Haki 2003

Bioresource Technology
89 (2003) 1734
Review Paper
Developments in industrially important thermostable enzymes: a

review
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
Abstract
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: rakshit@ait.ac.th (S.K. Rakshit).
G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734
18
0960-8524/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-8524(03)00033-6
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

denaturation
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
Source
Microorganism
Enzyme
References
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
a-Amylase
a-Amylase
a-Amylase
Pullulanase
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
Lipase
Endochitinase
Dong-Woo et al. (1999)

Kenji et al. (1998)
Compost
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.
b-N-acetylhexosaminidase
b-Amylase
Alkaline phosphatase
a-Amylase-like pullulanase
Xylanase
Inulinase
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

Enzyme
Temperature range (C)
Bioconversions
Applications
a-Amylase (bacterial)
90100
Starch!dextrose syrups
a-Amylase (fungal)
Pullulanase
Xylanase
Chitinase
5060
5060
4565, 105a
6575b
Craft pulp!xylan+lignin
Chitin!chitobiose
Starch hydrolysis, brewing, baking,

detergents
Production of maltose
Production of glucose syrups
Pulp and paper industry
Food, cosmetics, pharmaceuticals,
agrochemicals
Cellulase
4555, 95c
Chitin!N-acetyl glucosamine
(chitibiase)
N-acetyl glucosamine!
glucosamine (deacetylation) Chitin!
chitosan (deacetylase)
Cellulose!glucose
Protease
6585
Protein!amino acids and peptides
Lipase
3070
Fat removal, hydrolysis,

interesterification, alcholysis, aminolysis
DNA polymerase
9095
a
Xylanase from Thermotoga sp.
b
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.
4.
19
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
industry
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.
5.
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
20
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
non-reducing
end,
producing
oligo
and/or
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
Enzymes
Organism
Enzyme properties
Optimal temperature
C) Optimal
(
pH
References
a-Amylase
Bacillus amyloliquefaciens
Bacillus licheniformis
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
70
100
7080
70
70
55
100
100
100
65
100
90
95
85
60
80
7.0
6.06.5
5.06.0
7.0
5.5
5.6
5.5
6.57.5
5.0
5.5
5.5
4.06.3
4.87.8
5.6
4.05.0
Underkofler (1976)
Viara et al. (1993)
Vihinen and Mantsala (1990)
Jeayoung et al. (1989)
Aguilar et al. (2000)
Ramkrishna et al. (1993)
Laderman et al. (1993a,b)
Koch et al. (1991)
Haseltine et al. (1996)
Estelle et al. (1997)
Estelle et al. (1997)
Jensen and Olsen (1991)
Kwak et al. (1998)
b-Amylase
Bacillus circulans
60
Poonam and Dalel (1995)
Bacillus cereus var. mycoides

Bacillus sp.
Clostridium thermosulphurogenes
Clostridium thermosulfurogenes
50
50
75
60
7.5
5.5
5.86.0
Takasaki (1976a,b)
Young et al. (2001)
Shen et al. (1988)
Nipkow et al. (1989)
Bacillus sp.
60
Takasaki (1976a,b)
Pyrococcus furiosus
Pyrococcus woesi
Thermococcus aggregans
Thermus caldophilus GK24
Thermococcus celer
Thermococcus hydrothermalis
Thermotoga maritima MSB8
98
100
100
75
90
95
98
90
5.5
5.56.0
6.5
5.5
5.5
5.5
5.5
6.0

Rudiger et al. (1995)
Kim et al. (1996)
Gantlet and Duchiron (1998)
Bibel et al. (1998)
Pullulanase
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,
2000).
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
21
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).
22

Enzyme
Microorganism
Application temperature
range (C)
Application pH
range
Minimum Ca2 dosage

(ppm)
Bacterial mesophilic a-amylase
Bacillus subtilis
8085
6.07.0
150
Bacterial thermophilic a-amylase
95105
6.07.0
20
5570
5565
5565
4.05.0
3.55.0
3.55.0
50
0
0
Fungal a-amylase
Aspergillus oryzae
Amyloglucosidase
Aspergillus niger
Pullulanase
Bacillus acidopolluliticus
Source: Hans (1995).
Table 5
Source microorganisms and properties of thermostable xylanases
Organism
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 neapolitana
Thermotoga sp. strain FjSS3-B1
Thermotoga thermarum
80
80
6075
75
50
75
90
55
100
102
65
60
105
60, 70
7075
50
92
95
75
95
85
102
80
105
115
80
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) (
6.87.0
6.07.0
8.09.0
6.0
6.0
8.5
6.07.0
5.05.5
6.0
6.0
6.0
5.3
7.0, 6.7
6.5
5.0
6.2
6.07.5
6.2
6.0
5.5
5.5
7.0
6.87.8
5.3
6.6
References
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.,
2002).
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
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
23
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,
1992).
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
24
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
Organism
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) (
References
Optimal pH
Anaerocellu thermophilum
Bacillus subtilis
Pyrococcus furiosus
Pyrococcus horicoshi
Rhodothermus marinus
Thermotoga maritema MSB8
Thermotoga neapoltana
(EndocellulaseA)
Thermotoga neapoltana
(EndocellulaseB)
8590
6570
102105
97
95
95
95
5.06.6
5.06.5
6.58.0
6.07.0
6.0
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)
106
6.06.6
Bok et al. (1998)
cosmetics,
paper
production,
textile
finishes,
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
extreme
thermophilic
anaerobic
arachaeon
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
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
of
protease
activity
excreted
from
Bacillus
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).
25
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
Organism
Bacillus brevis
Bacillus sp. JB-99
Bacillus stearothermophilus TP26
Bacillus sp. no. AH-101
Bacillus thermoruber
Pyrococcus sp. KODI
Staphylothermus marinus
Thermoacidophiles
(archeal and bacterial origin)
Thermococcus aggreganes
Thermococcus celer
Thermotoga maritema
Enzyme properties
Optimal temperature
C) ( Optimal pH
References
60
70
60
85
80
75
80
45
100
6070
10.5
9.0
612
12.013.0
9
7
9
7.08.5
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)
90
95
85
95
7.0
7.5
8.5
9.5
Klingberg et al. (1991)

subcutaneous fat in the leather industry (Pandey et al.,
26
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,
1998).
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
Organism
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
a
Enzyme properties
Optimal temperature
C) ( Optimal pH
70
50
60
68
6070
7075
70
65
90
90
100
97
95
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
8.09.0
8.0
8.09.0
7.5
9.0
9.6
11.0
5.6
7.0
References
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
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.,
1991).
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
Polymerases
Organism
BstI pol
Deep Vent Pol
Pfu pol
Pwo pol
Taq pol I
Tfi pol
Tfl pol
Tth pol
Pyrococcus sp. GB-D
Pyrococcus furiosus
Pyrococcus woesei
Thermus aquaticus
Thermus filiformis
Thermus flavus
Thermus thermophilus
Tma pol
Thermotoga maritema
Vent pol
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
27
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
neapolitana
genes
encoding
for
xylanases
(Velikodvorskaya et al., 1997), Protease genes from
PCR reactions
References
HF
LF
LF
LF
HF
NI
NI
HF
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)
LF
LF
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,
28
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.
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Bioresource Technology

Developments in industrially important thermostable enzymes: a

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

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

and biotechnological and industrial applications of

2. Geothermal sites as sources of extremophiles

and Methanopyrus. Within the bacteria, Thermotoga

3. Resistance of thermophiles to high temperatures and

Shaw et al. (1995)

Bacillus thermoleovocans ID-1

Dong-Woo et al. (1999)

Bacillus stearothermophilus CH-4

Kenji et al. (1994)

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

Temperature range (C)

Starch hydrolysis, brewing, baking,

Protein!amino acids and peptides

Fat removal, hydrolysis,

from Thermotoga sp.

isms maximum temperature for growth. Thermophiles also

General advantages of enzymes from thermophiles

Thermostable enzymes are gaining wide industrial and

Cellulose hydrolysis, polymer

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

The starch industry is one of the largest users of

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

linkages in a random fashion and leads to the formation

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

atinization, hence, among other things avoiding a lot of

Poonam and Dalel (1995)

Bacillus cereus var. mycoides

Brown and Kelly (1993)

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

The pullulanases that were produced in earlier work

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

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

Minimum Ca2 dosage

Bacterial mesophilic a-amylase

Bacterial thermophilic a-amylase

These enzymes have offered a major step in the

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

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

benefits. However, scaling up of the enzyme production

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

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

Chitin, the second most abundant natural biopolymer

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

Zverlov et al. (1998)

Bok et al. (1998)

consumption are the muscle tissues. The resulting waste

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

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

There is considerable current interest on the exploration

Banerjee et al. (1999)

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

degumming processes of oil refineries and thereby

Active at water immiscible solvents.

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

facilitated by the availability of thermostable DNA

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