% of the worlds enzyme consumption (Van
der Maarel et al., 2002).
The enzymatic conversion of all starch includes
gelatinization, which involves the dissolution of starch
granules thereby forming a viscous suspension,
liquefaction, which involves partial hydrolysis and loss
in viscosity, and saccharification, involving the
production of glucose and maltose via further hydrolysis.
Gelatinisation is achieved by heating starch with water,
and starch is water-soluble only at high temperatures
which are dependent on the source (Rakshit, 1998). For
hydrolysis of the starch to proceed immediately after gel-

Andersson et al., 2002; Carlsen et al., 2002; Dijkhuizen et

al., 2002; Miura et al., 2002) to meet the requirements of
the starch industry. Table 3 shows thermostable microbial
starch hydrolyzing enzymes and their properties.
Thermostable a-amylases were isolated a long time ago
from Bacillus subtilis, Bacillus amyloliquefaciens and
Bacillus licheniformis (Underkofler, 1976). The
commercially available b-amylases with a catalytic activity
up to 60 C were isolated from various Bacillus species
(Brown, 1987). They can be used in conjunction with
debranching enzymes to produce pure maltose syrups.

Table 3

atinization, hence, among other things avoiding a lot of

cooling time, the enzyme has to be thermostable.
A number of attempts were made to isolate and
characterize amylolytic enzymes from diversified sources
(Daniel, 1979; Norman, 1979; Fogarty and Kelly, 1995;
Medda and Chandra, 1980; Morgan and Priest, 1981;
Fogarty, 1983; Krishnan and Chandra, 1983; Rolfsmeir and
Blum, 1995; Piller et al., 1996; Crab and Mitchinson, 1997;
Rama et al., 1998; Jin et al., 2001; Min Ha et al., 2001;
Stamford et al., 2001; Wojciechowski et al., 2001;
Source microorganisms and properties of thermostable starch hydrolyzing enzymes



Enzyme properties
Optimal temperature
C) Optimal



Bacillus amyloliquefaciens
Bacillus licheniformis
Bacillus stearothermophilus
Bacillus stearothermophilus
Bacillus subtilis
Lactobacillus manihotivorans
Myceliophthora thermophila
Pyrococcus furiosus
Pyrococcus woesei
Staphylothermus marinus
Sulfolobus solfataricus
Thermococcus aggreganes
Thermococcus celer
Thermococcus fumicolans
Thermococcus hydrothermalis
Thermomyces lanuginosus
Thermococcus profoundus






Underkofler (1976)
Viara et al. (1993)
Vihinen and Mantsala (1990)
Jeayoung et al. (1989)
Canganella et al. (1994)
Aguilar et al. (2000)
Ramkrishna et al. (1993)
Laderman et al. (1993a,b)
Koch et al. (1991)
Canganella et al. (1994)
Haseltine et al. (1996)
Canganella et al. (1994)
Canganella et al. (1994)
Estelle et al. (1997)
Estelle et al. (1997)
Jensen and Olsen (1991)
Kwak et al. (1998)


Bacillus circulans


Poonam and Dalel (1995)

Bacillus cereus var. mycoides

Bacillus sp.
Clostridium thermosulphurogenes
Clostridium thermosulfurogenes



Takasaki (1976a,b)
Young et al. (2001)
Shen et al. (1988)
Nipkow et al. (1989)

Bacillus sp.


Takasaki (1976a,b)

Pyrococcus furiosus
Pyrococcus woesi
Thermococcus aggregans
Thermus caldophilus GK24
Thermococcus celer
Thermococcus hydrothermalis
Thermococcus litoralis
Thermotoga maritima MSB8



Brown and Kelly (1993)

Rudiger et al. (1995)
Canganella et al. (1994)
Kim et al. (1996)
Canganella et al. (1994)
Gantlet and Duchiron (1998)
Brown and Kelly (1993)
Bibel et al. (1998)


G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

The pullulanases that were produced in earlier work

on different microorganisms were not very suitable for
operation under the conditions prevailing in the industry
(Nakamura et al., 1975; Konishi et al., 1979; De Mot et
al., 1984a,b; Takizawa and Muroorka, 1985). However,
many thermophilic microorganisms have since been
found to produce pullulanases (Suzuki and Chishoro,
1983; Jensen and Norman, 1984; Plant et al., 1986;
Antranikian et al., 1987; Koch et al., 1987; Saha et al.,
1988; Canganella et al., 1994; Swamy and Seenayya,
1996) and b-amylases (Shen et al., 1988; Nipkow et al.,
1989; Swampy et al., 1994; Rina and Geeta, 1996; Rama
et al., 1999; Erra-Pujada et al., 2001).
Termamyl and Fungamyl are two well-known
amylolyic enzymes which are now available
commercially. These enzymes are used world-wide for
the production of glucose syrups and syrups with
different level of dextrose equivalent. Heterooligosaccharides have been synthesized by Termamyl
obtained from B. licheniformis (Chitradon et al., 2000).
In the presence of soluble starch and added non-starch
sugars, oligosaccharides were produced by the reversed
catalytic reaction.
One of the concerns of the starch industry is the
calcium requirement of the a-amylase enzymes and the
formation of calcium oxalate, a substance that may block
process pipes and heat exchangers. Besides, such
accumulation in some products like beer is not
acceptable. Its precipitation can be reduced through a
decrease in calcium ion requirement of enzymes and
lowering of the pH of the production process. Activities
of a number of the starch hydrolyzing enzymes, however,
are calcium dependent although the degree of utilization
varies (Table 4). Production of calcium independent and
acid stable a-amylases has been attempted by protein
engineering and as a result Termamyl LC e, which was
produced via site-directed mutagenesis, has shown high
calcium independence (Hashida and Bisgaard-Frantzen,
Although, raw starch dominates in nature there are
few enzymes that can catalyze its hydrolysis efficiently
(Hamilton et al., 1999). Saccharification of liquefied
starch is carried out at low pH values. However,
currently used thermostable a-amylases are not stable at


Table 4
Calcium requirement of industrially important starch degrading enzyme

such low pH (Crab and Mitchinson, 1997). An

economical process could be attained through the use of
amylases stable at the saccharification stage. In addition
to this, a one-step process of starch hydrolysis using
amylolytic enzymes would decrease the costs of glucose
production. The anticipated diversity of species of
thermophiles within high temperature environments
(Huber and Stetter, 1998) and the requirements of the
new and improved technological operations for enzymes
with novel and fitting characteristics (Emmanuel et al.,
2000) have placed a challenge both for the scientific and
business community.

6. Thermostable xylanases
Xylan, which is the dominating component of
hemicelluloses, is one of the most abundant organic
substances on earth. It has a great application in the pulp
and paper industry (Dekker and Linder, 1979; Chen et
al., 1997; Lee et al., 1998). The wood used for the
production of the pulp is treated at high temperature and
basic pH, which implies that the enzymatic procedures
require proteins exhibiting a high thermostability and
activity in a broad pH range (Jacques et al., 2000).
Treatment with xylanase at elevated temperatures
disrupts the cell wall structure. This, as a result,
facilitates lignin removal in the various stages of
bleaching. Xylanases for such a purpose (1) must lack
cellulytic activity to avoid hydrolysis of the cellulose
fibers, (2) need to be of low molecular mass to facilitate
their diffusion in the pulp fibers, and (3) most
importantly, high yields of enzyme must be obtained at a
very low cost (Niehaus et al., 1999). According to these
authors all commercially available xylanases can only
partially fulfill these requirements, and the optimum
temperature for the activity of most xylanases is reported
to be 5060 C with a half-life of about 1 h at 55 C
(Jacques et al., 2000). However, some xylanases have
been reported to exhibit higher thermal stability and
optimal activity ranging from 80 to 100 C (Saul et al.,
1995; Zverlov et al., 1996; Morris et al., 1998).


G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734



Application temperature
range (C)

Application pH

Minimum Ca2 dosage


Bacterial mesophilic a-amylase

Bacillus subtilis




Bacterial thermophilic a-amylase

Bacillus licheniformis







Fungal a-amylase
Aspergillus oryzae
Aspergillus niger
Bacillus acidopolluliticus
Source: Hans (1995).
Table 5
Source microorganisms and properties of thermostable xylanases

Baccillus amyloliquefaciens
Bacillus circulans
Bacillus sp.
Bacillus sp. strain SPS-0
Bacillus subtilis
Clostridium abosum
Dictyoglomus sp. strain B1
Fusarium proliferatum
Pyrococcus furiosus
Pyrococcus furiosus
Scytalidium thermophilum
Streptomyces sp. strain S38
Sulfolobus solfataricus
Teheromyces lanuginosus (wild and mutant)
Teheromyces lanuginosus-SSBP
Thermoascus aurantiacus
Thermotoga maritima MSB8
Thermotoga maritima MSB8
Thermotoga maritima MSB8
Thermotoga neapolitana
Thermotoga neapolitana
Thermotoga neapolitana
Thermotoga sp. strain FjSS3-B1
Thermotoga sp. strain FjSS3-B1
Thermotoga sp. strain FjSS3-B1
Thermotoga thermarum

60, 70

These enzymes have offered a major step in the

reduction of chlorine consumption in the bleaching
process of kraft pulp, thus, lowering environmental
pollution by organic halogens (Vikari et al., 1994;
Eriksson and Heitmann, 1998). The enzymes are
generally classified in to two families. Family 10 groups
(EXs 10) having high molecular weights (>30 Kda) and
Family 11 (EXs 11) which are low molecular weight
(<30 Kda) xylanases (Henrissat and Bairoch, 1993;
Dominguez et al., 1995). Among the two families, the
EX 10-fold is found to be more thermostable (Fontes et
al., 1995). Due to their capacity to enhance the bleaching
of kraft paper, however, EXs 11 have remained more
attractive (Clarke et al., 1997; Morris et al., 1998; Georis
et al., 2000).
Thermostable xylanase were isolated from a number
of bacterial and fungal sources (Table 5). Members of the
Bacillus sp., Streptomyces sp., Thermoascus aurantiacus
and Fusarium proliferatum have been reported to produce
xylanases which are active at temperatures between 50

Enzyme properties
Optimal temperature
C) (

7.0, 6.7

Breccia et al. (1997)
Dhillon and Khanna (2000)
Gessesse (1998)
Bataillon et al. (2000)
Paula et al. (2002)
Swaroopa and Krishna (2000)
Marjaana et al. (1994)
Badal (2002)
Bauer et al. (1999)
Kengen et al. (1993)
Nazan and Zumrut (2000)
Georis et al. (2000)
Nucci et al. (1993)
Bakalova et al. (2002)
Johnson et al. (1999)
Kalegoris et al. (1998)
Winterhalter and Liebl (1995)
Bronnenmeier et al. (1995)
Gabelsberger et al. (1993)
Bok et al. (1998)
Bok et al. (1994)
Zverlov et al. (1996)
Ruthersmith and Daniel (1991)
Ruthersmith and Daniel (1991)
Simpson et al. (1991)
Sunna et al. (1996a)

and 80 C. While the Dictyoglomus sp. were described to

produce xylanases operating at an optimum temperature
of 90 C, a number of Thermotogales sp. were reported to
secrete thermostable xylanases which can function at
higher temperatures. Alkaliphilic and cellulase-free
xylanases with an optimum temperature of 65 C from
Thermoactinomyces thalophilus subgroup C (Kohilu et
al., 2001) and cellulase-free xylanases from Clostridium
abusonum CFR-702 (Swaroopa and Krishna, 2000) were
also reported recently. From one Thermotoga sp. an
enzyme active at 115 C was produced and characterized
(Simpson et al., 1991). It was also possible to produce
xylanases in solid-state fermentation, where a decreased
cultivation time and an increased concentration of basal
medium improved its production. This is a promising
aspect from an economic point of view (Park et al.,
The pulp and paper technology is one of the fastest
growing industries and the use of thermostable xylanases
seems attractive since they provide global environmental

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

benefits. However, scaling up of the enzyme production

from the respective microorganisms to the level required
by the industry remains to be seen. It is also worth
mentioning that, extreme thermophiles that are able to
secrete xylanase are few. The search for a thermophile with
high yield of enzyme and the desired characteristics is still
being pursued.
7. Thermostable cellulases
Cellulose, the most abundant organic source of feed,
fuel and chemicals (Spano et al., 1975) consists of glucose
units linked by b-1,4-glycosidic bonds in a linear mode.
The difference in the type of bond and the highly ordered
crystalline form of the compound between starch and
cellulose make cellulose more resistant to digest and
hydrolyze. The enzymes required for the hydrolysis of
cellulose include endoglucanases, exoglucanases and bglucosidases (Matsui et al., 2000). While cellulase is an
endoglucanase that hydrolyses cellulose randomly,
producing oligosacaccharides, cellobiose and glucose,
exoglucanases hydrolyze b-1,4-D-glucosidic linkages in
cellulose releasing cellobiose from the nonreducing end.
On the other hand, b-glycosidases of thermophilic origin,
which have received renewed attention in the
pharmaceutical industry hydrolyze cellobiose to glucose.
In the current industrial processes, cellulolytic enzymes
are employed in the color extractions of juices, in
detergents causing color brightening and softening, in the
biostoning of jeans, in the pretreatment of biomass that
contains cellulose to improve nutritional quality of forage
and in the pretreatment of industrial wastes (Buchert et al.,
1997; Niehaus et al., 1999; Bhat, 2000; Nakamura et al.,
2001; Van wyk et al., 2001; Zhou et al., 2001). In order to
attack the native crystalline cellulose, which is water
insoluble and occurs as fibers of densely packed structures,
however, thermostable cellulases active at high temperature
and high pH are required.
Thermostable cellulases of archaeal origin include those
isolated from Pyrococcus furiousus (Kengen et al., 1993)
and Pyrococcus horikoshi (Ando et al., 2002). While the
latter has an optimum temperature of 97 C, the enzyme
from Pyrocuccus furiousus has shown optimal activity at
102105 C (Table 6). Sulfolobus solfataricus MT4,
Sulfolobus acidocaldarius and Sulfolobus shibatae were
also described as producers of b-gucosidases (Grogan,
1991). From Thermotoga maritema MSB8 optimally active
cellulase acting at 95 C and between pH 6.0 and 7.0 was
reported (Bronnenmeier et al., 1995). Other endocellulases
(CelA and CelB) from Thermotoga neapolitana, were
purified and characterized (Bok et al., 1998). Optimal pH
for CelA is 6.0 at 95 C, and the pH


Table 6
Source microorganisms and properties of thermostable cellulases

range of CelB is broad (6.06.6) at 106 C. CelA, with the

ability to hydrolyze microcrystalline cellulose, was
isolated from the extremely thermophilic bacterium
Anaerocellum thermophilum (Zverlov et al., 1998) and
maximal activity of this enzyme was observed at pH 5.0
6.0 and 8595 C. A highly thermostable cellobiose (115
C at pH 6.87.8) was also produced from Thermotoga sp.
FjSS3-B1 (Ruthersmith and Daniel, 1991,
It is obvious that, the successful utilization of cellulose
is dependent on the development of economically
feasible technologies for the production of cellulase. The
biopolishing process of cotton in the textile industry, for
example, requires cellulase stable at high temperature
close to 100 C (Ando et al., 2002). Presently used
enzymes for this purpose, however, are active only at 50
55 C. Cellulase production is also found to be the most
expensive step during ethanol production from cellulosic
biomass, and accounted for approximately 40% of the
total cost (Spano et al., 1975). In the food industry,
degradation of cellulose by acids is still unsatisfactory
and results in the decomposition of the sugars. Finally,
even though many cellulolytic enzymes of thermophillic
origin are known their function under physiological
condition remains unclear.

8. Thermoactive chitinases


G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

Chitin, the second most abundant natural biopolymer

after cellulose, consists of a linear b-1,4-homopolymer of
N-acetylglucoseamine residues. In nature, it is usually
found attached to other polysaccharides and proteins. The
covering layer of insects, cell walls of various fungi and
crab and shrimp wastes are the main sources of chitin
(Majeti and Kumar, 2000; Nwe and Stevens, 2002;
Suntornsuk et al., 2002). The major applications of
chitosan include wastewater clearing, preparation of


of endochitinases (L, M and S) were purified and

characterized. They had different molecular masses (71,
62 and 53 Kda) and showed temperature optima of 75, 65
and 75 C at a pH of 6.5, 5.5 and 5.5 respectively. The isoelectric points were 5.3, 4.8, and 4.7. Thermostable
exochitinases were also isolated from Bacillus
stearothermophilus CH-4, isolated from a compost of
organic solid wastes (Kenji et al., 1998).
The basic parts of shrimp and prawns used for

Enzyme properties
Optimal temperature
C) (


Optimal pH
Anaerocellu thermophilum
Bacillus subtilis
Pyrococcus furiosus
Pyrococcus horicoshi
Rhodothermus marinus
Thermotoga maritema MSB8
Thermotoga neapoltana
Thermotoga neapoltana




Zverlov et al. (1998)

Mawadza et al. (2000)
Kengen et al. (1993)
Ando et al. (2002)
Hreggvidsson et al. (1996)
Bronnenmeier et al. (1995)
Bok et al. (1998)



Bok et al. (1998)

photographic products, cements, heavy metal chelating
agents and for medical and veterinary purposes (Spindler
et al., 1990; Georgopapadakou and Tkacz, 1995; Hirano,
1996; Cohen and Chet, 1998; Majeti and Kumar, 2000).
The production of chitosan from chitin involves a
deacetylation reaction that is done using 40% sodium
hydroxide solution. This is a highly corrosive stream which
is difficult to control. Attempts are thus being made to do
this conversion using deacetylase enzyme. Perhaps a
thermostable deacetylase enzyme will help in increasing
yields and conversion rates and allow reuse of the enzyme.
Endo-acting chitin hydrolase chitinaseA, the exoacting
hydrolase chitinaseB and N-acetyl-D-glucoseaminidase
are responsible for chitin degradation (Kenji et al., 1994).
However, chitin is not easily accessible to chitinases and
chitin deacetylases. Evidences from X-ray diffraction
studies have shown that chitin is a highly ordered
crystalline structure and does not dissolve in water
(Roberts, 1992). This property of chitin has shown the
need for thermostable enzymes.
The thermophilic organisms Bacillus licheniformis
X7u (Takayanagi et al., 1991), Bacillus sp. BG-11
(Bharat and Hoondal, 1998) and Strteptomyces
thermoviolaceus OPC-520 were reported to be the major
sources of chitinases (Tsujibo et al., 1995). Chitinase and
Nacetyl-glucosaminidase were produced from the
Thermococcus chitinophagus (Huber et al., 1995). A
thermophilic bacterium strain producing three different
endochitinases in its culture fluid was isolated from
chitin-containing compost (Kenji et al., 1994). The
strains belonged to the genus Bacillus and three isoforms

consumption are the muscle tissues. The resulting waste

of the products, thus, is vast (Healey et al., 1994). In
some developing countries it was reported that more than
2.5 million tones of shrimp and prawns are produced per
year (Anon., 1996). Therefore, there is a high need to
develop methods of producing value added products
from this waste. As the major way to exploit the potential
from this waste is the production of chitin and its
derivative chitosan, improved and economically feasible
chemical, enzymatic and novel biotechnological
conversion bioprocesses are required. Chitin together
with its derivatives can also be used as agrochemicals
(Kenji et al., 1998). However, there is only little effort to
clarify its utilization in the food industry.
9. Thermostable proteases
Proteases, which are generally classified into two
categories (exopeptidases, that cleave off amino acids from
the ends of the protein chain and endopeptidases, which
cleave peptide bonds within the protein) are becoming
major industrial enzymes, and constitute more than 65% of
the world market (Rao et al., 1998). These enzymes are
extensively used in the food, pharmaceutical, leather and
textile industries (Cowan, 1996; Fan et al., 2001; Mozersky
et al., 2002). The applications will keep increasing in the
future as will the need for stable biocatalysts capable of
withstanding harsh conditions of operation.
Relative ease of isolation of Bacilli from diverse sources
has made these organisms the focus of attention in
biotechnology (Johnevelsy and Naik, 2001). So far,
however, few thermophilic Bacillus sp. that produce
proteases have been isolated, the earliest isolate being
Bacillus stearothermophilus (Salleh et al., 1977) which is

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

stable at 60 C. Another Bacillus sp. has produced a

thermostable protease that has an optimum activity at 60 C
(Razak et al., 1993), while a different Bacillus
stearothermophilus sp. produced an alkaline and
thermostable protease which is optimally active at 85 C
(Razak et al., 1995, 1997; Rahman et al., 1994). A species
of Bacillus stearothermophilus TP26 that has been isolated
produces an extra cellular protease having an optimum
temperature of 75 C (Gey and Unger, 1995). Enhancement
stearothermophilus had also been possible using
economical chemical additives in the proteolysis reactions
involved in waste activated sludge (Kim et al., 2002a,b). In
a chemically defined medium, thermophilic and alkaliphilic
Bacillus sp. JB-99 was also reported to produce
thermostable alkaline proteases (Johnevelsy and Naik,
2001). Dominant producers of proteases in fact, are the
microorganisms of the genera Pyrococcus, Thermococcus
and Staphylothermus (Table 7). Extremely thermostable
serine proteases are produced by the hyperthermophilic
archaeum Desulfurococcus strain (Hanazawa et al., 1996),
and thermostable metalloproteases are reported from a
gram-negative thermophilic bacterium (Pawinee and
Wipapat, 1996).
Major area of focus in the future concerning the
production of proteases is the optimization of media
(Johnevelsy and Naik, 2001). Synthetic media have a great
advantage over complex media in that consistency of
processes and production is enhanced through avoiding the
variability of complex substrates. Thus, synthetic media
provides better control and monitoring, improved product
recovery and quality, and simplified purification systems
(Zhang and Greasham, 1999). It is for this reason that
reports on the production of protease enzymes using
synthetic media are available recently (Mao et al., 1992;
Ferrero et al., 1996).


There is considerable current interest on the exploration

of proteases that can catalyze reactions in cold water
(Demirijan et al., 2001). This will allow their use in
detergents which can be used in normal tap water without
the requirement for increasing the temperature of the water.
The search for such enzymes is very much a challenge at
this time.
10. Heat stable lipases
Lipases of microbial origin are the most versatile
enzymes and are known to bring about a range of
bioconversion reactions (Vulfson, 1994), which includes
hydrolysis, interesterification, esterification, alcoholysis,
acidolysis and aminolysis (Jaeger et al., 1994; Pandey et
al., 1999; Nagao et al., 2001; Kim et al., 2002a,b). Their
unique characteristics include substrate specificity,
stereospecificity, regioselectivity and ability to catalyze a
heterogeneous reaction at the interface of water soluble and
water insoluble systems (Brockman and Borgstorm, 1984;
Jaeger and Reetz, 1998).
The esters produced play a relevant role in the food
industry as flavor and aroma constituents (Gandhi et al.,
1995; Pandey et al., 1999). Whereas long chain methyl and
ethyl esters of carboxylic acid moieties provide valuable
oleo chemical species that may function as fuel for diesel
engines, esters of long chain carboxylic acid and alcohol
moieties (waxes) have applications as lubricants and
additives in cosmetic formulations (Linko et al., 1994).
Other applications include the removal of the pitch from
pulp produced in the paper industry, for the hydrolysis of
milk fat in the dairy industry, removal of non-cellulosic
impurities from raw cotton before further processing into
dyed and finished products, drug formulations in the
pharmaceutical industry and in the removal of

Table 7
Source microorganisms and properties of thermostable proteolytic enzymes

Bacillus brevis
Bacillus licheniformis
Bacillus stearothermophilus
Bacillus stearothermophilus
Bacillus sp. JB-99
Bacillus stearothermophilus TP26
Bacillus sp. no. AH-101
Bacillus thermoruber
Pyrococcus sp. KODI
Staphylothermus marinus
(archeal and bacterial origin)
Thermococcus aggreganes
Thermococcus celer
Thermococcus litoralis
Thermotoga maritema

Enzyme properties
Optimal temperature
C) ( Optimal pH







Banerjee et al. (1999)

Manchini and Foretina (1998)
Salleh et al. (1977)
Rahman et al. (1994)
Johnevelsy and Naik (2001)
Gey and Unger (1995)
Takami et al. (1989)
Manchini et al. (1988)
Fujiwara et al. (1996)
Mayer et al. (1996)
Kocabiyik and Erdem (2002)



Klingberg et al. (1991)

Klingberg et al. (1991)
Klingberg et al. (1991)
Klingberg et al. (1991)

subcutaneous fat in the leather industry (Pandey et al.,


G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

1999; Traore and Buschle-Diller, 2000). A biodiesel was

derived from vegetable oils using immobilized Candida
antarctica lipase (Shimada et al., 1999).
Most of the industrial processes in which lipases are
employed function at temperatures exceeding 45 C. The
enzymes, thus, are required to exhibit an optimum
temperature of around 50 C (Sharma et al., 2002).
According to some reports there are fats exhibiting
higher melting points and which are able to inhibit
enzymatic reactions at a low temperature (Dong-Woo et
al., 1999). Some enzymatic processes for the physical
refining of seed oils have four distinct requirements.
These include pH of 5.0 and optimal temperature of
around 65 C, adding an enzyme solution and enzyme
reaction followed by the separation of the
lysophosphatide from the oil at about 75 C (Klaus, 1998).
These reactions, therefore, are enhanced through the
utilization of thermo-tolerant lipases.
Lipases are of widespread occurrence throughout the
earths flora and fauna. More abundantly, however, they
are found in bacteria, fungi and yeasts (Wu et al., 1996).
Several Bacillus sp. were reported to be the main source
of lypolytic enzymes (Kim et al., 1994; Schmidt et al.,
1994; Luisa et al., 1997). While most of these enzymes
are active at a temperature of 60 C and pH of 7.0, lipases
from Bacillus thermoleovorans and a thermophilic
Rhizopus oryzae strain can moderately function at
extreme pH and temperature values (Dong-Woo et al.,
1999; Abel et al., 2000). The pH and temperature optima
for the catalytic activity of some thermostable lipases are
given in Table 8.
Reports of thermostable lipases from archaeal origin,
however, are very few. Phospholipase A2 which was
secreted from the archaea Pyrococcus horikoshii

degumming processes of oil refineries and thereby

reduces wastewater problems and running costs (Klaus,
Among the desirable characteristics that commercially
important lipases should exhibit, alkali tolerance and
thermostability are the main (Kulkurani and Gadre,
1999). To meet this end, there is a continuous search for
sources of highly active lipolytic enzymes with specific
stability to pH, temperature, ionic strength and organic
solvents (Lee and Rhee, 1993). Although, few lipases
exist which are able to operate at 100 C, their half-lives
are reported to be short (Rathi et al., 2000).
The other problem associated with the production of
lipases and which requires improvements is its secretion
at the late stage of growth (Tan and Gill, 1985). Proteases
secreted in the mean-time either change the properties of
the lipase produced or degrade it (Cordenons et al.,
1996). Perhaps it is for this reason that only few lipases
can be obtained in industrial quantities (Wu et al., 1996).
Hence the need for a more stable lipase enzyme produced
in large quantities.
11. Thermostable DNA polymerases
The polymerase chain reaction (PCR) process has led
to a huge advance in genetic engineering due to its
capacity to amplify DNA. The three successive steps in
this process include denaturation or melting of the DNA
strand (separation) obtained at a temperature of 9095 C,
renaturation or primer annealing at 55 C followed by
synthesis or primer extension at around 75 C (Mullis et
al., 1986; Erlich et al., 1988; Saiki et al., 1988).
Development in this process has been to a large extent

Table 8
Source microorganisms and properties of thermostable lipases

Bacillus acidocaldariusa (esterase)
Bacillus sp. RSJ-1
Bacillus strin J 33a
Bacillus stearothermophilusa
Bacillus thermocatenletusa
Bacillus thermoleovorans ID-1
Geobacillus sp.
Pseudomonas sp.
Pseudomonas sp.
Pyrobaculum calidifontis
Pyrococcus furiosusa (esterase)
Pyrococcus horikoshii
Pyrococcus horikoshii

Enzyme properties
Optimal temperature
C) ( Optimal pH


Active at water immiscible solvents.

(Yan et al., 2000) was involved in crude oil refining. This

enzyme was found to optimally react at 95 C and pH of
7.0. Phospholipase A2 has a better performance in the




Manco et al. (1998)
Sharma et al. (2002)
Nawani et al. (1998)
Gupta et al. (1999)
Klibanov (1983)
Dong-Woo et al. (1999)
Abdel-Fattah (2002)
Kulkurani and Gadre (1999)
Rathi et al. (2000)
Hotta et al. (2002)
Ikeda and Clark (1998)
Ando et al. (2002)
Yan et al. (2000)

facilitated by the availability of thermostable DNA

polymerases, which catalyse the elongation of the primer
DNA strand (Mullis and Faloona, 1987).
In earlier PCR procedures, DNA polymerases which
were isolated from Escheria coli were utilized (Mullis et

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

al., 1986; Erlich et al., 1988). These enzymes, however,

lost their enzymatic activities at elevated temperatures and,
thus, adding a new polymerase enzyme after each cycle
following the denaturation and primer hybridization steps
was necessary. This process made the thermal cycling a
time-consuming and costly procedure.
Taq polymerase from the bacterium Thermus aquaticus
was the first thermostable DNA polymerase characterized
(Chien et al., 1976; Kaledin et al., 1980). Repeated
exposure to 98 C in a reaction buffer had little effect on the
enzyme activity and significant activity remained after
exposure to 99 C. Although, Taq polymerase exhibits a 5 0
30 exonuclease activity, a 3050 exonuclease activity was
not detected. Thus, the base insertion fidelity is low as the
enzyme is unable to correct misincorporated nucleotides
(Longley et al., 1990; Ling et al., 1991). It had also been
possible to determine the error rates of some polymerases
in terms of base pairs (Alkami Biosystems, 1999).
Table 9 shows a variety of thermostable polymerases
with 3050-exonuclease-dependent proof reading activity
required from a high fidelity polymerase. Optimization of
the PCR procedure can be done by mixing a standard
polymerase, such as Taq polymerase with high fidelity
polymerases (Pfu, Vent and Deep Vent) (Ling et al.,
While the PCR process is developing rapidly through
the invention of better instruments (Mariella, 2001), lack of
fidelity has remained to be a serious challenge. The five
distinct activities in which errors can occur are the rate of
phosphodiester bond formation, the binding of dNTP by
the polymerase, the rate of pyrophospahte release,
contamination after misincorporation and the
Table 9
Thermostable DNA polymerases in PCR
BstI pol
Deep Vent Pol
Pfu pol
Pwo pol
Taq pol I
Tfi pol
Tfl pol
Tth pol

Bacillus stearothermophilus
Pyrococcus sp. GB-D
Pyrococcus furiosus
Pyrococcus woesei
Thermus aquaticus
Thermus filiformis
Thermus flavus
Thermus thermophilus

Tma pol
Thermotoga maritema
Vent pol
Thermococcus litoralis
HF and LF, high and low fidelity; NI, not identified.

3050-exonuclease proof reading capacity of the enzyme

(Alkami Biosystems, 1999). Some issues that nowadays
have stimulated scientists in the area thus, are
improvements required in the fidelity of the PCR,
development of strategies for more economical use of the
enzymes and ensuring rapid multiplication from fewer
strands. A thermostable DNA polymerase having these


characteristics will indeed improve the results obtained by

PCR machine.

12. Molecular cloning of thermophilic genes into

mesophilic hosts
Genetic and protein engineering are the modern
techniques for the commercial production of enzymes of
improved stability to high temperatures, extremes of pH,
oxidizing agents and organic solvents. Cloning and
expression of genomic information available in a
thermophile in a suitable and faster growing mesophilic
host has also provided possibilities of producing the
specific thermostable enzyme required for a particular
biotransformation process (Blackebrough and Birch, 1981;
William and Dennis, 1988; James, 1995; Ikeda and Clark,
1998; Hough and Danson, 1999).
Genes encoding a-amylase (amyA), pullulanase (pulA),
maltodextrin phosphrylase (agpA) and a-glucosidase
(aglA) were identified in the gene library of Thermotoga
maritema (Bibel et al., 1998). The enzymes produced after
expression in Escheria coli are reported to demonstrate
extreme thermostability. Pyrococus furiosus a-amylase
gene, which was cloned by activity screening in Escheria
coli was reported to be optimally active at 100 C, pH 5.5
6.0 and not to require Ca2 for its activity (Dong et al.,
1997a). Genes encoding pullulanases from Pyrococus
furiosus (Dong et al., 1997b) and Ferividobacterium
pennavorans (Bertoldo et al., 1999), Thermotoga
(Velikodvorskaya et al., 1997), Protease genes from
PCR reactions



Mead et al. (1991)

Cline et al. (1996)
Lundberg et al. (1991)
Frey and Suppman (1995)
Jones and Foulkes (1989)
Perler et al. (1996)
Kaledin et al. (1980)
Myers and Gelfand (1991),
Pantazaki et al. (2002)
Bost et al. (1994)
Perler et al. (1996)


Pyrobaculum aerophilum and Bacillus stearothermophilus

(Volkl et al., 1995; Kubo and Imanaka, 1988), lipases from
Bacillus thermocatenulatus (Schmidt et al., 1994), esterases
from Bacillus licheniformis (Alvarez-macarie et al., 1999)
and cellulase genes from the archaon Pyrococus furiosus
(Bauer et al., 1999) were expressed from an Escheria coli
recombinant strain. The production of cellulase enzyme
was further enhanced through DNA recombinant
technology by encoding the genes for cellulase in various
species of Clostridium (Beguin, 1992). In addition to this,


G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

the nucleotide sequence and the cloning of CelA and CelB

genes from C. thermocellum were studied and reported
(Bergquist et al., 1992). Cloning and expression of
chitinase genes was also successfully done (Duffner et al.,
1996; Chernin et al., 1997). In cases where a relevant gene
of a thermostable enzyme has been identified and once the
gene has been transferred into a mesophilic host the
resultant enzyme production can be facilitated through
appropriate incubation processes.

13. Conclusion
Recent investigations have demonstrated that
extremophilic archaea, bacteria and fungi have colonized
environments that were believed to be inhospitable for
survival. Their true diversity in fact, is not yet been fully
explored. The thermostable enzymes isolated from these
organisms have just started providing conversions under
conditions that are appropriate for industrial applications.
The conditions required by these thermostable enzymes
which bring about specific reactions not possible by
chemical catalysts are still mild and environmentally
benign, as compared to the temperatures and pressures
required for chemical conversions. Thus, with the
availability of thermostable enzymes a number of new
applications in the future are likely. Although, believed to
provide tremendous economical benefits, production of
the enzymes to the level required by the industries has
remained a challenge.
Abdel-Fattah, Y., 2002. Optimization of thermostable lipase production
from a thermophilic Geobacillus sp. using BoxBehnken
experimental design. Biotechnol. Lett. 24, 12171222.
Abel, H., Marie, D., Nathalie, R., Danielle, D., Louis, S., Louis, C.,
2000. Purification and characterization of an extracellular lipase
from a thermophilic Rhizopus oryzae strain isolated from palm
fruit. Enzyme Microb. Technol. 26, 421430.
Aguilar, G., Morlon-Guyot, J., Trejo-Aguilar, B., Guyot, J.P., 2000.
Purification and characterization of an extracellular alpha-amylase
produced by Lactobacillus manihotivorans an amylolytic lactic acid
bacterium. Enzyme Microb. Technol. 27, 406413. Alkami Biosystems,
1999. Alkami quick guide TM for PCR. Alkami Biosystems Inc., USA.
Alvarez-macarie, E., Augier-Magro, V., Baratti, J., 1999.
Characterization of a thermostable esterase activity from the
moderate thermophile Bacillus licheniformis. Biosci. Biotechnol.
Biochem. 63, 18651870.
Andersson, L., Rydberg, U., Larsson, H., Andersson, R., Aman, P.,
2002. Preparation and characterisation of linear dextrins and their
use as substrates in in vitro studies of starch branching enzymes.
Carbohyd. Polym. 47, 5358.
Ando, S., Ishida, H., Kosugi, Y., Ishikawa, K., 2002.
Hyperthermostable endoglucanase from Pyrococcus horikoshi.
Appl. Environ. Microbiol. 68, 430433.
Anon., 1996. World Review of Fisheries and Aquaculture. FAO, United
Antranikian, G., Herzberg, C., Gottschalk, G., 1987. Production of
thermostable a-amylase, pullulanase and a-glucosidase in

continuous culture by a new Clostridium isolate. Appl. Environ.

Microbiol. 53, 16681673.
Ashita, D., Gupta, J., Khanna, S., 2000. Enhanced production,
purification and characterization of novel cellulase-poor
thermostable, alkali tolerant xylanase from Bacillus circulans AB
16. Proc. Biochem. 35, 849856.
Badal, C., Gwo-Jenn, S., Kailash, C., Lloyd, W., Gregory, Z., 1989.
New thermostable a-amylase-like pullulanase from thermophilc
Bacillus sp. 3183. Enzyme Microb. Technol. 11, 760764.
Badal, C., 2002. Production, purification and properties of xylanase
from a newly isolated Fusarium proliferatum. Proc. Biochem. 37,
Bahrami, A., Shojaosadati, S., Mahbeli, G., 2001. Biodegradation of
dibenzothiophene by thermophilic bacteria. Biotechnol. Lett. 23,
Bakalova, N., Petrova, S., Atev, A., Bhat, M., Kolev, D., 2002.
Biochemical and catalytic properties of endo-1,4-b-xylanases from
Thermomyces lanuginosus (wild and mutant strains). Biotechnol.
Lett. 24, 11671172.
Banerjee, V., Saani, K., Azmi, W., Soni, R., 1999. Thermostable
alkaline protease from Bacillus brevis and its characterization as a
laundry additive. Proc. Biochem. 35, 213219.
Bataillon, M., Cardinali, A.-P., Castillon, N., Duchiron, F., 2000.
Purification and characterization of a moderately thermostable
xylanase from Bacillus sp. strain SPS-0. Enzyme Microb. Technol.
26, 187192.
Bauer, M., Driskil, L., Callen, W., Snead, M., Mathur, E., Kelly, R.,
1999. An endoglucanase EglA, from the hyperthermophilic
archaeon Pyrococcus furiosus hydrolyzes a-1,4 bonds in mixed
linkage (1!3), (1!4)-b-D-glucans and cellulose. J. Bacteriol. 181,
Becker, P., 1997. Determination of the kinetic parameters during
continuous cultivation of the lipase producing thermophile Bacillus
sp. IHI-91 on olive oil. Appl. Microb. Biotechnol. 48, 184190.
Beguin, P., 1992. Molecular biology of cellulose degradation. In:
Herbert, R., Sharp, R. (Eds.), Molecular Biology and Biotechnology
of Extremophiles. Chapman and Hall, NY.
Bergquist, P., Love, D., Croft, J., Streiff, M., Daniel, R., Morgan, H., 1992.
Cloning of genes involved in cellulose hydrolysis. In: Herbert, R.,
Sharp, R. (Eds.), Molecular Biology and Biotechnology of
Extremophiles. Chapman and Hall, NY.
Bertoldo, C., Duffner, F., Jorgensen, P., Antrakianin, G., 1999. Pullulanase
type I from Ferividobacterium pennavorans Ven5: cloning,
sequencing, expression of the gene and biochemical characterization
of the recombinant enzyme. Appl. Environ. Microbiol. 65, 20842091.
Bharat, B., Hoondal, G., 1998. Isolation, purification and properties of
thermostable chitinase from an alkalophilic Bacillus sp. BG-11.
Biotechnol. Lett. 20, 157159.
Bhat, M., 2000. Cellulases and related enzymes in biotechnology.
Biotechnol. Adv. 18, 355383.
Bibel, M., Bretel, C., Gosslar, U., Kriegshauser, G., Liebl, W., 1998.
Isolation and analysis of genes for amylolytic enzymes of the
hyperthermophilic bacterium Thermotoga maritema. FEMS Microbiol.
Lett. 158, 915.
Blackebrough, N., Birch, G., 1981. Enzymes of Food Processing. Applied
Science Publishers, London.
Bok, J., Goers, S., Eveleigh, D., 1994. Cellulase and xylanase systems of
Thermotoga neapolitana. ACS Symp. Ser. 566, 5465.
Bok, J., Dienesh, A., Yernool, D., Eveleigh, D., 1998. Purification,
characterization and molecular analysis of thermostable cellulases
CelA and CelB from Thermotoga neapolitana. Appl. Environ.
Microbiol. 64, 47744781.
Bost, D., Stoffel, S., Landre, P., Lawyer, F., Akers, J., Abramson, R.,
Gelfand, D., 1994. Enzymatic characterization of Thermotoga
maritema DNA polymerase and a truncated form, Ultma DNA
polymerase. FASEB J., A1395.

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

Breccia, J., Sineriz, F., Baigori, M.D., Guillermo, R.C., Hatti-Kaul, R.,
1997. Purification and characterisation of a thermostable xylanase
from Bascillus amyloliquefaciens. Enzyme Microb. Technol. 22, 42
Brockman, H., Borgstorm, B., 1984. Lipases. Elsevier, Amsterdam.
Bronnenmeier, K., Kern, A., Libel, W., Staudenbauer, W., 1995.
Purification of Thermotoga maritema enzymes for the degradation of
cellulose materials. Appl. Environ. Microbiol. 61, 13991407.
Brown, S., Kelly, R., 1993. Characterization of amylolytic enzymes
having both a 1,4 and a 1,6 hydrolytic activity from the thermophilic
archaea Pyrococcus furiosus and Thermococcus litoralis. Appl. Environ.
Microbiol. 59, 26142621. Brown, C., 1987. Enzyme technology. In:
Wilkinson, J. (Ed.), Introduction to Biotechnology. Black Well Scientific,
Buchert, J., Pere, J., Oijusluoma, L., Rahkamo, L., Viikari, L., 1997.
Cellulasestools for modification of cellulosic materials. In: Niches in
the World of Textiles World Conference of the Textile Institute,
Manchester, England, pp. 284290. Burrows, W., 1973. Text Book of
Microbiology. W.B. Saunders Company, Ontario.
Canganella, F., Andrade, C., Antranikian, G., 1994. Characterization of
amylolytic and pullulytic enzymes from thermophilic archaea and
from a new Ferividobacterium species. Appl. Microb. Biotechnol. 42,
Carlsen, M., Nielsen, J., Jorgensen, S.B., Zangirolami, T.C., 2002. Growth
and enzyme production during continuous cultures of a high amylaseproducing variant of Aspergillus oryzae. Braz. J. Chem. Eng. 19, 55
Chen, C., Adolphson, R., Dean, F., Eriksson, K., Adamas, M.,
Westpheling, J., 1997. Release of lignin from kraft pulp by a
hyperthermophilic xylanase from Thermotoga maritema. Enzyme
Microb. Technol. 20, 3945.
Chernin, L.S., de la Fuente, L., Sobolev, V., Haran, S., Vorgias, C.E.,
Oppenheim, B.A., Chet, I., 1997. Molecular cloning, structural
analysis and expression in E. coli of a chitinase gene from
Enterobacter agglomerans. Appl. Environ. Microbiol. 63, 834839.
Chien, A., Edgar, D., Trela, J., 1976. Deoxyribonucleic acid polymerase
from the extreme thermophilile Thermus aquaticus. J. Bacteriol. 127,
Chitradon, L., Mahakhan, P., Bucke, C., 2000. Oligosaccharide synthesis
by reversed catalysis using alpha-amylase from Bacillus licheniformis.
J. Mol. Catal. B: Enzym. 10, 273280.
Clarke, J., Rixon, J., Ciruela, A., Gilbert, H., Hazlewood, G., 1997.
Family-10 and Family-11 xylanases differ in their capacity to enhance
the bleachability of hardwood and softwood paper pulps. Appl.
Microb. Biotechnol. 48, 177183.
Cline, J., Braman, J., Hogrefe, H., 1996. PCR fidelity of Pfu DNA
polymerase and other DNA polymerases. Nucleic Acids Res. 24,
Cohen, K.R., Chet, I., 1998. The molecular biology of chitin digestion.
Curr. Opin. Biotechnol. 9, 270277.
Cordenons, A., Gonzalez, R., Kok, R., Hellingwerf, K., Nudel, C., 1996.
Effect of nitrogen sources on the regulation of extracellular lipase
production in Acinetobacter calcoaceticus strains. Biotechnol. Lett. 18,
Cowan, D., 1996. Industrial enzyme technology. Trends Biotechnol. 14
(6), 177178.
Crab, W., Mitchinson, C., 1997. Enzymes involved in the processing of
starch to sugars. Trends Biotechnol. 15, 349352.
Daniel, L., 1979. Fermentation and Enzyme Technology. John Willey and
Sons, NY.
Danson, M., Hough, D., Lunt, G., 1992. The Archaebacteria: Biochemical
and Biotechnology. Portland, London.
De Mot, R., Andries, K., Verachtert, H., 1984a. Comparative study of
starch degradation and amylase production by Ascomycetes yeast
species. Syst. Appl. Microbiol. 5, 106118.


De Mot, R., Van Oudendijck, E., Verachert, H., 1984b. Production of

extracellualar debranching activity by amylolytic yeasts. Biotechnol.
Lett. 6, 581586.
De Rosa, M., Morana, A., Riccio, A., Gambacorta, A., Trincone, A.,
Incani, O., 1994. Lipids of the archaea: a new tool for bioelectronics.
Biosens. Bioelectr. 9, 669675.
Dekker, R., Linder, W., 1979. Bioconversion of hemicellulose: aspects of
hemicellulase production by Trichoderma reesi QM9414 and enzymic
sacchrification of hemicellulose. S. Afr. J. Sci. 75, 6571.
Demirijan, D., Moris-Varas, F., Cassidy, C., 2001. Enzymes from
extremophiles. Curr. Opin. Chem. Boil. 5, 144151.
Dhillon, A., Khanna, S., 2000. Production of a thermostable alkalitolerant
xylanase from Bacillus circulans AB16 grown on wheat straw. World
J. Microbiol. Biotechnol. 27, 325327.
Diane, W., Stephan, R., Wolfgang, Z., 1997. Application of thermostable
enzymes for carbohydrate modification. In: Contribution of the Fourth
International Workshop on Carbohydrate as Organic Raw Materials,
March 2021, 1997. WUV-Universitatverlag, Vienna.
Dijkhuizen, L., Van der Maarel, M., Van der Veen, B.U., 2002. Properties
and applications of starch-converting enzymes of the alpha-amylase
family. J. Biotechnol. 94, 137155.
Dominguez, R., Souchon, H., Spinelli, S., Dauter, Z., Wilson, K.S.,
Chauvaux, S., Beguin, P., Alzari, P.M., 1995. A common protein fold
and similar active site in two distinct families of betaglycanases. Nat.
Struct. Biol. 2, 569576.
Dong, G., Vieille, C., Savachenko, A., Zeikus, G., 1997a. Cloning,
sequencing, and expression of the gene encoding extracellular
aamylase from Pyrococcus furiosus and biochemical characterization
of the recombinant enzyme. Appl. Environ. Microbiol. 63, 3569
Dong, G., Vieille, C., Savachenko, A., Zeikus, G., 1997b. Cloning,
sequencing, and expression of the gene encoding amylopullulanase
from Pyrococcus furiosus and biochemical characterization of the
recombinant enzyme. Appl. Environ. Microbiol. 63, 35773584.
Dong-Woo, L., You-Seok, K., Ki Jun, K., Byung-Chan, K., HakJong, C.,
Doo-sik, K., Maggy, T., Yu-ryang, P., 1999. Isolation and
characterisation of thermophilic lipase from Bacillus thermoleovorans
ID-1. FEMS Microbiol. Lett. 179, 393400.
Duffner, F., Christodoulou, E., Vorgias, C.E., 1996. Expression and
correct folding of a thermostable chitinase in E. coli. In: First
International Congress on Extremophiles, 26 June. Estoril,
Eichler, J., 2001. Biotechnological uses of archaeal extremozymes.
Biotechnol. Adv. 19, 61278.
Emmanuel, L., Stefan, J., Bernard, H., Abdel, B., 2000. Thermophilic
archaeal amylolytic enzymes. Enzyme Microb. Technol. 26, 314.
Eriksson, L., Heitmann, J., 1998. Applications of enzyme technology in
the paper industry. In: Proceedings of the Technical Association of
the Pulp and Paper Industry, vol. 3, Montreal.
Erlich, H., Gelfand, D., Saiki, R., 1988. Specific DNA amplification
product review. Nature 33, 461462.
Erra-Pujada, M., Chang, F., Debeire, P., Duchiron, F., ODonohue, M.,
2001. Purification and properties of the catalytic domain of the
thermostable pullulanase type II from
hydrothermalis. Biotechnol. Lett. 23, 12731277.
Estelle, L., Ladrat, C., Ann, G., Georges, B., Francis, D., 1997.
Thermostable amylolytic enzymes of thermophilic microorganisms
from deep-sea hydrothermal vents. Animal Biology. CR Acad. Sci.
Paris 320, 893898.
Everly, C., Alberto, J., 2000. Stressors, stress and survival: overview.
Front. Bioscien. 5, 780786.
Fan, Z., Zhu, Q., Dai, J., 2001. Enzymatic treatment of wool. J. Dong Hua
University (English edition) 18, 112115.
Ferrero, M., Castro, G., Abate, M., Baigori, M., Sinerz, F., 1996.
Thermostable alkaline protease of Bacilus licheniformis MIR 29:


G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

isolation, production and characterization. Appl. Microbiol.

Technol. 45, 327332.
Fitter, J., Herrmann, R., Hauss, T., Lechner, R.E., Dencher, N.A., 2001.
Dynamical properties of alpha-amylase in the folded and unfolded
state: the role of thermal equilibrium fluctuations for
conformational entropy and protein stabilization. Physica B:
Condens. Matter. 301, 17.
Fogarty, W., Kelly, C., 1995. Amylases, amyloglucosidases and related
glucanases. In: Enzyme and Microbiol Technology. In: Enzyme and
Microbial Systems Involved in Starch Processing, vol. 17, pp. 770
Fogarty, M., 1983. Microbial Enzymes and Biotechnology. Applied
Science Publishers, England.
Fontes, C., Hazlewood, G.P., Morag, E., Hall, J., Hirst, B.H., Gilbert,
H.J., 1995. Evidence for a general role for non-catalytic domains in
xylanases from thermophilic bacteria. Biochem. J. 307, 151158.
Franks, F., 1993. Protein Biotechnology: Isolation, Characterization, and
Stabilization. Humana, Totowa, NJ.
Fredrich, A., Antrakian, G., 1996. Keratin degradation by
Fervidobacterium pennavorans, a novel thermophilic anaerobic
species of the order Thermotogales. Appl. Environ. Microbiol. 62,
2875 2882.
Frey, B., Suppman, B., 1995. Demonstration of the expand PCR
systems greater fidelty and higher yields with a lacI-based fidelty
assay. Biochemica 2, 3435.
Fujiwara, S., Okuyama, S., Imanaka, T., 1996. The world of archea:
genome analysis, evolution and thermostable enzymes. Gene 179,
Gabelsberger, J., Liebl, W., Scheleifer, K., 1993. Cloning and
characterization of b-galactoside and b-glucoside hydrolysing
enzymes of Thermotoga maritema. FEMS Microbiol. Lett. 109,
Gandhi, N., Sawant, S., Joshi, J., 1995. Studies on the lipozymecatalyzed
synthesis of butyl laurate. Biotechnol. Bioeng. 46, 112.
Gantlet, H., Duchiron, F., 1998. Purification and properties of a
thermoactive and thermostable pullulanase from Thermococcus
hydrothermalis, a hyperthermophilic archaeon isolated from a deep
sea hydrothermal vent. Appl. Microbiol. Biotechnol. 49, 770777.
Georgopapadakou, N., Tkacz, J., 1995. The fungal cell wall as a drug
target. Trends Microbiol. 3, 98104.
Georis, J., Giannotta, F., De Buyl, E., Granier, B., Frere, J., 2000.
Purification and properties of three endo-beta-1,4-xylanases
produced by Streptomyces sp. strain S38 which differ in their
capacity to enhance the bleaching of kraft pulp. Enzyme Microb.
Technol. 26, 177183.
Gessesse, A., 1998. Purification and properties of two thermostable
alkaline xylanases from an alkaliphilic Bacillus sp. Appl. Environ.
Microbiol. 64, 35333535.
Gey, M., Unger, K., 1995. Calculation of the molecular masses of two
newly synthesized thermostable enzymes isolated from
thermophillic microorganismas. J. Chromatogr. B 166, 188193.
Groboillot, A., 1994. Immobilization of cells for application in the food
industry. Crit. Rev. Biotechnol. 14, 75107.
Grogan, W., 1991. Evidence that b-galactosidase of Sulfolobus
solfactoricus is only one of several activities of a thermostable bDglycosidase. Appl. Environ. Microbiol. 57, 16441649.
Gupta, R., Gupta, K., Sexena, R., Khan, S., 1999. Bleach-stable,
alkaline protease from Bacillus sp. Biotechnol. Lett. 21, 135138.
Hamilton, M., Kelly, C., Fogarty, W., 1999. Production and properties
of the raw starch-digesting a-amylase of Bacillus sp. IMD 435.
Proc. Biochem. 35, 2731.
Hanazawa, S., Hoaki, T., Jannasch, H., Maruyama, T., 1996. An
extremely thermostable serine protease from hyperthermophilic
archaeum Desulfurococcus strain sy, isolated from a deep sea
hydrothermal vent. J. Mar. Biotechnol. 4, 121126. Hans, S., 1995.

Enzymes in Food Processing, A Reprint from Biotechnology, second,

completely revised ed. Weinheim.
Haseltine, C., Rolfsmeier, M., Blum, P., 1996. The glucose effect and
regulation of a-amylase synthesis in the hyperthermophilic
archaeaon Sulfolobus solfataricus. J. Bacteriol. 178, 945950.
Hashida, M., Bisgaard-Frantzen, H., 2000. Protien engineering of new
industrial amylases. Trends Glycosci. Glycotechnol. 12, 389401.
Healey, M.G., Romo, C.R., Bustos, R., 1994. Bioconversion of marine
crustacean shell waste. Resour. Conserv. Recycl. 11, 139147.
Henrissat, B., Bairoch, A., 1993. New families in the classification of
glycosyl hydrolases based on amino acid sequence similarities.
Biochem. J. 293, 781788.
Herbert, R., Sharp, R., 1992. Molecular Biology and Biotechnology of
Extremophiles. Chapman and Hall, NY.
Hirano, S., 1996. Chitin biotechnological applications. In: Raafat
ElGewely, M. (Ed.), Biotechnogical Annual Review, vol. 2.
Elsevier Press.
Holst, O., Manelius, A., Krahe, M., Markl, H., Raven, N., Sharp, M.,
1997. Thermophiles and fermentation technology. Comp. Biochem.
Physiol. 118A, 415422.
Hotta, Y., Ezaiki, S., Atomi, H., Imanaka, T., 2002. Extremely stable
and versatile carboxyl esterase from a hyperthermophilic archaeon.
Appl. Environ. Microbiol. 68, 39253931.
Hough, D., Danson, M., 1999. Extremozymes. Curr. Opin. Chem. Boil.
3, 3946.
Hreggvidsson, G.O., Kaiste, E., Holst, O., Eggertsson, G., Palsdottir,
A., Kristjansson, J.K., 1996. An extremely thermostable cellulase
from the thermophilic eubacterium Rhodothermus marinus. Appl.
Environ. Microbiol. 62, 30473049.
Huber, H., Stetter, K., 1998. Hyperthermophiles and their possible
potential in biotechnology. J. Biotechnol. 64, 3952.
Huber, R., Stoohr, J., Hohenhaus, S., Rachel, R., Burgraff, S., Jannsch,
H., Stetter, K., 1995. Thermococcus chitonophagus sp. Nov., a
novel chitin degrading, hyperthermophilic archeum from the deep
sea hydrothermal vent environment. Arch. Microbiol. 164, 255
Ikeda, M., Clark, D., 1998. Molecular cloning of extremely
thermostable esterase gene from hyperthermophilic archaeon
Pyrococcus furiosus in Escherhia coli. Biotechnol. Bioeng. 57,
Jacques, G., Frederic, D.L., Joste, L.B., Viviane, B., Bart, D., Fabrizio, G.,
Benoit, G., Jean-marie, F., 2000. An additional aromatic interaction
improves the thermostability and thermophilicity of a mesophilic
family 11 xylanase: structural basis and molecular study. Protein Sci.
9, 466475.
Jaeger, K., Reetz, T., 1998. Microbial lipases from versatile tools for
biotechnology. TIBTECH. 16, 396403.
Jaeger, K., Ransac, S., Dijktra, C., Colson, C., Heuvel, M., Misset, O.,
1994. Bacterial lipases. FEMS Microbiol. Rev. 15, 2963.
Jakob, K., 1989. Thermophilic organisms as sources of thermostable
enzymes. TIBTECH. 7, 349353.
James, A., 1995. Is thermophily a transferable property in bacteria. Crit.
Rev. Microbiol. 21, 165174.
Jean-Jacques, A., Gladys, L., Sadok, K., Jacques, C., 1987. Isolation and
characterization of thermophilic bacterial strains with inulinase
activity. Appl. Environ. Microbiol. 53, 942945.
Jeayoung, K., Takashi, N., Ryu, S., 1989. Thermostable, rawstarchdigesting amylase from Bacillus stearothermophilus. Appl.
Environ. Microbiol. 55, 16381639.
Jensen, B., Norman, B., 1984. Bacillus acidopullulyticus pullulanase
application and regulatory aspects for use in the food industry. Proc.
Biochem. 19, 129134.
Jensen, B., Olsen, J., 1991. Physicochemical properties of a purified
alpha-amylase from the thermphilic fungus Thermomyces
lanuginosus. Enzyme Microb. Technol. 14, 112116.

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

Jin, F., Li, Y., Zhang, C., Yu, H., 2001. Thermostable a-amylase and agalactosidase production from the thermophilic and aerobic Bacillus
sp. JF strain. Proc. Biochem. 36, 559564.
Johnevelsy, B., Naik, G., 2001. Studies on production of thermostable
alkaline protease from thermophilic and alkaliphilic Bacillus sp. JB-99
in a chemically defined medium. Proc. Biochem. 37, 139144.
Johnson, L., Larr, M., Suren, S., Balakrishna, P., 1999. Characteristics of
b-D-xylanase from a thermophilic fungus, Thermomyces lanuginosusSSBP. Biotechnol. Appl. Biochem. 30, 7379.
Jones, M., Foulkes, N., 1989. Reverse transcription of mRNA by Thermus
aquaticus DNA polymerase. Nucleic Acids Res. 17, 8387 8388.
Kaledin, A., Sliusarenko, A., Gorodetski, S., 1980. Isolation and properties
of DNA polymerase from extreme thermophilic bacteria Thermus
aquqticus YT-1. Biochimiya 45, 644651.
Kalegoris, E., Christakopoulos, D., Kekos, D., Macris, B., 1998. Studies
on the solid-state production of thermostable endoxylanases from
Thermoascus aurantiacus: characterization of two isoenzymes. J.
Biotechnol. 60, 155163.
Kengen, S., Luesink, E., Stams, A., Zehnder, A., 1993. Purification and
characterization of an extremely thermostable b-glucosidase from the
hyperthermophilic archaeon Pyrococccus furiosus. Eur. J. Biochem.
213, 305312.
Kenji, S., Akira, Y., Hajime, K., Mamoru, W., Mitsuaki, M., 1994.
Purification and characterization of three thermostable bNacetylhexoseaminidase of Bacillus stearothermophilus CH-4 isolated
from chitin-containing compost. Appl. Environ. Microbiol. 60, 2911
Kenji, S., Akira, Y., Hajime, K., Mamoru, W., Mitsuaki, M., 1998.
Purification and characterization of three thermostable endochitinases
of a noble Bacillus strain, MH-1, isolated from chitincontaining
compost. Appl. Environ. Microbiol. 64, 33973402.
Kim, H., Sung, M., Kim, M., Oh, T., 1994. Occurrence of thermostable
lipase in thermophilic Bacillus sp. Strain 398. Biosci. Biotech.
Biochem. 58, 961962.
Kim, C., Nashiru, O., Ko, J., 1996. Purification and biochemical
characterization of pullulanase type I from Thermus caldophilus Gk24. FEMS Microbiol. Lett. 138, 147152.
Kim, I., Kim, H., Lee, K., Chung, S., Ko, S., 2002a. Lipase-catalyzed
acidolysis of perilla oil with caprylic acid to produce structured lipids.
JAOCS 79, 363367.
Kim, Y., Bae, J., Oh, B., Lee, W., Choi, J., 2002b. Enhancement of
proteolytic enzyme activity excreted from Bacillus stearothermophilus
for a thermophilic aerobic digestion process. Biores. Technol. 82, 57
Klaus, D., 1998. An enzyme process for the physical refining of seed oils.
Chem. Eng. Technol. 21, 278281.
Klibanov, A., 1983. Immobilized enzymes and cells as practical catalysts.
Science 219, 722727.
Klingberg, M., Hashwa, F., Antrakikian, G., 1991. Properties of extremely
thermostable proteases from anaerobic hyperthermophilic bacteria. Appl.
Microbiol. Biotechnol. 34, 715719. Knor, D., 1987. Food biotechnology.
In: Tannenbaum, S.R., Walstra, P. (Eds.), Food Science and Technology.
Dekker, New York.
Kocabiyik, S., Erdem, B., 2002. Intracellular alkaline proteases produced
by thermoacidophiles: Detection of protease heterogeneity by gelatin
zymography and polymerase chain reaction (PCR). Biores. Technol.
84, 2933.
Koch, R., Zabalowski, P., Antrakianin, G., 1987. Highly active and
thermostable amylases and pullulanases from various anaerobic
thermophiles. Appl. Microbiol. Biotechnol. 27, 192198.
Koch, R., Spreinat, K., Lemke, K., Antranikan, G., 1991. Purification and
Properties of a hyperthermoactive a-amylase from the
archaeobacterium Pyrococcus woesei. Arch. Microbiol. 155, 572
Kohilu, U., Nigam, P., Singh, D., Chaudhary, K., 2001. Thermostable,
alkaliphilic and cellulase free xylanases production by


Thermoactinomyces thalophilus subgroup C. Enzyme Microb.

Technol. 28, 606610.
Konishi, Y., Amemura, A., Tanabe, S., Harada, T., 1979. Immunological
study of pullulanase from Klebsiela strains and the occurrence of this
enzyme in the Enterobacteriaceae. Int. J. Syst. Bacteriol. 29, 1318.
Krahe, M., Antranikian, G., Markel, H., 1996. Fermentation of
extremophilic microorganisms. FEMS Microbiol. Rev. 18, 271 285.
Krishnan, T., Chandra, A., 1983. Purification and characterization of aamylase from B. licheniformis. Appl. Environ.Microbiol. 46, 430
Kubo, M., Imanaka, T., 1988. Cloning and nucleotide sequence of the
highly thermostable neutral protease gene from Bacillus
stearothermophilus. J. Gen. Microbiol. 134, 18831892.
Kulkurani, N., Gadre, R., 1999. A novel alkaline, thermostable, protease
free lipase from Pseudomonas sp. Biotechnol. Lett. 21, 897899.
Kumar, S., Nussinov, R., 2001. How do thermophilic proteins deal with
heat? A review. Cell. Mol. Life Sci. 58, 12161233.
Kumar, H.D., Swati, S., 2001. Modern Concepts of Microbiology, second
revised ed. Vikas Publishing House Pvt. Ltd., New Delhi.
Kwak, Y., Akeba, T., Kudo, T., 1998. Purification and characterization of
enzyme from hyperthermophilic archaeon Thermococcus profundus,
which hydrolyses both a-1-4 and a-1-6 glucosidic linkages. J. Ferment.
Bioeng. 86, 363367.
Laderman, K., Davis, B., Krutzsch, H., Lewis, M., Griko, Y., Privalov, P.,
Anfinsen, C., 1993a. The purification and characterization of an
extremely thermostable a-amylase from the hyperthermophilic
archaebacterium Pyrococcus furiosus. J. Biol. Chem. 268, 24394
Landerman, K., Asada, K., Uemori, T., Mukai, H., Taguchi, Y., Kato, I.,
Anfinsen, C., 1993b. Alpha amylase from the hyperthermophilic
archaebacterium Pyrococcus furious. Cloning and sequencing of the
gene and expression in E. coli. J. Biol. Chem. 268, 2440224407.
Lasa, I., Berenguer, J., 1993. Thermophilic enzymes and their
biotechnological potential. Microbiologia 9, 7789.
Lee, S., Rhee, J., 1993. Production and partial purification of a lipase from
Pseudomonas putida 3 SK. Enzyme Microbiol. Technol. 15, 617623.
Lee, Y., Lowe, S., Zeikus, J., 1998. Molecular biology and biochemistry
of xylan degradation by thermoanaerobes. In: Kalegoris, E.,
Christakopoulos, D., Kekos, D., Macris, B. (Eds.), Studies on the
solid-state production of thermostable endoxylanases from
Thermoascus aurantiacus: characterization of two isoenzymes. J.
Biotechnol. 60, 155163.
Leuschner, C., Antranikan, G., 1995. Heat stable enzymes from
extremely thermophilic and hyperthermophilic microorganisms.
World J. Microbiol. Biotechnol. 11, 95114.
Ling, L., Keohavong, P., Dias, C., Thilly, W., 1991. Optimization of the
polymerase chain reaction with regard to fidelty: modified T7, Taq,
and Vent DNA polymerases. PCR Meth. Appl. 1, 63
Linko, Y., Lamsa, M., Huhatala, A., Linko, P., 1994. Lipase catalyzed
transesterification of rapeseed oil snf 2-ethyl-1-hexanol. JAOCS 71,
Longley, M., Bennet, S., Mosbaugh, D., 1990. Characterization of the
50300 exonuclease associated with Thermus aquqticus DNA
polymerase. Nucleic Acids Res. 18, 73177322.
Lopez, G., 1999. DNA supercoiling and temperature adaptation: a clue to
early diversification of life. J. Mol. Evol. 46, 439452.
Luisa, M.R., Schmidt, C., Wahl, S., Sprauer, A., Schmid, R., 1997.
Thermoalkalophilic lipase of Bacillus thermocatenulatus. Large
scale production, purification and properties: aggregation behavior
and its effect on activity. J. Biotechnol. 56, 89102.
Lundberg, K., Shoemaker, D., Adams, M., Short, J., Sorge, J., Marthur,
E., 1991. High-fidelty amplification using a thermostable
polymerase isolated from Pyrococcus furiosus. Gene 108, 16.


G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

Majeti, N., Kumar, R., 2000. A review of chitin and chitosan applications.
React. Funct. Polym. 46, 127.
Mamo, G., Gessese, A., 1999. A highly thermostable amylase from a
newly isolated thermophilic Bacillus sp. WN11. J. Appl. Microbiol.
86, 557560.
Manchini, P., Foretina, M., 1998. Production in seawater of
thermostable alkaline protease by a halotolerant strain of Bacillus
licheniformis. Biotechnol. Lett. 20, 565568.
Manchini, P., Foretina, M., Parini, C., 1988. Thermostable alkaline
protease produced by Bacillus thermorubber: a new species of
Bacillus. Appl. Microbiol. Biotechnol. 28, 409413.
Manco, G., Adinolf, E., Pisani, F., Ottolina, G., Carea, G., Rossi, M.,
1998. Overexpression and properties of a new thermophilc and
thermostable esterase from Bacillus acidocaldarius with sequence
similarity to hormone-sensitive lipase subfamily. Biochem. J. 332,
Mao, W., Pan, R., Freedman, D., 1992. High production of alkaline
protease by Bacillus licheniformis in fed-batch fermentation using
synthetic medium. J. Ind. Microbiol. 11, 16.
Mariella, R., 2001. Development of a battery-powered, hand-held, realtime PCR instrument. Proc. SPIEInt. Soc. Opt. Eng. 4265, 5864.
Marjaana, R., Indra, M., Birgitte, A., Liisa, V., 1994. Application of
thermostable xylanase of Dictyoglomus sp. in enzymatic treatment
of kraft pulps. Appl. Microbiol. Biotechnol. 41, 130139.
Matsui, I., Sakai, Y., Matsui, E., Kikuchi, H., Kawarabayasi, Y., Honda,
K., 2000. Novel substrate specificity of a membrane-bound bglycosidase from the hyperthermophilic archaeon Pyrococcus
horikoshi. FEBS Lett. 467, 195200.
Mawadza, C., Hatti-Kaul, R., Zvauya, R., Mattiasson, B., 2000.
Purification and characterization of cellulases produced by two
Bacillus strains. J. Biotechnol. 83, 177187.
Mayer, J., Lupas, A., Kellerman, J., Eckerskorn, C., Baumeister, W.,
Peters, J., 1996. A hyperthermostable protease of the subtilisin
family bound to the surface of the layer of the archaeon
Staphylothermus marinus. Curr. Biol. 6, 739749.
Mead, D., McClary, J., Luckey, J., Kostichka, A., Witney, F., Smith, L.,
1991. Bst DNA polymerase permits rapid sequence analysis from
nanogram amounts of template. Biotechniques 11, 7678.
Medda, S., Chandra, K., 1980. New strains of B. licheniformis and B.
coagulans producing thermostable a-amylases active at alkaline pH.
J. Appl. Bacteriol. 48, 4758.
Min Ha, Y., Lee, D., Yoon, J., Park, Y., Kim, Y., 2001. Rapid and simple
purification of a novel extracellular b-amylase from Bacillus sp.
Biotechnol. Lett. 23, 14351438.
Miura, S., Kubota, N., Kawakita, H., Saito, K., Sugita, K., Watanabe,
K., Sugo, T., 2002. High-throughput hydrolysis of starch during
permeation across alpha-amylase-immobilized porous hollow-fiber
membranes. Radiat. Phys. Chem. 63, 143149.
Morgan, F., Priest, F., 1981. Characterization of thermostable aamylases
from B. licheniformis. J. Appl. Bacteriol. 50, 107114.
Morris, D., Gibbs, M., Chin, C., Koh, H., Wong, K., Allison, R.,
Nelson, P., Bergquist, P., 1998. Cloning of the xynB gene form
Dictyoglomus thermophilum Rt46B.1 and action of the gene
product on kraft pulp. Appl. Environ. Microb. 64, 17591765.
Mozersky, S., Marmer, W., Dale, A.O., 2002. Vigorous proteolysis:
Relining in the presence of an alkaline protease and bating
(PostLiming) with an extremophile protease. JALCA 97, 150155.
Mozhaev, V., 1993. Mechanism-based strategies for protein
thermostabilization. Trends Biotechnol. 11, 8895.
Mullis, K., Faloona, F., 1987. Specific synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction. Meth. Enzymol. 155, 335
Mullis, K., Faloona, F., Saiki, R., Horn, G., Erlich, H., 1986. Specific
enzymatic amplification of DNA in vitro: the polymerase chain
reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263273.

Myers, T., Gelfand, D., 1991. Reverse transcription and DNA

amplification by a Thermus thermophilus DNA polymerase.
Biochemistry 30, 76617665.
Nagao, T., Shimada, Y., Sugihara, A., Murata, A., Komemushi, S.,
Tominaga, Y., 2001. Use of thermostable Fusarium heterosporum
lipase for production of structured lipid containing oleic and
palmitic acids in organic solvent-free system. JAOCS 78, 167172.
Nakamura, N., Watanabe, K., Horikoshi, K., 1975. Purification and
some properties of alkaline pullulanases from a strain of Bacillus.
Biochem. Biophys. Acta 379, 188193.
Nakamura, H., Kubota, H., Kono, T., Isogai, A., Onabe, F., 2001.
Modification of pulp properties by cellulase treatment and
application of cellulase to wastepaper deinking and mechanical
pulp refining. In: 68th Pulp and Paper Research Conference.
Proceedings of the Pulp and Paper Research Conference, 1819/06,
Japan, pp. 25.
Nawani, N., Dosanjh, N., Kaur, J., 1998. A novel thermostable lipase
from a thermophilic Bacillus sp.: characterization and esterification
studies. Biotecnol. Lett. 20, 9971000.
Nazan, A., Zumrut, B., 2000. A vicel adsorbable endoglucanase
production by the thermophilic fungus Scytalidium thermophilum
type culture Torula thermophila. Enzyme Microb. Technol. 27,
Niehaus, F., Bertoldo, C., Kahler, M., Antranikian, G., 1999.
Extremophiles as a source of novel enzymes for industrial
applications. Appl. Microbiol. Biotechnol. 51, 711729.
Nipkow, A., Shen, G., Zeikus, J., 1989. Continuos production of
thermostable b-amylase with Clostridium thermosulfurogenes:
effect of culture conditions and metabolite levels on enzyme
synthesis and activity. Appl. Environ. Microbiol. 55, 689694.
Norman, B., 1979. The application of polysaccharide degrading
enzymes in starch industry. In: Berkley, R. et al. (Eds.), Microbial
Polysaccharides. Academic Press, New York, pp. 339376.
Nucci, R., Moracci, M., Vaccaro, C., Vespa, N., Rossi, M., 1993.
Exoglucosidase activity and substrate specificity of the bglucosidase isolated from the extreme thermophilic Sulfolobus
solfataricus. Biotechnol. Appl. Biochem. 17, 239250.
Nwe, N., Stevens, W., 2002. Production of fungal chitosan by solid
substrate fermentation followed by enzymatic extraction.
Biotechnol. Lett. 24, 131134.
Pandey, A., Benjamin, S., Soccol, C., Nigam, P., Krieger, N., Soccol, V.,
1999. The realm of microbial lipases in biotechnology: a review.
Biotechnol. Appl. Biochem. 29, 119131.
Pantazaki, A., Prista, A., Kyriakidis, D., 2002. Biotechnologically relevant
enzymes from Thermus thermophilus. Appl. Microbiol. Biotechnol.
58, 112.
Park, Y., Kang, S., Lee, J., Hong, S., Kim, S., 2002. Xylanase production
in solid state fermentation by Aspergillus niger mutant using statistical
experimental designs. Appl. Microbiol. Biotechnol. 58, 761766.
Paula, S., Alexandra, M., Jose, C., Maria, R., Maria, C., 2002. Rapid
detection of thermostable cellulase free xylanases by a strain of
Bacillus subtilis and its properties. Enzyme Microbiol. Technol. 30,
Pawinee, K., Wipapat, K., 1996. Thermostable metalloproteases excreted
by gram-negative thermophillic bacterium. In: Poster Presentation:
10th International Biotechnology symposium. Sydney, Australia.
Perler, F., Kumar, S., Kong, H., 1996. Thermostable DNA polymerases.
Adv. Protein Chem. 48, 377435.
Peter, W., Alexandra, T., Klaus, D., 2001. Conversions of glycerol to 1,3propandiol by a newly isolated thermophilic strain. Biotechnol. Lett.
23, 463466.
Piller, K., Daniel, R., Petach, H., 1996. Properties and stabilization of an
extracellular a-glucosidase from the extremely thermophilic archaebacteria Thermococcus strain AN1: enzyme activity at 130 C.
Biochem. Biophys. Acta 1292, 197205.

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

Plant, A., Morgan, H., Daniel, R., 1986. A highly stable pullulanase from
Thermus aquaticus. Enzyme Microb. Technol. 8, 668672.
Poonam, N., Dalel, S., 1995. Enzyme and microbial systems involved in
starch processing. Enzyme Microb. Technol. 17, 770778.
Rahman, R., Razak, C., Ampon, K., Basri, M., Yunus, W., Salleh, A.,
1994. Purification and characterisation of a heat stable protease from
Bacillus stearothermophilus F1. Appl. Microbiol. Biotechnol. 40, 822
Rakshit, S.K., 1998. Utilization of starch industry wastes. In: Martin, A.
(Ed.), Bioconversion of Waste Materials to Industrial Products, second
ed. Blackie Academic and Professional, London.
Rama, M., Swamy, M., Seenayya, G., 1998. Purification and
characterization of thermostable b-amylase and pullulanase from high
yielding Clostridium thermosulfurogenes SV2. World J. Microbiol.
Biotechnol. 14, 8994.
b-amylase and pullulanase in the presence of surfactants by
Clostridium thermosulfurogenes SV2. Proc. Biochem. 34, 8792.
Ramkrishna, S., Samir, K., Chakrabarty, S., 1993. Immobilization of aamylase from Mycelophthora thermophila D-14 (ATCC 48104).
Enzyme Microbiol. Technol. 15, 801804.
Rao, M., Tankasale, A., Ghatge, M., Desphande, V., 1998. Molecular and
biotechnological aspects of microbial proteases. Microbiol. Mol. Biol.
Rev. 62, 597634.
Rathi, P., Sapna, B., Sexena, R., Gupta, R., 2000. A hyperthermostable,
alkaline lipase from Pseudomonas sp. with the property of thermal
activation. Biotechnol. Lett. 22, 495498.
Razak, C., Samad, M., Basri, M., Yunus, W., Ampon, K., Salleh, A., 1993.
Thermostable extracellular protease by B. stearothermophilus. World
J. Microbiol. Biotechnol. 10, 260263.
Razak, C., Rahman, R., Salleh, A., Yunus, W., Ampon, K., Basri, M.,
1995. Production of a thermostable protease from a new high pH
isolate of Bacillus stearothermophilus. J. Biosci. 6, 94100.
Razak, C., Tang, S., Basri, M., Salleh, A., 1997. Prelimenary study on the
production of extracellular protease from a newly isolated Bacillus sp.
(no. 1) and the physical factors affecting its production, Pertanika. J.
Sci. Technol. 5, 169177.
Rina, R., Geeta, N., 1996. Microbial b-amylase: Biosynthesis,
characterization and industrial applications. Crit. Rev. in Microbiol.
22, 181199.
Roberts, G., 1992. Physical structures. In: Roberts, G.A.F. (Ed.), Chitin
Chemistry, first ed. Macmillan, London, pp. 2035.
Rolfsmeir, M., Blum, P., 1995. Purification and characterization of a
maltose from the extremely thermophilic crenarchaeote Sulfolobus
solfataricus. J. Bacteriol. 177, 482485.
Rothschild, L., Manicineli, R., 2001. Life in extreme environments.
Nature 409, 10921101.
Rudiger, A., Jorgensen, P., Antranikan, G., 1995. Isolation and
characterization of a heat stable pullulanase from the
hyperthermophilic Archeon Pyrococcus woesei after cloning and
expression of its gene in Escherichia coli. Appl. Environ. Microbiol.
61, 567575.
Ruthersmith, L., Daniel, R., 1991. Thermostable cellobiohydrolase from
the thermophilic eubacterium Thermotoga sp. Strain FjSS3B.1.
Purification and properties. Biochem. J. 277, 887890.
Ruthersmith, L., Daniel, R., 1992. Cellulytic and hemicellulytic enzymes
functional above 100 C. Ann. NY Acad. Sci. 672, 137
Saboto, D., Nucci, R., Rossi, M., Gryczynski, I., Gryczyniski, Z.,
Lakowicz, J., 1999. The b-glycosidase from the hyperthermophilic
archaeon Sulfolobus solfataricus: enzyme activity and conformational
dynamics at temperatures above 100 C. Biophys. Chem. 81, 2331.
Saha, B., Mathupala, S., Zeikus, J., 1988. Purification and characterization
of a highly thermostable novel pullulanase from Clostridium
thermohydrosulfuricum. Biochem. J. 252, 343348.


Saiki, R., Gelfand, D., Stoffe, S., Scharf, S., Higuchi, R., Horn, G., Mullis,
K., Erlich, H., 1988. Primer directed enzymatic amplification of DNA
with thermostable DNA polymerase. Science 239, 487491.
Salleh, A.B., Basri, M., Razak, C., 1977. The effect of temperature on the
protease from Bacillus stearothermophilus strain F1. Mal. J. Biochem.
Mol. Biol. 2, 3741.
Sarikaya, E., Higassa, T., Adachi, M., Mikami, B., 2000. Comparison of
degradation abilities of a- and b-amylases on raw starch granules.
Proc. Biochem. 35, 711715.
Satosi, H., Seigo, O., Kenji, T., Kazuhisa, K., Tetsuo, K., Toshiaki, K.,
Hitosi, K., 2001. Chemo-enzymatic synthesis of 3-(2-naphtyl)Lalanine by an amino transferase from the extreme thermophiles
Thermococcus profoundus. Biotechnol. Lett. 23, 589591.
Saul, D., Williams, L., Reeves, R., Gibbs, M., Bergquist, P., 1995.
Sequence and expression of a xylanase gene from the
characterization of the recombinant enzyme and its activity on Kraft
pulp. Appl. Environ. Microb. 61, 41104113.
Schmidt, C., Sztajer, H., Stocklein, W., Menge, U., Schimid, R., 1994.
Screening, purification and properties of a thermophilic lipase from
Bacillus thermocatelnulatus. Biochem. Biophys. Acta 1214, 4353.
Sebastien, Z., Jean-Luk, R., Dider, F., Yannik, G., Joseph, B., Jacques, D.,
2001. Characterization of highly thermostable alkaline phosphatase
from the Euryarchaeon Pyrococcus abyssi. Appl. Environ. Microbiol.
67, 45044511.
Sharma, R., Soni, S., Vohra, R., Gupta, L., Gupta, J., 2002.
Purification and characterization of a thermostable alkaline lipase
from a new thermophilic Bacillus sp. RSJ-1. Proc. Biochem. 37,
Shaw, J., Pang, L., Chen, S., Chen, I., 1995. Purification and properties of
an extracellular a-amylase from Thermus sp. Bot. Bull. Acad. Sci. 36,
Shen, G., Saha, B., Lee, Y., Bhatnagar, L., Zeikus, J., 1988. Purification
and characterization of a novel thermostable bamylase from
Clostridium thermosulfurogenes. Biochem. J. 254, 835840.
Shimada, Y., Watanabe, Y., Samukawa, T., Sugihara, A., Noda, H.,
Fukuda, H., Tominaga, Y., 1999. Conversion of vegetable oil to
biodiesel using immobilized Candida antarctica lipase. JAOCS 76,
Simpson, H., Haufler, U., Daniel, K., 1991. An extremely thermostable
xylanase from the thermophilic eubacterium Thermotoga. Biochem.
J. 277, 177185.
Spano, L., Medeiros, J., Mandels, M., 1975. In: Enzymatic Hydrolysis
of Cellulosic Waste to Glucose (Pollution Abatement Div., Food
Svcs. Lab, US Army Natick Labs), Natick, MA.
Spindler, K., Spindler, B.M., Londershausen, M., 1990. Chitin
metabolism: a target for drugs against parasites. Parasitol. Res. 76,
Stamford, T., Stamford, N., Coelho, L., Araujo, J., 2001. Production and
characterization of thermostable. Endophyte of yam bean aamylase
from Nocardiopsis sp. Biores. Technol. 76, 137141.
Stetter, K., 1999. Extremophiles and their adaptation to hot environments.
FEBS Lett. 452, 2225.
Sunna, A., Moracci, M., Rossi, M., Antranikian, G., 1996a. Glycosyl
hydrolases from hyperthermophiles. Extremophiles 1, 213.
Suntornsuk, W., Pochanakanich, P., Suntornsuk, L., 2002. Fungal
chitosan production on food processing by-products. Proc.
Biochem. 37, 727729.
Suzuki, Y., Chishoro, M., 1983. Production of extracellular
thermostable pullulanase by an amylolytic obligatory thermophilic
soil bacterium B. stearothermophilus. Eur. J. Appl. Biotechnol. 17,
24 29.
Swampy, M., Sai Ram, M., Seenayya, G., 1994. b-amylase from
Clostridium thermocellum SS8a thermophilic, anaerobic,
cellulytic bacterium. Lett. Appl. Microbiol. 18, 301304.


G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734

Swamy, M., Seenayya, G., 1996. Thermostable pullulanase and

aamylase activity from Clostridium thermosulfurogenes SV9optimization of culture conditions from enzymes production. Proc.
Biochem. 31, 157162.
Swaroopa, D., Krishna, N., 2000. Production of thermostable cellulasefree xylanases by Clostridium absonum CFR-702. Proc. Biochem.
36, 355362.
Takami, H., Akiba, T., Horikoshi, K., 1989. Production of extremely
thermostable alkaline protease from Bacillus sp. Appl. Microbiol.
Biotechnol. 30, 120124.
Takasaki, Y., 1976a. Production and utilization of b-amylase and
pullulanase from Bacillus cereus var. mycoides. Agri. Biol. Chem.
40, 15151522.
Takasaki, Y., 1976b. Purification and enzyme properties of b-amylase
and pullulanase from Bacillus cereus var. mycoides. Agri. Biol.
Chem. 40, 15231530.
Takayanagi, T., Ajisaka, K., Takiguchi, Y., Shimahara, K., 1991.
Isolation and characterization of thermostable chitinases from
Bacillus lichiniformis X_7u. Biochem. Biophys. Acta 1078, 404
Takizawa, N., Muroorka, Y., 1985. Cloning of the pullulanase gene in E.
coli and Klebsiella aerogenes. Appl. Environ. Microbiol. 49, 294
Tan, K., Gill, C., 1985. Effect of culture conditions on batch growth of
Pseudomonas fluorescens on olive oil. Appl. Microbiol. Biotechnol.
23, 2732.
Traore, M.K., Buschle-Diller, G., 2000. Environmentally friendly
scouring processes. Textile Chem. Colorist Am. Dye Stuff Report.
32, 4043.
Tsujibo, H., Endo, H., Miyamoto, K., Inamori, Y., 1995. Expression in
Escherichia coli of a gene encoding a thermostable chitinase from
Streptomyces thermoviolaceus OPC-520. Biosci. Biotechnol.
Biochem. 59, 145146.
Underkofler, L., 1976. Microbial enzymes. In: Miller, B., Litsky, W.
(Eds.), Industrial Microbiology. McGraw-Hill, New York.
Van der Maarel, M., Van der Veen, B., Uitdehaag, H., Leemhuis, H.,
Dijkhuizen, L., 2002. Properties and applications of
starchconverting enzymes of the a-amylase family. J. Biotechnol.
94, 137155.
Van wyk, J., Mogale, A., Seseng, T., 2001. Bioconversion of wastepaper to
sugars by cellulase from Aspergillus niger, Trichoderma viride and
Penicillium funiculosum. J. Solid Waste Technol. Manage. 27, 8286.
Velikodvorskaya, T., Volkov, I., Vasilenko, V., Zverlov, V., Pirusian, E.,
1997. Purification and some properties of Thermotoga neapolitana
thermostable xylanase B expressed in E. coli cells. Biochemistry
62, 6670.
Viara, N., Elena, P., Elka, I., 1993. Purification and characterization of a
thermostable alpha-amylase from Bacillus licheniformis. J.
Biotechnol. 28, 277289.
Vihinen, M., Mantsala, P., 1990. Characterization of a thermostable
Bacillus stearothermophilus alpha-amylase. Biotechnol. Appl.
Biochem. 12, 427435.

Vikari, L., Kantelinen, A., Sundquist, J., Linko, M., 1994. Xylanases in
bleaching: from an idea to the industry. FEMS Microbiol. Rev. 13,
Volkl, P., Stetter, K., Miller, J., 1995. The sequence of a subtilin-type
protease (aerolysin) from the hyperthermophilic archaeon
Pyrobaculum aerophilum reveals sites important to thermostability.
Protein Sci. 3, 13291340.
Vulfson, E., 1994. Industrial applications of lipases. In: Wooley, P.,
Peterson, S.B. (Eds.), Lipases: Their Structure, Biochemistry, and
Application. Cambridge University Press, Cambridge, UK, pp.
William, C., Dennis, C., 1988. Food Microbiology, international ed.
McGraw-Hill, NY.
Winterhalter, C., Liebl, W., 1995. Two extremely thermostable
xylanases of the hyperthemophilic bacterium. Thermotoga
maritema MSB8. Appl. Environ. Microbiol. 61, 18101815.
Woese, C., Kandler, O., Wheelis, M., 1990. Towards a natural system of
organisms: Proposal for the domains Archaea, Bacteria, and Eucara.
Proc. Natl. Acad. Sci. USA 87, 45764579.
Wojciechowski, P.M., Koziol, A., Noworyta, A., 2001. Iteration model
of starch hydrolysis by amylolytic enzymes. Biotechnol. Bioeng.
75, 530539.
Wu, X., Jaaskelainen, S., Linko, Y., 1996. An investigation of crude lipase
for hydrolysis, esterification, and transesterification. Enzyme Microb.
Technol. 19, 226231.
Yan, F., Yong-Goe, J., Kazuhiko, I., Hiroyasu, I., Susumu, A., Tohru, Y.,
Hiroshi, N., Shugui, C., Ikuo, M., Yoshitsugu, K., 2000. Thermophilic
phospholipase A2 in the cytosolic fraction from the archaeon
Pyrococcus horikoshii. JAOCS 77, 10751084.
Young, M., Dong, G., Jung-Hoon, Y., Yong-Ha, P., Young, J., 2001. Rapid
and simple purification of a novel b-amylase from Bacillus sp.
Biotechnol. Lett. 23, 14351438.
Zeikus, J., Lee, C., Lee, Y., Saha, B., 1998a. Thermostable saccharides:
new sources, uses and biodesigns. In: Kalegoris, E., Christakopoulos,
D., Kekos, D., Macris, B. (Eds.), Studies on the Solid-State Production
of Thermostable Endoxylanases from Thermoascus aurantiacus:
Characterization of Two Isoenzymes. J. Biotechnol. 60, 155163.
Zeikus, J., Vielle, C., Savachenko, A., 1998b. Thermozymes:
biotechnology and structure-function relationships. Extremophiles 2,
179 183.
Zhang, J., Greasham, R., 1999. Chemically defined media for commercial
fermentations. Appl. Microbiol. Biotechnol. 51, 407 421.
Zhou, L., Yeung, K, Yuen, C., 2001. Combined cellulase and wrinklefree
treatment on cotton fabric. J. Dong Hua University 18, 11
Zverlov, V., Piotukh, K., Dakhova, O., Velikodvorskaya, G., Borris, R.,
1996. The multidomain xylanase A of hyperthermophilic bacterium
Thermotoga neapolitana is extremely thermoresistant. Appl.
Microbiol. Biotechnol. 45, 245247.
Zverlov, V., Riedel, K., Bronnenmeier, K., 1998. Properties and gene
structure of a bifunctional cellulytic enzyme (CelA) from the extreme
thermophile Anaerocellum thermophilum with separate glycosyl
hydrolase family 9 and 48 catalytic domains. Microbiology 144, 457