Industrial relevance of thermophilic Archaea
Introduction
Microbial enzymes already occupy a prominent position in modern biotechnology, optimizing or even replacing processes that already exist. The application of enzymes and microorganisms to the sustainable production of chemicals, biopolymers, materials and fuels from renewable resources, also defined as industrial (white) biotechnology, offers great opportunities for the chemical and pharmaceutical industries. White biotechnology aims to reduce waste, energy input and raw material to improve a processes environmental friendliness.
The majority of the industrial enzymes known to date have been derived from Bacteria and fungi. The global annual enzyme market is around five billion Euros. The situation is different in the case of Archaea, which represent the third domain of life. Only a few archaeal enzymes have found their way to the market. Extremophilic Archaea have been subject to intensive investigations only in the past two decades [1]. As many representatives of this group can grow optimally under extreme conditions they are, however, an interesting source of stable enzymes. Based on the unique stability of their enzymes at high temperature, extremes of pH and high pressure, combined with their salt, organic solvent and metal tolerance, they are expected to be a powerful tool in industrial biotransformation processes that run at harsh conditions. The benefits of using enzymes from extremophiles (extremozymes) are manifold, including reduced risk of contamination, improved transfer rates, lower viscosity and higher solubility of substrates. The recent exciting results in the field of extremophile research, the high demands of the biotech industries for tailor-made novel biocatalysts, and the rapid development of new techniques such as genomics, proteomics, metabolomics, directed evolution and gene shuffling will stimulate the development of new industrial processes on the basis of biocatalysts from extremophiles. To meet the future challenges, innovative technologies are needed.
In this review we will focus on enzymes that are derived from extremophilic Archaea, especially thermophiles, and their relevance for industrial application.
Section snippets
Microbial life at elevated temperatures
Extreme environments such as geothermal sites (80–121 °C), polar regions (−20–20 °C), acidic (pH < 4) and alkaline (pH > 8) springs, saline lakes (2–5 M NaCl) and the cold pressurized depths of the oceans (<5 °C) are promising sources of unique microorganisms and enzymes. Most terrestrial biotopes, including hot springs with neutral pH, acidic pH or those that are rich in iron, are volcanically and geothermally heated. Submarine hydrothermal systems, situated at both shallow and abyssal depths, include
Biocatalysis under extreme conditions
Owing to the unique features of Archaea, their potential applications in biotechnology are far reaching, ranging from bioremediation of toxic compounds to the production of substances of medical use [3••]. There are many advantages to running processes at elevated temperature, including increased solubility of many polymeric substrates, decreased viscosity, increased bioavailability, faster reaction rates and the decreased risk of contamination. Stable enzymes allow the use of organic solvents
Starch-processing enzymes
Many archaeal enzymes involved in carbohydrate metabolism, particularly those of the glycosyl hydrolase family, are of great industrial interest. The starch-processing industry, which converts starch into more valuable products such as dextrins, glucose, fructose and trehalose, can profit from thermostable enzymes. In all starch-converting processes, high temperatures are required to liquefy starch and to make it accessible to enzymatic hydrolysis. The synergetic action of thermostable
Cellulose-degrading enzymes
Cellulose, the most abundant organic biopolymer in nature, can be hydrolyzed to glucose by the synergetic action of endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91) and β-glucosidase (EC 3.2.1.21). Several β-glucosidases have been detected in Sulfolobales species and were compared with the β-glucosidase from P. furiosus [7•]. The pyrococcal enzyme is stable and is optimally active at 103 °C. Using a mixture of these β-glucosidases, an ultra-high temperature process for the enzymatic
Xylan-degrading enzymes
Commercial interest in the applications of xylanases, which degrade the most abundant form of hemicellulose (xylan) from the plant cell wall fraction, has grown significantly. Xylanases play a key role as quality- and yield-improving agents in food, feed, pulp and paper industries. The complete degradation of xylan requires the action of the endo-β-1,4-xylanase (EC 3.2.1.8) and β-1,4-xylosidase (EC 3.2.1.37). Among the thermophilic Archaea, a xylanase has been produced from Pyrodictium abyssi
Chitin-degrading enzymes
Chitin, a highly insoluble linear β-1,4 homopolymer of N-acetyl-glucosamine residues, is produced in enormous amounts in the marine environment. To date, only three hyperthermophilic Archaea — Thermococcus chitonophagus [16], Thermococcus kodakaraensis KOD1 [17, 18] and P. furiosus [19] — have been shown to grow on chitin. An extremely thermostable endochitinase (EC 3.2.1.14) from Thermococcus chitonophagus, which has 50% activity even after 1 h at 120 °C, is also resistant to urea- and sodium
Proteolytic enzymes
Proteases and proteasomes play a key role in the cellular metabolism of Archaea, and only few enzymes have been characterized in detail (Table S1 in the supplementary material online). It has been found that most proteases from extremophilic Archaea belong to the serine type and are stable at high temperatures, even in the presence of high concentrations of detergents and denaturing agents [7•]. These properties are well illustrated by the extracellular enzyme from Thermococcus stetteri, which
DNA-processing enzymes
One of the most important advances in molecular biology during the past twenty years is the development of polymerase chain reaction (PCR) [23, 24, 25]. Thermostable DNA polymerases (EC 2.7.7.7) play a major role in a variety of molecular biological applications, for example in DNA amplification, sequencing or labeling. Archaeal polymerases from Pyrococcus or Thermococcus species, which have stringent proofreading abilities, are of widespread use. The recombinant KOD1 DNA polymerase from T.
Alcohol dehydrogenases
Dehydrogenases are enzymes that belong to the class of oxidoreductases. Within this class, alcohol dehydrogenases (ADHs) (EC 1.1.1.1) represent an important group of biocatalysts because they can be used efficiently in the synthesis of optically active alcohols, which are key building blocks for the chemical industry [37]. Thermoactive ADHs are also suitable for the synthesis of cofactors such as NAD and NADP that are used in various processes [38•]. ADHs are widely distributed among various
Esterases
In the field of biotechnology, esterases are gaining increasing attention because of their application in the biosynthesis of optically pure compounds. Several archaeal esterases were successfully cloned and expressed in mesophilic hosts. Esterases from Aeropyrum pernix, Pyrobaculum calidifontis and Sulfolobus tokodaii exhibit high thermoactivity and thermostability and are active in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide [7•]. The
CC bond forming enzymes
Synthetic building blocks that bear hydroxylated chiral centers are important targets for biocatalysis. CC bond forming enzymes, such as aldolases and transketolases, have been investigated for new applications, and various strategies for the synthesis of sugars and related oxygenated compounds have been developed [42]. The increased accessibility of these compounds led to the development of new therapeutics and diagnostics. The aldolase from S. solfataricus catalyses the CC bond synthesis with
Nitrile-degrading enzymes
Nitrile-degrading enzymes are of considerable importance in industrial biotransformations, and to date several processes have been developed that involve these. Thermoactive amidases and nitrilases are gaining more attention, especially in enzymatic processes at high temperatures or in mixtures of organic solvents. The stereoselective amidase (EC 3.5.1.4) from S. solfataricus has a broad substrate spectrum and is active at 95 °C [7•]. Recently, in our laboratory, a thermostable nitrilase from
Aminoacylases
Owing to their chiral specificity in the synthesis of acylated amino acids, aminoacylases (EC 3.5.1.14) are attractive candidates for application in fine chemistry [44]. The enantioselective L-aminoacylase from T. litoralis has a broad substrate spectrum towards N-acylated α-amino acids, with respect to both side chains and N-acyl groups. The substrate preference for N-benzoyl groups over N-acetyl groups has been demonstrated. The application of this enzyme in commercial biotransformation
Other applications of Archaea
Some archaeal metabolites such as peptides, osmotically active substances (compatible solutes), exopolysaccharides and lipids are also of industrial interest. Archaeal lipids are an excellent source for the formation of liposomes that have remarkable thermostability and tightness against solute leakage. They could be a superior alternative as delivery systems for drugs, genes or cancer-imaging agents [46]. Owing to their bipolar tetraether structure, archaeal lipids have been proposed as
Conclusions
Owing to their properties such as activity over a wide temperature and pH range, substrate specificity, stability in organic solvents, diverse substrate range and enantioselectivity, in the future, biocatalysts from extremophilic microorganisms will have countless applications in industry. Their importance is increasing day by day in several fields, such as in the production of food additives, detergents, chemicals and pharmaceuticals. The growing demand for more robust biocatalysts has shifted
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We thank DBU (German Federal Environmental Foundation) and BMBF (Federal Ministry of Education and Research) for financial support.
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