Microbial Cell Factories BioMed Central Review Open Access Potential and utilization of thermophiles and thermostable enzymes in biorefining Pernilla Turner, Gashaw Mamo and Eva Nordberg Karlsson* Address: Dept Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Email: Pernilla Turner - [email protected]; Gashaw Mamo - [email protected]; Eva Nordberg Karlsson* - [email protected] * Corresponding author Published: 15 March 2007 Received: 4 January 2007 Accepted: 15 March 2007 Microbial Cell Factories 2007, 6:9 doi:10.1186/1475-2859-6-9 This article is available from: http://www.microbialcellfactories.com/content/6/1/9 © 2007 Turner et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts. Background general acceptance. Most thermophilic bacteria character- Thermostable enzymes and microorganisms have been ised today grow below the hyperthermophilic boundary topics for much research during the last two decades, but (with some exceptions, such as Thermotoga and Aquifex the interest in thermophiles and how their proteins are [5]) while hyperthermophilic species are dominated by able to function at elevated temperatures actually started the Archaea. as early as in the 1960's by the pioneering work of Brock and his colleagues [1]. Microorganisms are, based on their Use and development of molecular biology techniques, optimal growth temperatures, divided into three main permitting genetic analysis and gene transfer for recom- groups, i.e. psychrophiles (below 20°C), mesophiles binant production, led to dramatically increased activities (moderate temperatures), and thermophiles (high tem- in the field of thermostable enzymes during the 1990's. peratures, above 55°C) [2]. Only few eukaryotes are This also stimulated isolation of a number of microbes known to grow above this temperature, but some fungi from thermal environments in order to access enzymes grow in the temperature range 50 – 55°C [3]. Several years that could significantly increase the window for enzy- ago Kristjansson and Stetter [4], suggested a further divi- matic bioprocess operations. One of the early successful sion of the thermophiles and a hyperthermophile bound- commercialised examples was analytical use of a ther- ary (growth at and above 80°C) that has today reached mostable enzyme, Taq-polymerase, in polymerase chain Page 1 of 23 (page number not for citation purposes) Microbial Cell Factories 2007, 6:9 http://www.microbialcellfactories.com/content/6/1/9 reactions (PCR) for amplification of DNA, and a number bilities for prolonged storage (at room temperature), of other DNA modifying enzymes from thermophilic increased tolerance to organic solvents [14], reduced risk sources have, since then, been commercialised in this area of contamination, as well as low activity losses during [6-8]. Another area of interest has been the prospecting for processing (when staying below the Tm of the enzyme) industrial enzymes for use in technical products and proc- even at the elevated temperatures often used in raw mate- esses, often in a very large scale. Enzymes can be advanta- rial pre-treatments. geous as industrial catalysts as they rarely require toxic metal ions for functionality, hence creating the possibility Discovery and use of thermostable enzymes in combina- to use more environmentally friendly processing [9]. tion with recombinant production and development Thermostable enzymes offer robust catalyst alternatives, using site-directed and enzyme evolution technologies, able to withstand the often relatively harsh conditions of have erased some of the first identified hinders (e.g. lim- industrial processing. ited access and substrate specificity) for use in industrial biocatalysis. Today, a number of biotechnology compa- Conversion of biomass into sugars for e.g. energy utiliza- nies are continuously prospecting for new, and adapting tion was a topic of concern about 30 years ago. Renewed existing enzymes to reactions of higher volumes and more interest in biocatalytic conversions has recently emerged, severe process conditions [15]. Enzyme prospecting often with the growing concern on the instability and possible focuses on gene retrieval directly from Nature by molecu- depletion of fossil oil resources as well as growing envi- lar probing techniques, followed by recombinant produc- ronmental concern, and focus is again put on biorefining, tion in a selected host. Availability of genes encoding and the biorefinery concept. In biorefining, renewable stable enzymes, and knowledge on structural features in resources such as agricultural crops or wood are utilized the enzymes, can also be utilized in molecular develop- for extraction of intermediates or for direct bioconversion ment for enzyme improvement (Table 1). into chemicals, commodities and fuels [10,11]. Ther- mostable enzymes have an obvious advantage as catalysts In vitro evolution strategies can utilize genes encoding in these processes, as high temperatures often promote thermostable proteins as stable scaffolds. When develop- better enzyme penetration and cell-wall disorganisation ing thermostable enzyme scaffolds, the starting material is of the raw materials [12]. By the parallel development in an already stable backbone, thus creating a good possibil- molecular biology, novel and developed stable enzymes ity for evolution to optimize function at selected condi- also have a good chance to be produced at suitable levels. tions for activity. An example where this type of This review will discuss the potential and possibilities of development has been utilized is the diversification of the thermostable enzymes, developed or isolated from ther- binding specificity of a carbohydrate binding module, mophiles, including examples where whole cells are con- CBM4-2 originating from a xylanase from the ther- sidered, in bioconversions of renewable raw materials mophilic bacterium Rhodothermus marinus [30]. Carbohy- with a biorefining perspective. Examples of commercial drate binding modules allow fine-tuned polysaccharide thermostable enzymes acting on renewable raw materials recognition [31] and have potential as affinity handles in will be illustrated. different types of applications, as recently reviewed by Volkov and co-workers [32]. Using CBM4-2, which has Stability and development of thermostable both high thermostability and good productivity in E. coli enzymes expression systems, a single heat stable protein could be In industrial applications with thermophiles and ther- developed with specificity towards different carbohydrate mostable enzymes, isolated enzymes are today dominat- polymers [27], as well as towards a glycoprotein [33], ing over microorganisms. An enzyme or protein is called showing the potential of molecular biology for selective thermostable when a high defined unfolding (transition) specificity development of a single protein with overall temperature (Tm), or a long half-life at a selected high tem- desirable properties. perature, is observed. A high temperature should be a tem- perature above the thermophile boundary for growth In vitro evolution strategies are more commonly used to [>55°C]. Most, but not all proteins from thermophiles are increase stability (Table 1), often using genes encoding thermostable. Extracellular enzymes generally show high non-thermostable enzymes with desired activities, for thermostability, as they cannot be stabilised by cell-spe- development of better thermostability, and using the tem- cific factors like compatible solutes [13]. In addition, a perature of the screening assay as selection pressure [34- few thermostable enzymes have also been identified from 36]. This could for instance include development of ther- organisms growing at lower temperatures (see for exam- mostable cellobiohydrolases, which are uncommon ple B. licheniformis amylase below). Fundamental reasons among thermophiles, but beneficial for lignocellulose to choose thermostable enzymes in bioprocessing is of conversions. In addition, such strategies can be used to course the intrinsic thermostability, which implies