Send Orders for Reprints to [email protected] Current Biotechnology, 2014, 3, 45-59 45 Selected Enzymes from Extreme Thermophiles with Applications in Biotechnology Peter L. Bergquist*,1, Hugh W. Morgan2 and David Saul3 1Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Macquarie University, Sydney, New South Wales 2109, Australia; and Department of Molecular Medicine & Pathology, University of Auckland Medical School, Auckland 1142, New Zealand 2Department of Biological Sciences, Thermophile Research Unit, Waikato University, Hamilton 3240, New Zealand 3ZyGEM Corporation Ltd, Waikato Innovation Park, Hamilton 3216, New Zealand Abstract: Enzymes from extreme thermophiles that grow above 70 °C have a number of attractions in industrial applications. They are often highly resistant to denaturing conditions and are stable at elevated temperatures and over a range of pH values. There has been a widespread search for micro-organisms producing novel enzymes and where found, most publications (and research grant applications) promise that their superior properties would be suited to particular industries that operate at elevated temperatures - for example, bleaching of kraft pulp in the pulp and paper industry. Yet examination of the academic and patent literature reveals few of these proteins adopted in industrial enzymology. Most employed successfully have been as laboratory reagents, particularly Thermus aquaticus DNA polymerase, which made the polymerase chain reaction possible and revolutionized gene manipulation at a laboratory level. Extremozymes marketed for the laboratory are lower volume / higher value products and have been cloned and expressed (usually) in Escherichia coli. This review examines the characterization and application of thermophilic enzymes for several activities that have been identified and (usually) produced in recombinant bacteria. DNA polymerases, glycosyl hydrolases, lipases and proteases from extreme thermophiles are described and evaluated for their potential and actual applications in biotechnology. Some of the barriers to widespread industrial acceptance are described. Emphasis is placed on a number of examples illustrated by the anaerobic extreme thermophiles Caldicellulosiruptor sp. and Dictyoglomus sp. with which the authors are familiar. A number of attractive enzymes are not scalable economically in Escherichia coli or the organism from which the gene has been isolated is an obligate anaerobe and enzyme yields from the native organism are low. Consequently, attention has turned to commonly used ‘cell factories’ to provide suitable yields of enzymes used as bulk chemicals. These 'factories' are usually fungi such as Saccharomyces cerevisiae and Trichoderma reesei or bacteria such as Bacillus sp. A number of challenges, such as codon usage incompatibility, must be overcome to achieve economic yields. Keywords: Cell factories, DNA polymerases, extremozymes, glycosyl hydrolases, industrial enzymology, proteinases, pulp bleaching. INTRODUCTION small differences in amino acid sequence gave them remarkable comparative stabilities. The study of micro-organisms able to survive and multiply in extreme environments is a relatively recent Microorganisms are largely neglected in studies of phenomenon. Environments that were identified as having biodiversity yet are the most phylogenetically and extremes of temperature, salinity and pH, etc were physiologically diverse group on the planet. Figures vary, considered to be effectively sterile until the second half of but most microbiologists agree that we have probably the 20th century. The realization that these environments identified less than 1% of the total microbiota that exists in harboured a diverse assemblage of organisms was natural environments. For example, the total number of fascinating initially and there was curiosity and speculation bacterial species worldwide is conservatively estimated at as to how their macromolecular make-up was adapted to 13-50 million, yet to date, only 4,000 species have been these conditions. It was soon realised that these organisms described [1, 2]. Much of the problem has been caused by possessed proteins that were in many ways similar to those dependence on culture-based enrichment techniques that fail in the familiar more mesophilic microorganisms, but often to support most organisms [3]. The traditional techniques now have been augmented by a powerful array of DNA- based methods that have allowed the amplification of small *Address correspondence to this author at the Department of Chemistry and subunit (SSU) rRNA and other genes directly from natural Biomolecular Sciences, Biomolecular Frontiers Research Centre, Macquarie environments. Uncultured microorganisms make up the University, Sydney, New South Wales 2109, Australia; major part of the Earth’s biological diversity. In many Tel: +61 2 9850 8614; Fax: +61 29850 8313; environments, >99% of the microorganisms cannot be Email: [email protected] 2211-551X/14 $58.00+.00 © 2014 Bentham Science Publishers 46 Current Biotechnology, 2014, Volume 3, No. 1 Bergquist et al. cultured by standard techniques and the uncultured fraction the notion that the active site is more flexible than the whole includes diverse organisms that are only distantly related to protein and loss of activity occurs before denaturation. In the cultured ones. Therefore, cultivation-independent methods classical model, the two rate constants for the processes are essential to understand the genetic diversity, population (ΔG*cat and Δ G*inact ) will lead to a temperature optimum structure and ecological roles of the majority of (Topt) that is dependent on the length of assay because microorganisms. Metagenomics, the culture-independent thermal denaturation is time dependent [12, 13]. A change in genomic analysis of an assemblage of microorganisms, has the assay duration will change the apparent optimum the potential to answer many fundamental questions in temperature. The equilibrium model introduces new thermal microbial biodiversity and ecology [4, 5] and allows the parameter Teq where the concentrations of the active and examination of the total genomic diversity of a sample and inactive enzyme are equal and allows prediction of the avoids the over-representation of fast growing members of temperature at which the enzyme activity is maximal (Topt, the microbial community. ref. [12]). Accordingly, they propose that successful screening for high(er) temperature enzymes will need to take The stabilities discovered from both enrichment and into account both thermal stability and activity (via T ), metagenomic studies were not limited solely to temperature eq unlike the classical model which suggests both activity and but to a range of conditions which led to denaturation of resistance to thermal denaturation can be achieved by their mesophilic counterparts. The ability of some enzymes such as lipases to show activity in organic solvents selecting for thermal activity alone. Similarly, the equilibrium model teaches that in enzyme engineering encouraged their use in bio-transformations in the studies using rational or directed mutation procedures, an biopharmaceutical industry to perform reactions with regio- increase in thermostability will not necessarily lead to and enantio-specificity. The general ‘green’ trend for enhanced activity at high temperatures as the temperature of industrial chemistry that became evident towards the end of T must be shifted also [13]. These considerations will have the last century and the realization that enzymatic processes eq could be carried out under mild catalytic conditions, an effect also on enzyme reactions in a reactor to give a product, as the output at given times and temperatures will stimulated the discovery of new extremophile organisms, vary between the two models. Given the general lack of and by inference, their enzymes. This shift occurred against a understanding of enzyme kinetics by the average molecular background of improving techniques to examine biologist engaged in biotechnological outcomes, it is microbiological diversity without needing traditional culture possible that resources are being wasted from the point of methods. As a result, a number of enzymes that showed characteristics that could aid their incorporation into enzyme identification to product formation as many researchers appear to be unfamiliar with, or ignorant of, both industrial processes were isolated and their characteristics the classical and equilibrium models [15]. published. Many authors extrapolated (or imagined) the industrial benefits of their particular enzyme from laboratory One area that has adopted an enzymatic step is the pulp bench experiments and every enzyme that had even the and paper industry in the bleaching of pulp, largely as the remotest possibility of a commercial use was proposed as result of R&D by Viikari and collaborators [17]. The new being industrially-relevant in published papers and research process had the advantage of requiring effectively no grant applications. Yet the reality is that only a few of these alteration to the current procedures and infrastructure at kraft enzymes have found a place in industrial processes, largely mills apart from the addition of a metering pump. Kraft pulp as a result of cost, plant design and a general reluctance to comes out of the cook at temperatures between 70 °C and change a system that works. 100 °C and pH~13 and would be expected to be