Introduction

Catalysts are substances that reduce the energy barrier of chemical reactions, therefore producing a dramatic increase in reaction rates. Biocatalysts are catalysts of biological origin, which are extremely important, because they are highly specific, highly active under mild environmental conditions and biodegradable (Polastro, 1989; Benkovic and Ballesteros, 1997). These characteristics are highly desirable for the processing industry and a strong tendency exist to replace conventional chemical process and develop new process using this novel type of catalysts. Biocatalysts can be cellular (a whole cell which is used in all its catalytic potential) or acellular (one or more enzymes isolated from the producing cell). This article will refer to acellular biocatalysts.

Despite their obvious advantages as process catalysts, biocatalysts, as proteins, are labile molecules. Therefore biocatalyst stability and stabilization is a central issue of biotechnology today. In fact, biocatalyst operational stability will determine to a large extent the viability of the process, be it new or faced to compete with an already existing technology. Key aspects of biocatalyst stabilization will be reviewed, considering the production of intrinsically stable biocatalysts, strategies for operational stabilization of biocatalysts and mathematical modeling of biocatalyst inactivation during operation.

Enzyme stability is dictated by its three-dimensional configuration, which is in turn determined by genetic (primary structure) and environmental (interaction with the surroundings) factors. The former aspect will be now addressed.

Screening for intrinsically stable biocatalysts is a prominent area of research in biocatalysis. Research on extremophiles, i.e. organisms able to survive and thrive in extreme environmental conditions, as promising sources for highly stable enzymes is a very active research subject at present (Davis, 1998). Those conditions (extreme temperatures, extreme pHs and aggressive chemicals) may be present in the reaction medium, being then fruitful to produce enzymes suited to express and retain their activities in such conditions.

Biocatalyst thermostability allows a higher operation temperature, which is clearly advantageous because of a higher reactivity (higher reaction rate, lower diffusional restrictions), higher stability, higher process yield (increased solubility of substrates and products and favorable equilibrium displacement in endothermic reactions), lower viscosity and fewer contamination problems (Mozhaev, 1993).

Thermophiles represent an obvious source of thermostable enzymes, being reasonable to assume that such character will confer their proteins a high thermal stability. This is certainly so, as can be appreciated in the case of several biotechnologically relevant enzymes from the hyperthermophilic archaebacteria Pyrococcus furiosus and Thermotoga sp (Adams et al, 1995; Fischer et al, 1996, Adams and Kelly, 1998).

Table1. Thermostable enzymes from Pyrococcus furiosus and Thermotoga sp.

In fact, it has been claimed that enzymes from thermophiles are stable at temperatures higher in 20 °C to the optimum growth temperature for such organisms (Daniel, 1996), which represents temperatures even over 100 °C. By now, a high number of thermostable enzymes from thermophiles has been reported; most of them belong to eubacterial and archaebacterial kingdoms (Coolbear et al, 1992). However, the technological use of thermophiles still faces several challenges since knowledge on physiology and genetics of such organisms is poor, they are fastidious, grow slowly and are not recognized as safe. Therefore, even though thermal stability can be considered a rare event in mesophilic organisms, thermostable enzymes used by industry are still produced from mesophiles and commercial enzymes from thermophiles are still scarce, as can be appreciated in Table 2 (Kristjánsson, 1989, Coolbear et al, 1992). Opportunities for thermophilic enzymes in industrial processes has been recently highlighted (Caruana, 1997).

Table 2. Industrial thermostable enzymes, commercial enzymes from thermophiles and termophilic genes cloned in mesophilic hosts

It follows then the convenience of cloning termophilic genes into more suitable mesophilic hosts. Those systems will be highly productive and the enzymes produced will retain its original thermostability. In fact, in a number of cases thermophilic genes have been cloned and expressed in mesophilic hosts, producing enzymes highly active and stable at high temperatures. Some examples are in Table 2 (Kristjánsson, 1989; Adams et al, 1995; Halldórsdóttir, et al 1998; Rúa et al, 1998).

In some cases, remarkable similarities are observed between thermophilic enzymes and their mesophilic counterparts (Vieille and Zekus, 1996). This opens up the possibility of using protein engineering techniques (Imanaka et al, 1988) to produce point mutations in the mesophilic structural gene, which will result in the corresponding aminoacid substitution in the primary structure of the encoded protein (Daniel, 1996). Good results have been obtained in several cases when replacing amino acids for those corresponding to the thermophilic protein. Most effective zones for substitution will be the more flexible for being the more labile (Vieille and Zekus, 1996). However, homology between mesophilic enzymes and their thermophilic counterparts are usually between 30 and 50 % and no general strategy for converting mesophilic into thermophilic enzymes have emerged yet, making thermophiles or the genes derived from them the preferred source for thermostable enzymes in the foreseeable future (Adams and Kelly, 1998)

Genetic engineering and protein engineering are modern techniques already in use for the commercial production of biocatalysts of improved stability, not only to high temperatures, but also to extremes of pH, oxidizing agents and organic solvents. Cloning and expression in suitable hosts is being used routinely by major enzyme production companies to produce improved biocatalysts; this certainly applies to the cloning of thermostable enzyme genes. Protein engineering is also being used to obtain improved biocatalysts, the case of alkaline protease being a paradigm. Already in the market, thermostable proteases capable to withstand harsh washing conditions (high pH, high concentration of strong oxidants) are products of protein engineering produced by point aminoacid substitutions in the most labile region of the molecule (Anonymous, 1997; Anonymous, 1998). In the production of syrups from cornstarch, thermostability of a -amylase is severely reduced below pH 6, which poses the inconvenience of pH adjustment before and after starch liquefaction. A thermostable a -amylase from Bacillus licheniformis, active at low pH and low Ca⁺⁺ concentration has been recently patented (Crabb and Mitchinson, 1997). A thermostable glucose isomerase is a major challenge in the production of high-fructose corn syrup. Equilibrium is favored at high temperature, so that at 110 °C 55 % HFCS could be produced at the enzyme reactor stage, without the cumbersome process of sugar fractionation now used (Pedersen, 1993).

Strategies for the operational stabilization of biocatalysts

Biocatalysts are requested to perform in an environment quite different from its natural habitat. Most enzymatic reactions are performed in aqueous media, which favors inactivation. Water acts as a reactant in inactivation reactions and also as a lubricant in conformational changes associated with protein unfolding (Mozhaev, 1993). Therefore, biocatalyst stabilization under operation is a key issue of biocatalysis.

Several strategies are at hand to increase operational stability: chemical modification of enzyme structure, derivatization, immobilization, crystallization and medium engineering.

Stabilization by chemical modification of the protein molecule is attractive, but has not received much attention (Inada et al, 1986; O'Fágáin et al, 1988; Besson et al, 1995; Erarslan and Ertan, 1995). Increased stability has been obtained by the introduction of hydrophilic groups in the surface of the enzyme molecule that reduces the contact of hydrophobic regions with water, thereby preventing incorrect refolding after reversible denaturation (Mozhaev, 1993).

Derivatization with polymers is being increasingly proposed for the stabilization of soluble enzymes. Modification of proteases with carbohydrate polymers, like polymeric sucrose and dextran, has proven to stabilize them against inactivation induced by temperature and chaotropic agents (Sundaram and Venkatesh, 1998). Horseradish peroxidase has been recently stabilized with several methoxypolyethylene glycols (García and Marty, 1998).

Immobilization to solid carriers is perhaps the most used strategy to improve the operational stability of biocatalysts, other benefits being obtained as well, like better control of operation, flexibility of reactor design, and facilitated product recovery without catalyst contamination (Katchalsky-Katzir, 1993). Despite its great technological potential, few large-scale processes utilize immobilized enzymes. Severe restrictions may arise because of additional costs, activity losses and diffusional restrictions. In the last few years, improvement in carrier and immobilization techniques are opening new options for process development.

Cross-linked enzyme crystals (CLEC) are highly stable novel type biocatalysts. They are produced by stepwise crystallization and molecular cross-linking to preserve the crystalline structure. CLEC are extremely stable, not only with respect to temperature, but also to other inactivating agents like organic solvents and proteases. Specific activity is significantly higher for CLEC than for immobilized enzymes, although activity can be severely restricted by the low molecular flexibility of the enzyme and substrate size exclusion. Stability of CLEC in organic hydrophobic solvents and water-miscible co-solvents is remarkably high (Noritomi et al, 1998); therefore most applications of CLEC are being developed in connection with enzyme synthesis in organic media. Some examples are the production of chiral compounds, peptides and esters. Several CLEC are now on the market. Some of them are lipases, thermolysin, glucose isomerase and penicillin acylase, the last two of paramount commercial significance (Margolin, 1996). Industrial use of CLEC will depend on a positive cost-benefit analysis with respect to other biocatalyst forms.

A completely different approach for biocatalyst stabilization is medium engineering, i.e. the manipulation of reaction medium (Gupta, 1992). Since water is involved in enzyme inactivation, partial or almost total substitution of water might be beneficial for biocatalyst stability (Bell et al, 1995). In fact, numerous cases have been reported where remarkable enzyme stability has been obtained in such media (Koskinen and Klibanov, 1996). Until recently, the use of enzymes in non-aqueous media seemed unfeasible because of the very low activities obtained. However, its tremendous technological potential has been a powerful driving force for research and development in that area of biocatalysis, recent advances are outstanding (Klibanov, 1997) and guidelines have emerged for the proper selection of solvents (Rosell et al, 1998). Even though the main purpose of medium engineering in biocatalysis is associated with the utilization of robust commercial hydrolytic enzymes in organic synthesis (Halling, 1984), thermostability in organic media is an additional bonus of great significance in process economy. In recent years, proteases, lipases, acylases and glycosidases in organic media have been studied in the synthesis of peptides (Feliu et al, 1995; Gill et al, 1996, Sergeeva et al, 1997, Clapés et al, 1997; van Unen et al, 1998), esters (John and Abraham, 1991; Sarney and Vulfson, 1995, Coulon and Ghoul, 1998), oligosaccharides (Bucke, 1996) and glycosides (Stevenson et al, 1993; Scheckermann et al, 1997). Products have considerable pharmacological and industrial relevance and in all those cases thermal stability of the biocatalyst was a key issue.

Biocatalyst thermal stability is a fundamental aspect in reactor performance. Despite this, most information on biocatalyst stability, being gathered under non-reactive conditions, is of limited use, leaving aside modulation effects by substrates and products, which certainly play a role during catalysis. Only in few cases, the modulation of enzyme inactivation by reagents and products has been studied and made explicit in reactor modeling (Illanes et al, 1992; Houng et al, 1993, Illanes et al, 1996, Abu-Reesh and Faqir, 1996).

Based on such considerations, we have developed models to describe enzyme reactor performance considering enzyme inactivation under modulation (Illanes et al. 1994; Illanes et al, 1996; Illanes et al. 1998a). These models certainly represent a better approach to reactor design than conventional models based on stability data gathered under non-reactive conditions (Yang and Okos, 1989; Ortega et al, 1998). These models have been used to determine optimum temperature for reactor operation (Illanes et al. 1998b), which is the most important parameter to be optimized in any biocatalyzed process (Faqir and Abu-Reesh, 1998).

• Biocatalyst stability is a key issue for bioprocess economic viability. Industrial bioprocesses will keep increasing in the future as far as stable and robust biocatalysts are developed to withstand harsh conditions of operation.
• Research on intrinsically stable enzymes from extremophiles is very active and important outcomes for biocatalysis are expected in the near future.
• Gene cloning of such enzymes on mesophilic hosts and protein engineering of mesophilic enzymes are being exploited already by major enzyme companies to develop stable and robust biocatalysts. It is foreseeable that in the near future most industrial biocatalysts will be products of genetic and protein engineering.
• Stabilization of biocatalysts by conventional means, like immobilization, and new methodologies, like cross-linked enzyme crystals, is broadening the scope of biocatalysis.
• Increased stability of enzymes in non-aqueous media is a relevant technological asset for the development of biocatalysis in organic synthesis.
• Modeling of operational stability of biocatalysts, considering modulation factors, is required for the proper design of bioreactors. Temperature, as the key variable in any bioprocess, can be conveniently optimized through the use of such models.

References

Abu-Reesh, I. and Faqir, N. (1996). Simulation of glucose isomerase reactor: optimum operating temperature. Bioprocess Engineering 14:205-210

Adams, M., Perler, F., and Kelly, R. (1995). Extremozymes: expanding the limits of biocatalysis. Bio/Technology 13:662-668

Adams, M. and Kelly, R. (1998). Finding and using hyperthermophilic ezymes. Trends in Biotechnology. 16:329-332

Anonymous (1997). Una amplia gama de proteasas, una amplia gama de propiedades. BioTimes (Novo-Nordisk) 2:14-15

Anonymous (1998). Top llega al tope de Japón. BioTimes (Novo-Nordisk) 1:4-5

Bell, G., Halling, P., Moore, B., Partridge,J. And Rees, G. (1995). Biocatalyst behavior in low-water systems. Trends in Biotechnology 13:468-473

Benkovic, S. and Ballesteros, A. (1997). Biocatalysts- the next generation. Trends in Biotechnology 15:385-386

Besson, C., Favre-Bonvin, G., O'Fagain, C. and Wallach, J. (1995). Chemical derivation of Pseudomonas aeruginosa elastase showing increased stability. Enzyme and Microbial Technology 17:877-881

Bucke, C. (1996). Oligosaccharide synthesis using glycosidases. Journal of Chemical Technology and Biotechnology 67:217-220

Caruana, C. (1997). Enzymes tackle tough processing. Chemical Engineering Progress November, pp 13-20

Clapés, P., Pera, E. and Torres, J. (1997). Peptide bond formation by the industrial protease, neutrase, in aqueous media. Biotechnology Letters 19:1023-1026

Coolbear, T., Daniel, R. and Morgan, H. (1992). The enzymes from extreme thermophiles: bacterial sources, thermostability and industrial relevance. Advances in Biochemical Engineering/Biotechnology 45:57-98

Coulon, D. and Ghoul, M. (1998). The enzymatic synthesis of non-ionic surfactants: the sugar esters. Agro Food Industry Hi-Tech 9:22-26

Crabb, W. and Mitchinson, C. (1997). Enzymes involved in the processing of starch and sugars. Trends in Biotechnology 15:349-352

Daniel, R. (1996). The upper limits of enzyme stability. Enzyme and Microbial Technology 19:74-79

Davis, M. (1998). Making a living at the extremes. Trends in Biotechnology 16:102-104

Erarslan, A. and Ertan, H. (1995). Thermostabilization of penicillin G acylase obtained from a mutant of E.coli ATCC 11105 by bisimidoesters as homobifunctional cross-linking agents. Enzyme and Microbial Technology 17:629-635

Faqir, N. and Abu-Reesh, I. (1998). Optimum temperature operation mode for glucose isomerase reactor operating at constant glucose conversion. Bioprocess Engineering 19:11-17

Feliu, J., de Mas, C and López-Santín, J. (1995). Studies on papain in the synthesis of Gly-Phe in two-liquid phase media. Enzyme and Microbial Technology 17:882-887

Fischer, L, Bromann, R., Kengen, S., de Vos, W. and Wagner, F. (1996). Catalytic potency of b -glucosidase from the extremophile Pyrococcus furiosus in glucoconjugate synthesis. Bio/Technology 14:88-91

García, D. and Marty, J. (1998). Chemical modification of horseradish peroxidase with several methoxypolyethylene glycols. Applied Biochemistry and Bioengineering 73:173-184

Gill, I., López-Fandiño, R., Jorba, X. and Vulfson, E. (1996). Biologically active peptides and enzymatic approaches to their production. Enzyme and Microbial Technology 18:162-183

Gupta, M. (1992). Enzyme function in organic solvents. European Journal of Biochemistry 203:25-32

Haard, N. (1998). Specialty enzymes from marine microorganisms. Food Technology 52(7):64-67

Halldórsdóttir,S., Thórólfsdóttir, E., Spilliaert, R., Johansson, M., Thorbjarnardóttir, S., Palsdóttir, A., Hreggvidsson, G., Kristjánsson, J., Holst, O. and Eggertsson, G. (1998). Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosilhydrolase family 12. Applied Microbiology and Biotechnology 49:277-284

Halling, P. (1984). Effects of water on equilibria catalysed by hydrolytic enzymes in biphasic reaction systems. Enzyme and Microbial Technology 6:513-516

Henley,J. and Sadana, A. (1986). Deactivation theory. Biotechnology and Bioengineering. 28:1277-1285

Herbert, R. (1992). A perspective on the biotechnological potential of extremophiles. Trends in Biotechnology 7:349-353

Houng,J., Yu, H and Chen, K. (1993). Analysis of substrate protection of an immobilized glucose isomerase reactor. Biotechnology and Bioengineering 41:451-158

Illanes, A., Zuñiga, M., Contreras, S. and Guerrero, A. (1992). Reactor design for the enzymatic isomerization of glucose to fructose. Bioprocess Engineering 7:199-204

Illanes, A., Altamirano, C. and Cartagena, O. (1994). Enzyme reactor performance under thermal inactivation. In Advances in Bioprocess Engineering (Galindo, E., Ramírez, O. eds.) Kluwer Academic Publishers, Dordrecht, pp 467-472

Illanes, A., Altamirano, C. and Zuñiga, M. (1996). Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products. Biotechnology and Bioengineering 50:609-616

Illanes, A., Altamirano, C., Aillapán, A., Tomasello, G. and Zuñiga, M. (1998 a). Packed-bed reactor performance with immobilized lactase under thermal inactivation. Enzyme and Microbial Technology 23:3-9

Illanes, A. Wilson, L., Altamirano, C., and Aillapán, A. (1998b). Reactor performance under thermal inactivation and temperature optimization with chitin-immobilized lactase. Progress in Biotechnol. 15: 27-34.

Imanaka, T., Shibazaki, M. and Takagi, M. (1988). A new way of enhancing the thermostability of proteases. Nature 324:695-687

Inada, Y., Yoshimoto, T., Matsushima, A. and Saito, Y. (1986). Engineering physicochemical and biological properties of proteins by chemical modification. Trends in Biotechnology 4:68-73, 1986

John, V. and Abraham, G. (1991). Lipase catalysis and its applications. In: Biocatalysts for Industry (Dordick, J. Ed.) Plenum Press, New York, pp 193-217

Katchalsky-Katzir, E. (1993). Immobilized enzymes - learning from past successes and failures. Trends in Biotechnology 11:471-478

Khmelnitsky, Y., Levashov, A., Klyachko, N. and Martinek, K. (1988). Engineering biocatalytic systems in organic media with low water content. Enzyme and Microbial Technology 10:710-724

Klibanov, A. (1997). Why are enzymes less active in organic solvents than in water? Trends in Biotechnology 15:97-101

Koskinen, A. and Klibanov, A. Enzymatic Reactions in Organic Media. (1996). Blackie Academic& Professional, London, 314 pp.

Kristjánsson, J. (1989). Thermophilic organisms as sources of thermostable enzymes. Trends in Biotechnology 10:395-401

Margolin, A. (1996). Novel crystalline catalysts. Trends in Biotechnology 14:223-230

Marshall, C. (1997). Cold-adapted enzymes. Trends in Biotechnology 15:359-364

May, S. (1992). Biocatalysis in the 1990s: a perspective. Enzyme and Microbial Technology 14:80-84

Mozhaev, V. (1993). Mechanism-based strategies for protein thermostabilization. Trends in Biotechnology 11:88-95

Noritomi, H., Koyama, K., Kato, S. and Nagahama, K. (1998). Increased thermostability of cross-linked enzyme crystals of subtilisin in organic solvents. Biotechnology Techniques 12:467-469

O'Fágáin, C., Sheenan, H., O'Kennedy, R. and Kilty, C. (1988). Maintenance of enzyme structure. Possible methods for enhancing stability. Process Biochemistry 23:166-171

Ortega, N., Busto, M. and Pérez-Mateos, M (1998). Stabilisation of b -glucosidase entrapped in alginate and polyacrylamide gels towards thermal and proteolytic deactivation. Journal of Chemical Technology and Biotechnology 73:7-12

Pedersen, S. (1993). Industrial aspects of immobilized glucose isomerase. In: Industrial Application of Immobilized Biocatalysts (Tanaka, A., Tosa, T.,Kobayashi, T., eds). Marcel Dekker, New York, pp 185-208

Polastro, E. (1989). Enzymes in the fine-chemicals industry: dreams and realities. Bio/Technology 7:1238-1241

Rosell, C., Terreni, M., Fernández-Lafuente, R. and Guisán, J (1998). A criterion for the selection of monophasic solvents for enzymatic synthesis. Enzyme and Microbial Technology 23:64-69

Rúa, M., Atomi, H., Schmidt-Dannert, C. and Schmid, R. (1998). High-level expression of the thermoalkalophilic lipase from Bacillus thermocatenulatus in Escherichia coli. Applied Microbiology and Biotechnology 49:405-410

Sarney, D. and Vulfson, E. (1995). Application of enzymes to the synthesis of surfactants. Trends in Biotechnology 13:164-172

Scheckermann, C., Wagner, F. and Fischer, L. (1997). Galactosylation of antibiotics using the b -galactosidase from Aspergillus oryzae. Enzyme and Microbial Technology 20:629-634

Sergeeva, M., Paradkar, V. and Dordick, J,. (1997). Peptide synthesis using proteases dissolved in organic solvents. Enzyme and Microbial Technology 20:623-628

Somkuti, G. and Holsinger, V. (1997). Microbial technologies in the production of low lactose in dairy foods. Food Science and Technology International 3:163-169

Stevenson, D., Stanley, R. and Furneaux, R. (1993). Optimization of b -D-galactopyranoside synthesis from lactose using commercially availableb -galactosidases. Biotechnology and Bioengineering 42:657-666

Sundaram, P. and Venkatesh, R. (1998). Retardation of thermal and urea induced inactivation of a -chymotrypsin by modification with carbohydrate polymers. Protein Engineering 11:699-705

van Unen, D., Engbersen, J. and Reinhoudt, D. (1998). Large acceleration of a -chymotrypsin-catalyzed dipeptide formation by 18-Crown-6 in organic solvents. Biotechnology and Bioengineering 59:553-556

Vieille, C. and Zeikus, J. (1996). Thermoenzymes: identifying molecular determinants of protein structural and functional stability. Trends in Biotechnology 14:183-190

Wasserman, B. (1984). Thermostable enzyme production. Food Technology 38(2):78-88

Yang, S. and Okos, M. (1989). Effects of temperature on lactose hydrolysis by immobilized b -galactosidase in plug-flow reactor. Biotechnology and Bioengineering 33:873-885