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 specic 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 specic 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 The role of enzymes in many processes has been known for a long time. Their existence was associated with the history of ancient Greece where they were using enzymes from microorganisms in baking, brewing, alcohol production, cheese making etc. With better knowledge and purication of enzymes the number of applications has increased manyfold, and with the availability of thermostable enzymes a number of new possibilities for industrial processes have emerged. Thermostable enzymes, which have been isolated mainly from thermophilic organisms, have found a number of commercial applications because of their overall inherent stability (Demirijan et al., 2001). Advances in this area have been possible with the isolation of a large number of benecial thermophilic microorganisms from dierent exotic ecological zones of the earth and the subsequent extraction of useful enzymes from them (Burrows, 1973; Antranikian et al., 1987; Groboillot,

Corresponding author. Tel.: +66-2-524-6115; fax: +66-2-524-6111. E-mail address: rakshit@ait.ac.th (S.K. Rakshit).

1994; Bharat and Hoondal, 1998; Bauer et al., 1999; Kohilu et al., 2001). While the most widely used thermostable enzymes are the amylases in the starch industry (Poonam and Dalel, 1995; Crab and Mitchinson, 1997; Emmanuel et al., 2000; Sarikaya et al., 2000), a number of other applications are in various stages of development. In the food related industry, they have been used in the synthesis of amino acids (Satosi et al., 2001). In the petroleum, chemical and pulp and paper industries, for example, thermostable enzymes have been used for the elimination of sulphur containing pollutants through the biodegradation of compounds like dibenzothiophene (Bahrami et al., 2001), in the production of 1,3-propanediol from glycerol and in replacing polluting chemical reagents causing toxic products (Peter et al., 2001). Currently, a number of publications have extensively discussed developments in this area. Adaptation of extremophiles to hot environments (Danson et al., 1992; Stetter, 1999), production of heat-stable enzymes from thermophiles and hyperthermophiles (Knor, 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)

0960-8524/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0960-8524(03)00033-6

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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 ecient 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.

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 specic 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-

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 Examples of sites serving as sources of microorganisms which can provide thermo tolerant enzymes Source Hot spring Hot spring Deep sea hydrothermal vent Marine solfatare Decomposed plant samples from a lake Hot spring Compost of fermenting citrus peels, coee and tea extract residues Compost Korean salt fermented anchovy Deep sea hydrothermal vent Sediments of hot springs Garbage dump Compost treated with artichoke juice Microorganism Thermus sp. Bacillus sp. WN.11 Staphilothermus marinus Thermococcus litoralis Clostridium absonum CFR-702 Bacillus thermoleovocans ID-1 Bacillus strain MH-1 Enzyme a-Amylase a-Amylase a-Amylase Pullulanase Cellulase free xylanase Lipase Endochitinase References Shaw et al. (1995) Mamo and Gessese (1999) Canganella et al. (1994) Brown and Kelly (1993) Swaroopa and Krishna (2000) Dong-Woo et al. (1999) Kenji et al. (1998)

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)

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734 Table 2 Bioconversion reactions and applications of thermostable enzymes Enzyme a-Amylase (bacterial) a-Amylase (fungal) Pullulanase Xylanase Chitinase Temperature range (C) 90100 5060 5060 4565, 105a 6575b Bioconversions Starch ! dextrose syrups Starch ! dextrose syrups Starch ! dextrose syrups Craft pulp ! xylan + lignin Chitin ! chitobiose Chitin ! N -acetyl glucosamine (chitibiase) N -acetyl glucosamine ! glucosamine (deacetylation) Chitin ! chitosan (deacetylase) Cellulose ! glucose Protein ! amino acids and peptides Fat removal, hydrolysis, interesterication, alcholysis, aminolysis DNA amplication Applications Starch hydrolysis, brewing, baking, detergents Production of maltose Production of glucose syrups Pulp and paper industry Food, cosmetics, pharmaceuticals, agrochemicals

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Cellulase Protease Lipase

4555, 95c 6585 3070

DNA polymerase
a

9095

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

Xylanase from Thermotoga sp. b Within this range enzyme activity was high. c Cellulases 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 eect of structural uctuations 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 exibility 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.

(Leuschner and Antranikan, 1995; Fredrich and Antrakian, 1996; Diane 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 signicant inuence on the bioavailability and solubility of organic compounds and thereby provides ecient bioremediation (Becker, 1997). Other values of elevated process temperatures include higher reaction rates due to a decrease in viscosity and an increase in diusion coecient 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 specic thermostable enzymes, their sources, characteristics and application requirements of high temperature resistance.

4. 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

5. Amylolytic enzymes The starch industry is one of the largest users of enzymes for the hydrolysis and modication of this useful raw material. The starch polymer, like other such

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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 classied into endo-acting and exo-acting enzymes. a-amylase is an endo-acting enzyme and hydrolyses 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 saccharication, 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-

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 diversied 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; 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 (Underkoer, 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 Source microorganisms and properties of thermostable starch hydrolyzing enzymes Enzymes a-Amylase Organism 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 Bacillus circulans Bacillus cereus var. mycoides Bacillus sp. Clostridium thermosulphurogenes Clostridium thermosulfurogenes Bacillus sp. Pyrococcus furiosus Pyrococcus woesi Thermococcus aggregans Thermus caldophilus GK24 Thermococcus celer Thermococcus hydrothermalis Thermococcus litoralis Thermotoga maritima MSB8 Enzyme properties Optimal temperature (C) 70 100 7080 70 70 55 100 100 100 65 100 90 95 85 60 80 60 50 50 75 60 60 98 100 100 75 90 95 98 90 Optimal pH 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 7.5 5.5 5.86.0 5.5 5.56.0 6.5 5.5 5.5 5.5 5.5 6.0 Underkoer (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) Poonam and Dalel (1995) Takasaki (1976a,b) Young et al. (2001) Shen et al. (1988) Nipkow et al. (1989) Takasaki (1976a,b) 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) References

b-Amylase

Pullulanase

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The pullulanases that were produced in earlier work on dierent 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 dierent level of dextrose equivalent. Hetero-oligosaccharides 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 LCe, 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 eciently (Hamilton et al., 1999). Saccharication of liqueed starch is carried out at low pH values. However, currently used thermostable a-amylases are not stable at

such low pH (Crab and Mitchinson, 1997). An economical process could be attained through the use of amylases stable at the saccharication 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 tting characteristics (Emmanuel et al., 2000) have placed a challenge both for the scientic 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 bers, (2) need to be of low molecular mass to facilitate their diusion in the pulp bers, 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 fulll 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).

Table 4 Calcium requirement of industrially important starch degrading enzyme Enzyme Bacterial mesophilic a-amylase Bacterial thermophilic a-amylase Fungal a-amylase Amyloglucosidase Pullulanase Source: Hans (1995). Microorganism Bacillus subtilis Bacillus licheniformis Aspergillus oryzae Aspergillus niger Bacillus acidopolluliticus Application temperature range (C) 8085 95105 5570 5565 5565 Application pH range 6.07.0 6.07.0 4.05.0 3.55.0 3.55.0 Minimum Ca2 dosage (ppm) 150 20 50 0 0

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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 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 Enzyme properties Optimal temperature (C) 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 Optimal pH 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 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) References

These enzymes have oered 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 classied 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 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 benets. 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

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mode. The dierence 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 b-glucosidases (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 bers 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 puried and characterized (Bok et al., 1998). Optimal pH for CelA is 6.0 at 95 C, and the pH

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 5.06.0 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 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 cosmetics, paper production, textile nishes, photographic products, cements, heavy metal chelating agents

Table 6 Source microorganisms and properties of thermostable cellulases Organism Anaerocellu thermophilum Bacillus subtilis Pyrococcus furiosus Pyrococcus horicoshi Rhodothermus marinus Thermotoga maritema MSB8 Thermotoga neapoltana (EndocellulaseA) Thermotoga neapoltana (EndocellulaseB) Enzyme properties Optimal temperature (C) 8590 6570 102105 97 95 95 95 106 Optimal pH 5.06.6 5.06.5 6.58.0 6.07.0 6.0 6.06.6 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) References

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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 dicult 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 diraction 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 N acetyl-glucosaminidase were produced from the extreme thermophilic anaerobic arachaeon Thermococcus chitinophagus (Huber et al., 1995). A thermophilic bacterium strain producing three dierent endochitinases in its culture uid was isolated from chitin-containing compost (Kenji et al., 1994). The strains belonged to the genus Bacillus and three isoforms of endochitinases (L, M and S) were puried and characterized. They had dierent 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 iso-electric 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 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 eort to clarify its utilization in the food industry.

9. Thermostable proteases Proteases, which are generally classied into two categories (exopeptidases, that cleave o 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 dierent 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 dened 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 simplied purication 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).

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734 Table 7 Source microorganisms and properties of thermostable proteolytic enzymes Organism 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 Thermoacidophiles (archeal and bacterial origin) Thermococcus aggreganes Thermococcus celer Thermococcus litoralis Thermotoga maritema Enzyme properties Optimal temperature (C) 60 70 60 85 80 75 80 45 100 6070 90 95 85 95 Optimal pH 10.5 9.0 612 12.013.0 9 7 9 7.08.5 7.0 7.5 8.5 9.5 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) Klingberg Klingberg Klingberg Klingberg et et et et al. al. al. al. (1991) (1991) (1991) (1991) References

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, interesterication, esterication, 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 specicity, stereospecicity, 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 avor 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 nished products, drug formulations in the pharmaceutical industry and in the

removal of subcutaneous fat in the leather industry (Pandey et al., 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 rening 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 ora 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

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

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) 70 50 60 68 6070 7075 70 65 90 90 100 97 95 Optimal pH 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)

Active at water immiscible solvents.

(Yan et al., 2000) was involved in crude oil rening. This enzyme was found to optimally react at 95 C and pH of 7.0. Phospholipase A2 has a better performance in the degumming processes of oil reneries 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 specic 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 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 rst thermostable DNA polymerase characterized (Chien et al., 1976; Kaledin et al., 1980). Repeated exposure to 98 C in a reaction buer had little eect on the enzyme activity and signicant activity remained after exposure to 99 C. Although, Taq polymerase exhibits a 50 30 exonuclease activity, a 30 50 exonuclease activity was not detected. Thus, the base insertion delity 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 30 50 -exonuclease-dependent proof reading activity required from a high delity polymerase. Optimization of the PCR procedure can be done by mixing a standard polymerase, such as Taq polymerase with high delity 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 delity has remained to be a serious challenge. The ve 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

G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734 Table 9 Thermostable DNA polymerases in PCR Polymerases BstI pol Deep Vent Pol Pfu pol Pwo pol Taq pol I T pol T pol Tth pol Tma pol Vent pol Organism Bacillus stearothermophilus Pyrococcus sp. GB-D Pyrococcus furiosus Pyrococcus woesei Thermus aquaticus Thermus liformis Thermus avus Thermus thermophilus Thermotoga maritema Thermococcus litoralis PCR reactions HF LF LF LF HF NI NI HF LF LF References 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)

27

HF and LF, high and low delity; NI, not identied.

30 50 -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 delity 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 specic 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 identied 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 5.56.0 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 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, 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 (Duner et al., 1996; Chernin et al., 1997). In cases where a relevant gene of a thermostable enzyme has been identied 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 specic 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 benets, production of the enzymes to the level required by the industries has remained a challenge.

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G.D. Haki, S.K. Rakshit / Bioresource Technology 89 (2003) 1734 Bergquist, P., Love, D., Croft, J., Strei, 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., Duner, 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, purication 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. Purication, characterization and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana. Appl. Environ. Microbiol. 64, 47744781. Bost, D., Stoel, 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. Breccia, J., Sineriz, F., Baigori, M.D., Guillermo, R.C., Hatti-Kaul, R., 1997. Purication and characterisation of a thermostable xylanase from Bascillus amyloliquefaciens. Enzyme Microb. Technol. 22, 4249. Brockman, H., Borgstorm, B., 1984. Lipases. Elsevier, Amsterdam. Bronnenmeier, K., Kern, A., Libel, W., Staudenbauer, W., 1995. Purication 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 Scientic, Oxford. Buchert, J., Pere, J., Oijusluoma, L., Rahkamo, L., Viikari, L., 1997. Cellulasestools for modication 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, 239245. Carlsen, M., Nielsen, J., Jorgensen, S.B., Zangirolami, T.C., 2002. Growth and enzyme production during continuous cultures of a high amylase-producing variant of Aspergillus oryzae. Braz. J. Chem. Eng. 19, 5567. 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.

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