Review Paper
Developments in industrially important thermostable enzymes: a review

https://doi.org/10.1016/S0960-8524(03)00033-6Get rights and content

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.

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 purification 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 beneficial thermophilic microorganisms from different exotic ecological zones of the earth and the subsequent extraction of useful enzymes from them (Burrows, 1973; Antranikian et al., 1987; Groboillot, 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, Zeikus et al., 1998b) 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.

Section snippets

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 0–3 or 10–12), or at a very high (5–30%) salt concentration (Herbert and Sharp, 1992).

In the last decade, a number of hyperthermophilic archaea, the least

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

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

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 α-amylases, glucoamylases or β-amylases and isoamylases or pullulanases (Poonam and Dalel, 1995). The enzymes are classified into endo-acting and exo-acting enzymes. α-amylase is an endo-acting enzyme and hydrolyses linkages in a random fashion and

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

Thermostable cellulases

Cellulose, the most abundant organic source of feed, fuel and chemicals (Spano et al., 1975) consists of glucose units linked by β-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 β-glucosidases (Matsui et al., 2000). While cellulase is an

Thermoactive chitinases

Chitin, the second most abundant natural biopolymer after cellulose, consists of a linear β-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

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

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, Kim et al., 2002b). Their unique characteristics include substrate specificity, stereospecificity, regioselectivity and ability to catalyze a heterogeneous reaction at the

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 90–95 °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

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

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

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