Available online at www.sciencedirect.com
ScienceDirect Extremozymes — biocatalysts with unique properties from extremophilic microorganisms Skander Elleuche, Carola Schro¨der, Kerstin Sahm and Garabed Antranikian Extremozymes are enzymes derived from extremophilic microorganisms that are able to withstand harsh conditions in industrial processes that were long thought to be destructive to proteins. Heat-stable and solvent-tolerant biocatalysts are valuable tools for processes in which for example hardly decomposable polymers need to be liquefied and degraded, while cold-active enzymes are of relevance for food and detergent industries. Extremophilic microorganisms are a rich source of naturally tailored enzymes, which are more superior over their mesophilic counterparts for applications at extreme conditions. Especially lignocellulolytic, amylolytic, and other biomass processing extremozymes with unique properties are widely distributed in thermophilic prokaryotes and are of high potential for versatile industrial processes. Addresses Institute of Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, D-21073 Hamburg, Germany Corresponding author: Antranikian, Garabed ([email protected])
Current Opinion in Biotechnology 2014, 29:116–123 This review comes from a themed issue on Cell and pathway engineering Edited by Tina Lu¨tke-Eversloh and Keith EJ Tyo
0958-1669/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2014.04.003
Introduction Biotechnology is omnipresent and has a greater impact than previously envisioned on several diverse industries, such as feed and food production, biofuel and energy generation as well as sustainable production of high-value chemical compounds (Figure 1). Conditions in an industrial process are often far from standard biocatalyst’s properties. Hence, there is considerable demand for a new generation of stable enzymes that are able to reach this goal by replacing or supplementing traditional chemical processes [1]. Enzyme properties such as temperature stability, selectivity, solvent-tolerance, or affinity to the substrate can be improved through genetic engineering. However, the task Current Opinion in Biotechnology 2014, 29:116–123
is tedious and lacks rational concepts. Extremozymes, enzymes derived from extremophilic microorganisms (Box 1), are an attractive alternative to tuning a given biocatalyst for a specific industrial application. They are capable of catalysing their respective reactions in nonaqueous environments, water/solvent mixtures, at extremely high pressures, acidic and alkaline pH, at temperatures up to 1408C, or near the freezing point of water [2]. These enzymes already contain properties that are generated in synthetically tailored enzymes by directed evolution, DNA shuffling, gene fusion, truncation, or rational protein synthesis [3,4,5]. However, the majority of enzymes that are currently used in the industry are obtained from fungi or mesophilic bacteria. Only a few Box 1 Extremophilic microorganisms Living organisms colonize almost all ecological niches on earth. Those that are metabolically active and grow under conditions unusual from a human perspective are termed extremophiles, because they seem to ‘love the extreme’. At temperatures above 628C only thermophilic prokaryotic organisms can survive and thrive and among hyperthermophilic organisms growing optimally at temperatures above 808C Archaea are dominating. From what is known today, Methanopyrus kandleri is able to survive the highest temperature of 1228C under 200 bar pressure [52]. Acidophiles growing optimally at pH values around or below 3.0 can be found in all three domains of life. Mesophilic acidophiles (Acidithiobacillus, Ferroplasma, Leptospirillum) have long been used for bioleaching. Some Archaea can grow at low pH and high temperature (thermoacidophiles). Picrophilus torridus is one of the most thermoacidophilic microorganisms, growing at pH values of zero and at temperatures of up to 658C. Hyperthermophilic thermoacidophiles growing at temperatures above 808C usually tolerate only pH values of 2.0–3.0 although some Sulfolobus species and Acidianus infernus can cope with pH values lower than 2.0 [53]. All cellular components (proteins, nucleic acids and lipids) of extremophiles have to be stable at specific extreme growth conditions. For chemical extremes like pH or salinity specific export systems, membrane adaptations or small molecules called compatible solutes can provide a near to normal intracellular milieu in some species. But even in these organisms, extracellular enzymes have to be stable in the extreme surrounding. No individual structural properties can be singled out to account for extremozyme stability. However, for increased temperature stability of proteins, some principles can be identified like highly charged exterior surfaces, rigid folds maintained by multiple ion-pair networks, tight hydrophobic core packing and overall more compact and densely packed protein structures, partly caused by an increased content of acidic and basic amino acids [54]. In addition to these structural features, highly effective protein-repair and DNA-repair systems and cytoplasmic membranes with specific characteristics are contributing to enable microorganisms to cope with extreme conditions.
www.sciencedirect.com
Industrial applications of extremozymes Elleuche et al. 117
Figure 1
using catalysis
ymes
z extremo
Bio
Biorefinery
New processes and products
Textiles Dairy -12 ºC
Temperature
122 ºC 20 ºC
pH
37 ºC Starch liquefaction
Detergent
pH 11
pH 2 pH 8
pH 5 Food
Solvents
0%
100 % Classic biocata al lysis
New processes and products
Two phase biotransformations
Current Opinion in Biotechnology
Extremozymes widen the scope of biocatalysis. Biocatalysts of extremophiles can withstand a broader range of temperature, pH, and solvents than enzymes from mesophilic organisms.
extremozymes have found their way to the market with DNA-polymerase from Thermus aquaticus and its relatives being the most prominent example. Nowadays, new challenges have broadened the range of successful utilization of extremozymes beyond their use in DNA replication. Besides utilization of traditional protein engineering approaches in mesophilic hosts, genetic improvements of extremophilic expression systems are currently under intense investigation. Improper folding and differences in codon usage generally interferes with the production of functional extremozymes in commonly used expression systems such as Escherichia coli or Bacillus sp. Therefore, it is an important prerequisite to establish fine regulated extremophilic hosts, efficient transformation approaches www.sciencedirect.com
and adequate expression vectors. Only a few functional systems have been presented mainly using different members of the genus Thermus or the hyperthermophilic crenarchaeotal species Sulfolobus solfataricus [6,7]. Even the application of extremophiles as cellular biocatalysts for biotransformation has attracted interest in recent years. Especially metabolic and regulatory networks of thermophiles have been manipulated to generate microbial cellular factories for biofuel production in a Consolidated Bioprocessing approach that might be superior in the future due to accelerated reaction rates, reduced energy input, and minimized contamination risk. The ability to utilize multiple carbohydrate sources inCurrent Opinion in Biotechnology 2014, 29:116–123
118 Cell and pathway engineering
Table 1 Heat-active hydrolases with potential industrial relevancea Enzyme (glycoside hydrolase family) a-Amylase a-Amylase (GH13) Pullulanase (GH13) Pullulanase/amylase (GH13) a-Glucosidase b-Amylase Endoglucanase (GH5) Endoglucanase (GH5) b-Glucosidase (GH1) b-Glucosidase (GH1) Endoxylanase (GH10) Endoxylanase (GH10) Endoxylanase (GH10) Chitinase Chitinase Lipase Lipase Protease a b
Source/organism
Maximal activity at
Stability (T1/2)
Specific activity
Refs
Halorubrum xinjiangense Pilot-plant biogas reactor Thermotoga neapolitana Thermococcus kodakarensis KOD1
708C (pH 8.5) 808C (pH 7.0) 808C (pH 5.0–7.0) 1008C (pH 5.6-6.0)
808C, 60 min 708C, >3 hours 808C, 88 min n.d. b
487 U/mg 1000 U/mg 25 U/mg 118 U/mg
[43] [18] [44] [16]
Geobacillus toebii E134 Salimicrobium halophilum LY20 Archaeal enrichment Dictyoglomus thermophilum Thermotoga thermarum DSM 5069T Hydrothermal spring metagenome Acidothermus cellulolyticus 11B Thermotoga thermarum Thermotoga petrophila Sulfolobus tokodaii Bacillus thuringiensis subsp. kurstaki Thermoanaerobacter thermohydrosulfuricus Metagenomic enrichment culture Coprothermobacter proteolyticus
708C (pH 6.8) 708C (pH 10.0) 1098C (pH 6.8) 60–858C (pH 5) 908C (pH 4.8) 908C (pH 6.5) 908C (pH 6.0) 958C (pH 7.0) 958C (pH 6.0) 708C (pH 2.5) 1108C (pH 9.0) 758C (pH 8.0) 708C (pH 8.0) 858C (pH 9.5)
n.d. b 708C, >24 hours 1008C, 4.5 hours 708C, 336 hours 908C, 2 hours 908C, >90 min 908C, 90 min 908C, 60 min 968C, 55 min n.d. b n.d. b 908C, >50 min 908C, >4 hours n.d. b
5 U/mg 573 U/mg 4 U/mg 5 U/mg 142 U/mg (Vmax) 3195 U/mg 350 U/mg 146 U/mg 2600 U/mg 0.08 U/mg 4.7 U/mg 12 U/mg 12 U/mg 4 U/mg
[45] [46] [27] [26] [47] [48] [28] [29] [49] [30] [31] [50] [51] [37]
Enzyme data from 2009 to 2014 publications were exclusively listed. n.d., not determined.
cluding starch, hemicellulose, and others contributes to the applicability of extremophilic species in industrial bioprocesses. Thermophilic bacteria with a potential to produce cellulosic solvents belong to species of the genera Thermoanaerobacter, Caldicellulosiruptor, and Clostridium. However, their unique features such as available information on efficient replication origins of plasmids, stable marker genes, and low permeable plasma membranes usually hinder the genetic manipulation of extremophilic microorganisms [8]. This short survey describes recent developments regarding extremozymes relevant for industrial utilizations. The main focus lies on the production and application of glycoside hydrolases such as starch and lignocellulose degrading enzymes relevant for the production of 1st and 2nd generation biofuels and other biomass processing biocatalysts.
Amylolytic enzymes The first enzyme that has been discovered in 1833 by the French chemist Anselme Payen was diastase, a starchdegrading enzyme. Starch is a ubiquitous reserve molecule in plants and one of nature’s most abundant energy sources. It is a heterogenous polysaccharide composed of glucose units, which are linked via a-1,4-glycosidic and a-1,6-glycosidic bonds forming the insoluble linear polymer amylose and the soluble branched component amylopectin. A combination of enzymatic actions is required for the complete degradation of starch into monomeric glucose. The respective glycoside hydrolases, such as a-amylases and b-amylases, glucoamylases, a-glucosidases, and pullulanases are widespread among Current Opinion in Biotechnology 2014, 29:116–123
microorganisms including extremophiles [5,9] (Table 1). Starch has attracted industrial attention in versatile processes with starch-hydrolysing enzymes accounting for 25% of the global enzyme market. This complex polysaccharide is mainly used as an additive in food or as a natural occurring ingredient. The conventional industrial conversion of starch to single glucose units is composed of a two-step industrial process: (i) liquefaction of the raw starch granules followed by (ii) saccharification. The key enzymes in the production of glucose from starch are typically a bacterial a-amylase and a fungal glucoamylase with the addition of a pullulanase to cleave a-1,6-linkages during saccharification [10]. This process sets off with a first indispensable heating step (1058C for 5 min and 958C for 1 hour at pH 6.0), to facilitate the liquefaction of dry starch, followed by a saccharification step (608C for 3 hours at pH 4.5) [11]. Because of the lack of enzymes that are active at higher temperatures and pH in the 2nd step, cooling and pH adjustment is necessary. This energy and time consuming step can be optimized by finding more suitable amylolytic enzymes [12]. These enzymes should be completely independent from calcium or other metallic ions for catalytic activity [13]. The first archaeal a-amylase with a temperature optimum around 1008C and residual activity at 1308C has been characterized in 1990 and was found in Pyrococcus furiosus [14]. A calcium-independent, acid-stable a-amylase has been recently investigated in detail and was shown to be a potential candidate for industrial starch hydrolysis with a www.sciencedirect.com
Industrial applications of extremozymes Elleuche et al. 119
Figure 2
FEEDSTOCKS
PLATFORM
CHEMICALS
Sugars
M
Cellulose
Glucose
Hemicellulose
Fructose
Lignin Pectin
Xylose Arabinose Sucrose
I C R O B I A L
Fatty acids Oil Glycerin
Energy C2-C6 compounds Ethanol
Biofungicide Butanol Bioinsecticide Lactic acid Emulsifiers Propionic acid
O
Succinic acid Polymers Glutamic acid
E
Textiles Acetoin Food Lysine
R
Chitin
Oligo saccharides
Feed
S
Vitamins
I
Methane
Environment Health
O N
NOVEL EXTREMOZYMES
Pharma ceuticals
Antioxidants
C
V Amino acids
Chemicals
Propanol
N
Protein
APPLICATION
Carbon dioxide
Hygiene Wastewater Current Opinion in Biotechnology
Development of sustainable biobased technologies. Schematic diagram illustrates different feedstock materials that can be processed by tailor-made extremozymes to produce platform chemicals for various industrial applications.
half-life of 30 min at 808C [15]. The most heat-active pullulanase with a temperature optimum at 1008C has been recently discovered in Thermococcus kodakarensis KOD1 [16]. One of the most heat-active starch-degrading enzymes is a GH57-family a-amylase from Methanococcus jannaschii with a temperature optimum at 1208C [17]. Novel genes encoding amylolytic enzymes were also identified from industrial processes containing mixed microbial cultures. A highly active a-amylase of unknown origin has been recently discovered in a pilot-plant biogas reactor [18]. Moreover, starch has attracted much industrial attention as a substrate for ethanol production, that is, as a fuel additive or starting material for chemicals. Since 1st generation biofuels are in direct competition with the www.sciencedirect.com
food industry, agricultural lignocellulose-containing biomass is a more appropriate material for the production of renewable biofuels to compete with fossil fuels.
Lignocellulose-degrading enzymes Low-value local feedstock, such as lignocellulose from straw, should preferably be used in a flexible zero-waste biorefinery to create profitable plants competing with existing industries (Figure 2) [19]. For the production of 2nd generation biofuels, the complex structure of lignocellulosic biomass must be disrupted to gain access to the polysaccharides and other polymers. The lignocellulose material, which is composed of cellulose, hemicellulose, and lignin, is nowadays decomposed by different methods, such as dilute acid treatment or ammonia freeze explosion [20]. However, hydrothermal preCurrent Opinion in Biotechnology 2014, 29:116–123
120 Cell and pathway engineering
treatment combined with moderate pressure is a more sustainable technology for the decomposition of various plant materials such as straw, since no additional chemicals are necessary [21]. Cellulose is composed of b-1,4-glycosidic linked D-glucose monomers. Hemicellulose is a heterogeneous polysaccharide that mainly consists of b-1,4-glycosidic linked D-xylose with other sugar monomers, such as D-mannose, D-glucose, D-arabinose, and D-galactose. These polysaccharides can be degraded to fermentable sugars by cellulases (endoglucanases, cellobiohydrolases, and bglucosidases) and hemicellulases (mainly endoxylanases and b-xylosidases) [22]. The complex structure of lignin can be subjected to pyrolysis to yield higher-value products, such as phenols, methane, and coal. Alternatively, laccases, peroxidases, and oxidases interact synergistically to degrade lignin [23]. Bioethanol production from lignocellulose does not operate as profitable as starch-based production, yet. However, efficiency can be increased by the combined saccharification of cellulose and hemicellulose to provide both glucose and xylose for subsequent fermentation. This, however, is based on the availability of yeast strains or alternative ethanol producers to metabolize xylose as well. Furthermore, combining hydrothermal pretreatment with enzymatic degradation at high temperatures under applied pressure is a proposed idea to optimize process designs [24]. Hereby, cooling steps or time for transfer is omitted. Additionally, high temperatures contribute to substrate accessibility and reduced viscosity. Shorter residence times, higher substrate loadings, and faster enzymatic degradation can be obtained. In order to achieve a more sustainable technology, an integrated biorefinery concept should be developed. The application of tailor-made extremozymes and the development of robust microorganisms with intelligent metabolic pathways will allow for the bioconversion of waste biomass to chemicals of industrial value such as alcohols, solvents, acids, emulsifiers, and biopolymers. The most expensive step in lignocellulose biorefineries is enzyme application, thus representing an enormous potential for improvement. Existing bioethanol producing plants employ fungal enzymes for polysaccharide containing biomass degradation at moderate temperatures. For the continuous operation at high temperatures, commercially available enzymes are not suitable [25]. Important cellulase and hemicellulase requirements for application in new concept biorefineries include high activity at elevated temperatures and high thermostability for extensive use. Protein engineering has become a powerful tool for enhancing stability and robustness. Nevertheless, the discovery of an endoglucanase from Dictyoglomus thermophilum, which Current Opinion in Biotechnology 2014, 29:116–123
exhibits a half-life of 336 hours at 708C, demonstrates the existence and availability of hyperthermophiles producing extremely thermostable cellulases [26]. In addition, Archaea and their enzyme portfolio are becoming a focus of research, because of their high thermostability. This was shown by an archaeal enrichment culture producing an endoglucanase with a half-life of 4.5 hours at 1008C [27]. Thermoactive and thermostable xylanases were lately discovered from many species of the genus Thermotoga (Table 1). The endoxylanases from Acidothermus cellulolyticus and from Thermotoga thermarum have a half-life at 908C of 1.5 hour and 1 hour, respectively [28,29]. Thus, prospective metagenomic investigations will give special attention to environments harbouring hyperthermophiles. By employing novel thermostable enzymes, biorefineries are getting closer to a sustainable as well as competitive fuel-generating technology.
Other industrial relevant enzymes Besides their application in starch and lignocellulose degradation, extremozymes are of tremendous interest for further industrial processes. Chitinases decompose the b-1,4-glycosidic linked N-acetylglucosamine units of chitin by the synergistic action of different enzymes. Since the exoskeleton of fungi, insects, and crustaceans is composed of this structural polysaccharide, chitinolytic enzymes are applied as biofungicide and bioinsecticide. Recently, a heat-active chitinase from the archaeon Sulfolobus tokodaii was characterized, showing optimal activity at 708C at pH values around 2.5 [30]. In contrast, a bacterial representative was found to exhibit highest activity at 1108C in alkaline environments with pH values between 9.0 and 12.0 [31]. Another field of application is the production of chitin-oligosaccharides or fertiliser from marine waste [32]. A chitinolytic extremozyme from the halophilic archaeon Halobacterium salinarum showed highest activity in the presence of 1.5 M NaCl, allowing for suitable degradation of oceanic chitin-containing residuals [33]. Another structural polysaccharide with a backbone of a1,4-linked galacturonic acid residues and heterogeneous side chains consisting of L-arabinose, D-galactose, L-rhamnose, and D-xylose is pectin. Because of different proportions of methoxyl groups, there is a need for various enzymes to degrade different pectins. These biocatalysts are suitable for numerous industrial sectors, such as the beverage industry, wastewater purification, or wine production [34]. Proteolytic enzymes are extensively used in the detergent, food, leather, pharmaceutical, and textile industry. Various possibilities of protease engineering approaches have been reviewed in the light of a variety of improved enzymes available [35]. Nevertheless, novel biocatalysts with properties such as temperature optima between 4 www.sciencedirect.com
Industrial applications of extremozymes Elleuche et al. 121
and 378C are still being discovered. These candidates are suitable for cold washing purposes [36]. Additionally, heat-active enzymes with optimal activity at high temperatures, such as a protease from the thermophilic Coprothermobacter proteolyticus, are also in demand for use under rough process conditions [37]. Lipolytic enzymes can be used for the synthesis or hydrolysis of fatty acids. Esterases prefer short chain acyl esters (10 carbon atoms). Multiple industrial applications favour the screening and identification of novel lipid-modifying enzymes from extreme environments. Because of their unique attributes including their enantioselectivity and regioselectivity, these enzymes are applied in the synthesis of drugs and other valuable chemical components [38]. Cold-active lipolytic enzymes, such as an esterase from Pseudoalteromas arctica, have been shown to exhibit residual activity even near 08C [39]. Moreover, a heatactive lipase (Topt: 708C) from Thermoanaerobacter thermohydrosulfuricus has recently been modified by incorporating non-canonical amino acids to widen the range of industrial applications by enhancing enzyme activation, activity, and substrate tolerance [40]. The identification of heat-active phytases from extremophiles is crucial for ensuring rich animal nutrition preparations and optimal ecology-minded monogastric stockbreeding. Phytate is found in grains, legumes, and oil seeds and consists of six phosphate molecules that can be enzymatically released stepwise to give inositol. The cyclic acid molecule can be utilized by ruminants but not by other animals. Hence, phosphorolytic enzymes, the so called phytases, are used for swine, poultry, and also fish diets to increase the phytate phosphorus uptake without external phosphate supplementation, thereby simultaneously reducing the faecal phosphorus excretion. Therefore, these hydrolases contribute on the one hand to reduce animal feed costs and on the other hand to environmental protection by minimizing animal waste pollution. Heat-active phytases are needed for various processes such as feed pelleting at high temperatures [41]. So far, no applicable phytases with high temperature optima were discovered, opening an up-to-date research field to handle the prospective demand of these enzymes [42].
challenges to be met, including the production of extremozymes in large-scale. Moreover, the establishment of efficient heterologous production systems based on extremophilic hosts is still at the beginning, but the advancing availability will pave the way for easier and faster production of extremozymes in the future. Additionally, a lot of work that has been done in the past 50 years using traditional laboratory workhorses such as E. coli and unicellular yeasts can be easily transferred to research on extremophiles. Moreover, the rapid development in the biological ‘-omics’ fields of (meta-) genomics, proteomics, and metabolomics and of directed evolution, multienzyme complexes and synthetic biology will soon provide tailor-made biocatalysts with desired properties leading to the development of a sustainable biobased industry.
Acknowledgements Mazen Rizk is thanked for critically reading the manuscript. Research at the institute is funded by BMBF (German Federal Ministry for Education and Research) through clusters Biokatalyse2021 (0315171) and Biorefinery2021 (0315559A).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Woodley JM: Protein engineering of enzymes for process applications. Curr Opin Chem Biol 2013, 17:310-316.
2.
Adams MW, Perler FB, Kelly RM: Extremozymes: expanding the limits of biocatalysis. Biotechnology (N Y) 1995, 13:662-668.
3.
Davids T, Schmidt M, Bottcher D, Bornscheuer UT: Strategies for the discovery and engineering of enzymes for biocatalysis. Curr Opin Chem Biol 2013, 17:215-220.
4.
Rizk M, Antranikian G, Elleuche S: End-to-end gene fusions and their impact on the production of multifunctional biomass degrading enzymes. Biochem Biophys Res Commun 2012, 428:1-5.
5.
Nisha M, Satyanarayana T: Recombinant bacterial amylopullulanases: developments and perspectives. Bioengineered 2013, 4. This review provides an excellent overview about GH13-family and GH57family amylopullulanases considering their distribution in microorganisms, catalytic functionality, and industrial applications. 6.
Albers SV, Jonuscheit M, Dinkelaker S, Urich T, Kletzin A, Tampe R, Driessen AJ, Schleper C: Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus. Appl Environ Microbiol 2006, 72:102-111.
7.
Leis B, Angelov A, Liebl W: Screening and expression of genes from metagenomes. Adv Appl Microbiol 2013, 83:1-68.
Conclusion and future prospects
8.
Because of economical awareness towards environmental protection and the need of sustainable biofuel alternatives, processes catalysed by enzymes are gradually replacing chemical routes in industry. So far, the majority of biocatalysts that are used in industry are derived from mesophilic fungi or bacteria, but the number of industrial relevant proteins of extremophilic origin is continuously increasing. Nevertheless, there are some essential
9.
www.sciencedirect.com
Lin L, Xu J: Dissecting and engineering metabolic and regulatory networks of thermophilic bacteria for biofuel production. Biotechnol Adv 2013, 31:827-837. A research review demonstrating genetic engineering techniques of thermophiles in detail. The authors give an excellent overview about carbohydrate utilization for biofuel production and take different stress responses into account. Sunna A, Moracci M, Rossi M, Antranikian G: Glycosyl hydrolases from hyperthermophiles. Extremophiles 1997, 1:2-13.
10. Kumar P, Satyanarayana T: Microbial glucoamylases: characteristics and applications. Crit Rev Biotechnol 2009, 29:225-255. Current Opinion in Biotechnology 2014, 29:116–123
122 Cell and pathway engineering
11. Bertoldo C, Antranikian G: Starch-hydrolyzing enzymes from thermophilic archaea and bacteria. Curr Opin Chem Biol 2002, 6:151-160. 12. Elleuche S, Antranikian G: Starch-hydrolyzing enzymes from thermophiles. In Thermophilic Microbes in Environmental and Industrial Biotechnology: Biotechnology of Thermophiles. Edited by Satyanarayana T, Littlechild J, Kawarabayasi Y. Springer; 2013:509-533. A comprehensive and up-to-date review, that offers deep insights into heat-stable amylolytic enzymes alongside with the production in appropriate hosts and possible biotechnological applications. 13. Antranikian G, Egorova K: Extremophiles: a unique source of biocatalysts for industrial biotechnology. In In Physiology and Biochemistry of Extremophiles. Edited by Gerday C, Glansdorff N. ASM Press; 2007:361-406. 14. Koch R, Zablowski P, Spreinat A, Antranikian G: Extremely thermostable amylolytic enzyme from the archaebacterium Pyrococcus furiosus. FEMS Microbiol Lett 1990, 71:21-26. 15. Sharma A, Satyanarayana T: Cloning and expression of acidstable, high maltose-forming, Ca2+-independent alphaamylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis. Extremophiles 2012, 16:515-522. 16. Han T, Zeng F, Li Z, Liu L, Wei M, Guan Q, Liang X, Peng Z, Liu M, Qin J et al.: Biochemical characterization of a recombinant pullulanase from Thermococcus kodakarensis KOD1. Lett Appl Microbiol 2013, 57:336-343. 17. Kim JW, Flowers LO, Whiteley M, Peeples TL: Biochemical confirmation and characterization of the family-57-like alphaamylase of Methanococcus jannaschii. Folia Microbiol (Praha) 2001, 46:467-473. 18. Jabbour D, Sorger A, Sahm K, Antranikian G: A highly thermoactive and salt-tolerant alpha-amylase isolated from a pilot-plant biogas reactor. Appl Microbiol Biotechnol 2013, 97:2971-2978. An amylase that tolerates elevated temperatures in combination with high concentrations of salt offers the potential to be applicable for starch degradation under harsh conditions. 19. Clark JH, Luque R, Matharu AS: Green chemistry, biofuels, and biorefinery. Annu Rev Chem Biomol Eng 2012, 3:183-207. 20. Alvira P, Tomas-Pejo E, Ballesteros M, Negro MJ: Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 2010, 101:4851-4861. 21. Thomsen MH, Thygesen A, Jorgensen H, Larsen J, Christensen BH, Thomsen AB: Preliminary results on optimization of pilot scale pretreatment of wheat straw used in coproduction of bioethanol and electricity. Appl Biochem Biotechnol 2006, 129–132:448-460. 22. Klippel B, Antranikian G: Lignocellulose converting enzymes from thermophiles. In Extremophiles Handbook. Edited by Horikoshi K, Antranikian , Bull AT, Robb FT, Stetter KO. Springer; 2011:443-474. 23. Martinez AT, Speranza M, Ruiz-Duenas FJ, Ferreira P, Camarero S, Guillen F, Martinez MJ, Gutierrez A, del Rio JC: Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin. Int Microbiol 2005, 8:195-204. 24. Kirsch C, Zetzl C, Smirnova I: Development of an integrated thermal and enzymatic hydrolysis for lignocellulosic biomass in fixed-bed reactors. Holzforschung 2011, 65:483-489. 25. Bhalla A, Bansal N, Kumar S, Bischoff KM, Sani RK: Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresour Technol 2013, 128:751-759. 26. Shi R, Li Z, Ye Q, Xu J, Liu Y: Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum. Bioresour Technol 2013, 142:338-344. A highly thermostable bacterial cellulase with a half-life of two weeks at high temperatures distinguishes this enzyme from other cellulolytic biocatalysts. Since the enzymatic supply is often the most cost-effective position, processes may run for more than two weeks using the same load of enzyme, thus, contributing to an enormous cost reduction. Current Opinion in Biotechnology 2014, 29:116–123
27. Graham JE, Clark ME, Nadler DC, Huffer S, Chokhawala HA, Rowland SE, Blanch HW, Clark DS, Robb FT: Identification and characterization of a multidomain hyperthermophilic cellulase from an archaeal enrichment. Nat Commun 2011, 2:375. An endoglucanase displays optimal activity at 1098C and exhibits an outstanding half-life of 5 hours at 1008C. This research emphasizes Archaea to become focus of research for prospective metagenomic studies on thermostable cellulases. 28. Barabote RD, Parales JV, Guo YY, Labavitch JM, Parales RE, Berry AM: Xyn10A, a thermostable endoxylanase from Acidothermus cellulolyticus 11B. Appl Environ Microbiol 2010, 76:7363-7366. 29. Shi H, Zhang Y, Li X, Huang Y, Wang L, Wang Y, Ding H, Wang F: A novel highly thermostable xylanase stimulated by Ca2+ from Thermotoga thermarum: cloning, expression and characterization. Biotechnol Biofuels 2013, 6:26. 30. Staufenberger T, Imhoff JF, Labes A: First crenarchaeal chitinase found in Sulfolobus tokodaii. Microbiol Res 2012, 167:262-269. 31. Kuzu SB, Guvenmez HK, Denizci AA: Production of a thermostable and alkaline chitinase by Bacillus thuringiensis subsp. kurstaki strain HBK-51. Biotechnol Res Int 2012, 2012:1354-1398. 32. Hamid R, Khan MA, Ahmad M, Ahmad MM, Abdin MZ, Musarrat J, Javed S: Chitinases: an update. J Pharm Bioallied Sci 2013, 5:21-29. 33. Garcia-Fraga B, da Silva AF, Lopez-Seijas J, Sieiro C: Functional expression and characterization of a chitinase from the marine archaeon Halobacterium salinarum CECT 395 in Escherichia coli. Appl Microbiol Biotechnol 2013, 98:2133-2143. 34. Cabeza MS, Baca FL, Mun˜oz Puntes E, Loto F, Baigorı´ MD, Morata VI: Selection of psychrotolerant microorganisms producing cold-active pectinases for biotechnological processes at low temperature. Food Technol Biotechnol 2011, 49:187-195. 35. Li Q, Yi L, Marek P, Iverson BL: Commercial proteases: present and future. FEBS Lett 2013, 587:1155-1163. 36. Saba I, Qazi PH, Rather SA, Dar RA, Qadri QA, Ahmad N, Johri S, Taneja SC, Shawl S: Purification and characterization of a cold active alkaline protease from Stenotrophomonas sp., isolated from Kashmir, India. World J Microbiol Biotechnol 2012, 28:1071-1079. 37. Toplak A, Wu B, Fusetti F, Quaedflieg PJ, Janssen DB: Proteolysin, a novel highly thermostable and cosolventcompatible protease from the thermophilic bacterium Coprothermobacter proteolyticus. Appl Environ Microbiol 2013, 79:5625-5632. 38. Sharma R, Thakur V, Sharma M, Birkeland N-K: Biocatalysis through thermostable lipases: adding flavor to chemistry. In Thermophilic Microbes in Environmental and Industrial Biotechnology: Biotechnology of Thermophiles. Edited by Satyanarayana T, Littlechild J, Kawarabayasi Y. Springer; 2013:905-927. 39. Al Khudary R, Venkatachalam R, Katzer M, Elleuche S, Antranikian G: A cold-adapted esterase of a novel marine isolate, Pseudoalteromonas arctica: gene cloning, enzyme purification and characterization. Extremophiles 2010, 14: 273-285. 40. Hoesl MG, Acevedo-Rocha CG, Nehring S, Royter M, Wolschner C, Wiltschi B, Budisa N, Antranikian G: Lipase congeners designed by genetic code engineering. ChemCatChem 2011, 3:213-221. 41. Yao MZ, Zhang YH, Lu WL, Hu MQ, Wang W, Liang AH: Phytases: crystal structures, protein engineering and potential biotechnological applications. J Appl Microbiol 2012, 112:1-14. 42. Lei XG, Weaver JD, Mullaney E, Ullah AH, Azain MJ: Phytase, a new life for an ‘‘old’’ enzyme. Annu. Rev. Anim. Biosci. 2013, 1:283-309. This review summarizes structural information about phytases, possibilities of application, new approaches of industry and academia as well as related issues and future directions. An overview covering all interests is given and recommended as greatly readable. www.sciencedirect.com
Industrial applications of extremozymes Elleuche et al. 123
43. Moshfegh M, Shahverdi AR, Zarrini G, Faramarzi MA: Biochemical characterization of an extracellular polyextremophilic alpha-amylase from the halophilic archaeon Halorubrum xinjiangense. Extremophiles 2013, 17:677-687. 44. Kang J, Park KM, Choi KH, Park CS, Kim GE, Kim D, Cha J: Molecular cloning and biochemical characterization of a heatstable type I pullulanase from Thermotoga neapolitana. Enzyme Microb Technol 2011, 48:260-266. 45. Cihan AC, Benli M, Cokmus C: Purification and characterization of intracellular and extracellular alpha-glucosidases from Geobacillus toebii strain E134. Cell Biochem Funct 2011, 30:6981. 46. Li X, Yu HY: Purification and characterization of novel organicsolvent-tolerant beta-amylase and serine protease from a newly isolated Salimicrobium halophilum strain LY20. FEMS Microbiol Lett 2012, 329:204-211. 47. Zhao L, Xie J, Zhang X, Cao F, Pei J: Overexpression and characterization of a glucose-tolerant b-glucosidase from Thermotoga thermarum DSM 5069T with high catalytic efficiency of ginsenoside Rb1 to Rd. J Mol Catal B Enzym 2013, 95:62-69. 48. Schro¨der C, Elleuche S, Blank S, Antranikian G: Characterization of a heat-active archael beta-glucosidase from a hydrothermal spring metagenome. Enzyme Microb Technol 2014, 57:48-54.
www.sciencedirect.com
49. ul Haq I, Hussain Z, Khan MA, Muneer B, Afzal S, Majeed S, Akram F: Kinetic and thermodynamic study of cloned thermostable endo-1,4-beta-xylanase from Thermotoga petrophila in mesophilic host. Mol Biol Rep 2012, 39:7251-7261. 50. Royter M, Schmidt M, Elend C, Hobenreich H, Schafer T, Bornscheuer UT, Antranikian G: Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis. Extremophiles 2009, 13:769-783. 51. Chow J, Kovacic F, Dall Antonia Y, Krauss U, Fersini F, Schmeisser C, Lauinger B, Bongen P, Pietruszka J, Schmidt M et al.: The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases. PLoS ONE 2012, 7:4766-4775. 52. Stetter KO: History of discovery of extremophiles. In Extremophiles Handbook. Edited by Horikoshi K, Antranikian G, Bull AT, Robb FT, Stetter KO. Springer; 2011:403-425. 53. Zaparty M, Siebers B: Physiology, metabolism and enzymology of thermoacidophiles. In Extremophiles Handbook. Edited by Horikoshi K, Antranikian G, Bull AT, Robb FT, Stetter KO. Springer; 2011:601-639. 54. Luo H, Robb FT: Thermophilic protein folding systems. In Extremophiles Handbook. Edited by Horikoshi K, Antranikian G, Bull AT, Robb FT, Stetter KO. Springer; 2011:583-599.
Current Opinion in Biotechnology 2014, 29:116–123
ScienceDirect Extremozymes — biocatalysts with unique properties from extremophilic microorganisms Skander Elleuche, Carola Schro¨der, Kerstin Sahm and Garabed Antranikian Extremozymes are enzymes derived from extremophilic microorganisms that are able to withstand harsh conditions in industrial processes that were long thought to be destructive to proteins. Heat-stable and solvent-tolerant biocatalysts are valuable tools for processes in which for example hardly decomposable polymers need to be liquefied and degraded, while cold-active enzymes are of relevance for food and detergent industries. Extremophilic microorganisms are a rich source of naturally tailored enzymes, which are more superior over their mesophilic counterparts for applications at extreme conditions. Especially lignocellulolytic, amylolytic, and other biomass processing extremozymes with unique properties are widely distributed in thermophilic prokaryotes and are of high potential for versatile industrial processes. Addresses Institute of Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, D-21073 Hamburg, Germany Corresponding author: Antranikian, Garabed ([email protected])
Current Opinion in Biotechnology 2014, 29:116–123 This review comes from a themed issue on Cell and pathway engineering Edited by Tina Lu¨tke-Eversloh and Keith EJ Tyo
0958-1669/$ – see front matter, # 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2014.04.003
Introduction Biotechnology is omnipresent and has a greater impact than previously envisioned on several diverse industries, such as feed and food production, biofuel and energy generation as well as sustainable production of high-value chemical compounds (Figure 1). Conditions in an industrial process are often far from standard biocatalyst’s properties. Hence, there is considerable demand for a new generation of stable enzymes that are able to reach this goal by replacing or supplementing traditional chemical processes [1]. Enzyme properties such as temperature stability, selectivity, solvent-tolerance, or affinity to the substrate can be improved through genetic engineering. However, the task Current Opinion in Biotechnology 2014, 29:116–123
is tedious and lacks rational concepts. Extremozymes, enzymes derived from extremophilic microorganisms (Box 1), are an attractive alternative to tuning a given biocatalyst for a specific industrial application. They are capable of catalysing their respective reactions in nonaqueous environments, water/solvent mixtures, at extremely high pressures, acidic and alkaline pH, at temperatures up to 1408C, or near the freezing point of water [2]. These enzymes already contain properties that are generated in synthetically tailored enzymes by directed evolution, DNA shuffling, gene fusion, truncation, or rational protein synthesis [3,4,5]. However, the majority of enzymes that are currently used in the industry are obtained from fungi or mesophilic bacteria. Only a few Box 1 Extremophilic microorganisms Living organisms colonize almost all ecological niches on earth. Those that are metabolically active and grow under conditions unusual from a human perspective are termed extremophiles, because they seem to ‘love the extreme’. At temperatures above 628C only thermophilic prokaryotic organisms can survive and thrive and among hyperthermophilic organisms growing optimally at temperatures above 808C Archaea are dominating. From what is known today, Methanopyrus kandleri is able to survive the highest temperature of 1228C under 200 bar pressure [52]. Acidophiles growing optimally at pH values around or below 3.0 can be found in all three domains of life. Mesophilic acidophiles (Acidithiobacillus, Ferroplasma, Leptospirillum) have long been used for bioleaching. Some Archaea can grow at low pH and high temperature (thermoacidophiles). Picrophilus torridus is one of the most thermoacidophilic microorganisms, growing at pH values of zero and at temperatures of up to 658C. Hyperthermophilic thermoacidophiles growing at temperatures above 808C usually tolerate only pH values of 2.0–3.0 although some Sulfolobus species and Acidianus infernus can cope with pH values lower than 2.0 [53]. All cellular components (proteins, nucleic acids and lipids) of extremophiles have to be stable at specific extreme growth conditions. For chemical extremes like pH or salinity specific export systems, membrane adaptations or small molecules called compatible solutes can provide a near to normal intracellular milieu in some species. But even in these organisms, extracellular enzymes have to be stable in the extreme surrounding. No individual structural properties can be singled out to account for extremozyme stability. However, for increased temperature stability of proteins, some principles can be identified like highly charged exterior surfaces, rigid folds maintained by multiple ion-pair networks, tight hydrophobic core packing and overall more compact and densely packed protein structures, partly caused by an increased content of acidic and basic amino acids [54]. In addition to these structural features, highly effective protein-repair and DNA-repair systems and cytoplasmic membranes with specific characteristics are contributing to enable microorganisms to cope with extreme conditions.
www.sciencedirect.com
Industrial applications of extremozymes Elleuche et al. 117
Figure 1
using catalysis
ymes
z extremo
Bio
Biorefinery
New processes and products
Textiles Dairy -12 ºC
Temperature
122 ºC 20 ºC
pH
37 ºC Starch liquefaction
Detergent
pH 11
pH 2 pH 8
pH 5 Food
Solvents
0%
100 % Classic biocata al lysis
New processes and products
Two phase biotransformations
Current Opinion in Biotechnology
Extremozymes widen the scope of biocatalysis. Biocatalysts of extremophiles can withstand a broader range of temperature, pH, and solvents than enzymes from mesophilic organisms.
extremozymes have found their way to the market with DNA-polymerase from Thermus aquaticus and its relatives being the most prominent example. Nowadays, new challenges have broadened the range of successful utilization of extremozymes beyond their use in DNA replication. Besides utilization of traditional protein engineering approaches in mesophilic hosts, genetic improvements of extremophilic expression systems are currently under intense investigation. Improper folding and differences in codon usage generally interferes with the production of functional extremozymes in commonly used expression systems such as Escherichia coli or Bacillus sp. Therefore, it is an important prerequisite to establish fine regulated extremophilic hosts, efficient transformation approaches www.sciencedirect.com
and adequate expression vectors. Only a few functional systems have been presented mainly using different members of the genus Thermus or the hyperthermophilic crenarchaeotal species Sulfolobus solfataricus [6,7]. Even the application of extremophiles as cellular biocatalysts for biotransformation has attracted interest in recent years. Especially metabolic and regulatory networks of thermophiles have been manipulated to generate microbial cellular factories for biofuel production in a Consolidated Bioprocessing approach that might be superior in the future due to accelerated reaction rates, reduced energy input, and minimized contamination risk. The ability to utilize multiple carbohydrate sources inCurrent Opinion in Biotechnology 2014, 29:116–123
118 Cell and pathway engineering
Table 1 Heat-active hydrolases with potential industrial relevancea Enzyme (glycoside hydrolase family) a-Amylase a-Amylase (GH13) Pullulanase (GH13) Pullulanase/amylase (GH13) a-Glucosidase b-Amylase Endoglucanase (GH5) Endoglucanase (GH5) b-Glucosidase (GH1) b-Glucosidase (GH1) Endoxylanase (GH10) Endoxylanase (GH10) Endoxylanase (GH10) Chitinase Chitinase Lipase Lipase Protease a b
Source/organism
Maximal activity at
Stability (T1/2)
Specific activity
Refs
Halorubrum xinjiangense Pilot-plant biogas reactor Thermotoga neapolitana Thermococcus kodakarensis KOD1
708C (pH 8.5) 808C (pH 7.0) 808C (pH 5.0–7.0) 1008C (pH 5.6-6.0)
808C, 60 min 708C, >3 hours 808C, 88 min n.d. b
487 U/mg 1000 U/mg 25 U/mg 118 U/mg
[43] [18] [44] [16]
Geobacillus toebii E134 Salimicrobium halophilum LY20 Archaeal enrichment Dictyoglomus thermophilum Thermotoga thermarum DSM 5069T Hydrothermal spring metagenome Acidothermus cellulolyticus 11B Thermotoga thermarum Thermotoga petrophila Sulfolobus tokodaii Bacillus thuringiensis subsp. kurstaki Thermoanaerobacter thermohydrosulfuricus Metagenomic enrichment culture Coprothermobacter proteolyticus
708C (pH 6.8) 708C (pH 10.0) 1098C (pH 6.8) 60–858C (pH 5) 908C (pH 4.8) 908C (pH 6.5) 908C (pH 6.0) 958C (pH 7.0) 958C (pH 6.0) 708C (pH 2.5) 1108C (pH 9.0) 758C (pH 8.0) 708C (pH 8.0) 858C (pH 9.5)
n.d. b 708C, >24 hours 1008C, 4.5 hours 708C, 336 hours 908C, 2 hours 908C, >90 min 908C, 90 min 908C, 60 min 968C, 55 min n.d. b n.d. b 908C, >50 min 908C, >4 hours n.d. b
5 U/mg 573 U/mg 4 U/mg 5 U/mg 142 U/mg (Vmax) 3195 U/mg 350 U/mg 146 U/mg 2600 U/mg 0.08 U/mg 4.7 U/mg 12 U/mg 12 U/mg 4 U/mg
[45] [46] [27] [26] [47] [48] [28] [29] [49] [30] [31] [50] [51] [37]
Enzyme data from 2009 to 2014 publications were exclusively listed. n.d., not determined.
cluding starch, hemicellulose, and others contributes to the applicability of extremophilic species in industrial bioprocesses. Thermophilic bacteria with a potential to produce cellulosic solvents belong to species of the genera Thermoanaerobacter, Caldicellulosiruptor, and Clostridium. However, their unique features such as available information on efficient replication origins of plasmids, stable marker genes, and low permeable plasma membranes usually hinder the genetic manipulation of extremophilic microorganisms [8]. This short survey describes recent developments regarding extremozymes relevant for industrial utilizations. The main focus lies on the production and application of glycoside hydrolases such as starch and lignocellulose degrading enzymes relevant for the production of 1st and 2nd generation biofuels and other biomass processing biocatalysts.
Amylolytic enzymes The first enzyme that has been discovered in 1833 by the French chemist Anselme Payen was diastase, a starchdegrading enzyme. Starch is a ubiquitous reserve molecule in plants and one of nature’s most abundant energy sources. It is a heterogenous polysaccharide composed of glucose units, which are linked via a-1,4-glycosidic and a-1,6-glycosidic bonds forming the insoluble linear polymer amylose and the soluble branched component amylopectin. A combination of enzymatic actions is required for the complete degradation of starch into monomeric glucose. The respective glycoside hydrolases, such as a-amylases and b-amylases, glucoamylases, a-glucosidases, and pullulanases are widespread among Current Opinion in Biotechnology 2014, 29:116–123
microorganisms including extremophiles [5,9] (Table 1). Starch has attracted industrial attention in versatile processes with starch-hydrolysing enzymes accounting for 25% of the global enzyme market. This complex polysaccharide is mainly used as an additive in food or as a natural occurring ingredient. The conventional industrial conversion of starch to single glucose units is composed of a two-step industrial process: (i) liquefaction of the raw starch granules followed by (ii) saccharification. The key enzymes in the production of glucose from starch are typically a bacterial a-amylase and a fungal glucoamylase with the addition of a pullulanase to cleave a-1,6-linkages during saccharification [10]. This process sets off with a first indispensable heating step (1058C for 5 min and 958C for 1 hour at pH 6.0), to facilitate the liquefaction of dry starch, followed by a saccharification step (608C for 3 hours at pH 4.5) [11]. Because of the lack of enzymes that are active at higher temperatures and pH in the 2nd step, cooling and pH adjustment is necessary. This energy and time consuming step can be optimized by finding more suitable amylolytic enzymes [12]. These enzymes should be completely independent from calcium or other metallic ions for catalytic activity [13]. The first archaeal a-amylase with a temperature optimum around 1008C and residual activity at 1308C has been characterized in 1990 and was found in Pyrococcus furiosus [14]. A calcium-independent, acid-stable a-amylase has been recently investigated in detail and was shown to be a potential candidate for industrial starch hydrolysis with a www.sciencedirect.com
Industrial applications of extremozymes Elleuche et al. 119
Figure 2
FEEDSTOCKS
PLATFORM
CHEMICALS
Sugars
M
Cellulose
Glucose
Hemicellulose
Fructose
Lignin Pectin
Xylose Arabinose Sucrose
I C R O B I A L
Fatty acids Oil Glycerin
Energy C2-C6 compounds Ethanol
Biofungicide Butanol Bioinsecticide Lactic acid Emulsifiers Propionic acid
O
Succinic acid Polymers Glutamic acid
E
Textiles Acetoin Food Lysine
R
Chitin
Oligo saccharides
Feed
S
Vitamins
I
Methane
Environment Health
O N
NOVEL EXTREMOZYMES
Pharma ceuticals
Antioxidants
C
V Amino acids
Chemicals
Propanol
N
Protein
APPLICATION
Carbon dioxide
Hygiene Wastewater Current Opinion in Biotechnology
Development of sustainable biobased technologies. Schematic diagram illustrates different feedstock materials that can be processed by tailor-made extremozymes to produce platform chemicals for various industrial applications.
half-life of 30 min at 808C [15]. The most heat-active pullulanase with a temperature optimum at 1008C has been recently discovered in Thermococcus kodakarensis KOD1 [16]. One of the most heat-active starch-degrading enzymes is a GH57-family a-amylase from Methanococcus jannaschii with a temperature optimum at 1208C [17]. Novel genes encoding amylolytic enzymes were also identified from industrial processes containing mixed microbial cultures. A highly active a-amylase of unknown origin has been recently discovered in a pilot-plant biogas reactor [18]. Moreover, starch has attracted much industrial attention as a substrate for ethanol production, that is, as a fuel additive or starting material for chemicals. Since 1st generation biofuels are in direct competition with the www.sciencedirect.com
food industry, agricultural lignocellulose-containing biomass is a more appropriate material for the production of renewable biofuels to compete with fossil fuels.
Lignocellulose-degrading enzymes Low-value local feedstock, such as lignocellulose from straw, should preferably be used in a flexible zero-waste biorefinery to create profitable plants competing with existing industries (Figure 2) [19]. For the production of 2nd generation biofuels, the complex structure of lignocellulosic biomass must be disrupted to gain access to the polysaccharides and other polymers. The lignocellulose material, which is composed of cellulose, hemicellulose, and lignin, is nowadays decomposed by different methods, such as dilute acid treatment or ammonia freeze explosion [20]. However, hydrothermal preCurrent Opinion in Biotechnology 2014, 29:116–123
120 Cell and pathway engineering
treatment combined with moderate pressure is a more sustainable technology for the decomposition of various plant materials such as straw, since no additional chemicals are necessary [21]. Cellulose is composed of b-1,4-glycosidic linked D-glucose monomers. Hemicellulose is a heterogeneous polysaccharide that mainly consists of b-1,4-glycosidic linked D-xylose with other sugar monomers, such as D-mannose, D-glucose, D-arabinose, and D-galactose. These polysaccharides can be degraded to fermentable sugars by cellulases (endoglucanases, cellobiohydrolases, and bglucosidases) and hemicellulases (mainly endoxylanases and b-xylosidases) [22]. The complex structure of lignin can be subjected to pyrolysis to yield higher-value products, such as phenols, methane, and coal. Alternatively, laccases, peroxidases, and oxidases interact synergistically to degrade lignin [23]. Bioethanol production from lignocellulose does not operate as profitable as starch-based production, yet. However, efficiency can be increased by the combined saccharification of cellulose and hemicellulose to provide both glucose and xylose for subsequent fermentation. This, however, is based on the availability of yeast strains or alternative ethanol producers to metabolize xylose as well. Furthermore, combining hydrothermal pretreatment with enzymatic degradation at high temperatures under applied pressure is a proposed idea to optimize process designs [24]. Hereby, cooling steps or time for transfer is omitted. Additionally, high temperatures contribute to substrate accessibility and reduced viscosity. Shorter residence times, higher substrate loadings, and faster enzymatic degradation can be obtained. In order to achieve a more sustainable technology, an integrated biorefinery concept should be developed. The application of tailor-made extremozymes and the development of robust microorganisms with intelligent metabolic pathways will allow for the bioconversion of waste biomass to chemicals of industrial value such as alcohols, solvents, acids, emulsifiers, and biopolymers. The most expensive step in lignocellulose biorefineries is enzyme application, thus representing an enormous potential for improvement. Existing bioethanol producing plants employ fungal enzymes for polysaccharide containing biomass degradation at moderate temperatures. For the continuous operation at high temperatures, commercially available enzymes are not suitable [25]. Important cellulase and hemicellulase requirements for application in new concept biorefineries include high activity at elevated temperatures and high thermostability for extensive use. Protein engineering has become a powerful tool for enhancing stability and robustness. Nevertheless, the discovery of an endoglucanase from Dictyoglomus thermophilum, which Current Opinion in Biotechnology 2014, 29:116–123
exhibits a half-life of 336 hours at 708C, demonstrates the existence and availability of hyperthermophiles producing extremely thermostable cellulases [26]. In addition, Archaea and their enzyme portfolio are becoming a focus of research, because of their high thermostability. This was shown by an archaeal enrichment culture producing an endoglucanase with a half-life of 4.5 hours at 1008C [27]. Thermoactive and thermostable xylanases were lately discovered from many species of the genus Thermotoga (Table 1). The endoxylanases from Acidothermus cellulolyticus and from Thermotoga thermarum have a half-life at 908C of 1.5 hour and 1 hour, respectively [28,29]. Thus, prospective metagenomic investigations will give special attention to environments harbouring hyperthermophiles. By employing novel thermostable enzymes, biorefineries are getting closer to a sustainable as well as competitive fuel-generating technology.
Other industrial relevant enzymes Besides their application in starch and lignocellulose degradation, extremozymes are of tremendous interest for further industrial processes. Chitinases decompose the b-1,4-glycosidic linked N-acetylglucosamine units of chitin by the synergistic action of different enzymes. Since the exoskeleton of fungi, insects, and crustaceans is composed of this structural polysaccharide, chitinolytic enzymes are applied as biofungicide and bioinsecticide. Recently, a heat-active chitinase from the archaeon Sulfolobus tokodaii was characterized, showing optimal activity at 708C at pH values around 2.5 [30]. In contrast, a bacterial representative was found to exhibit highest activity at 1108C in alkaline environments with pH values between 9.0 and 12.0 [31]. Another field of application is the production of chitin-oligosaccharides or fertiliser from marine waste [32]. A chitinolytic extremozyme from the halophilic archaeon Halobacterium salinarum showed highest activity in the presence of 1.5 M NaCl, allowing for suitable degradation of oceanic chitin-containing residuals [33]. Another structural polysaccharide with a backbone of a1,4-linked galacturonic acid residues and heterogeneous side chains consisting of L-arabinose, D-galactose, L-rhamnose, and D-xylose is pectin. Because of different proportions of methoxyl groups, there is a need for various enzymes to degrade different pectins. These biocatalysts are suitable for numerous industrial sectors, such as the beverage industry, wastewater purification, or wine production [34]. Proteolytic enzymes are extensively used in the detergent, food, leather, pharmaceutical, and textile industry. Various possibilities of protease engineering approaches have been reviewed in the light of a variety of improved enzymes available [35]. Nevertheless, novel biocatalysts with properties such as temperature optima between 4 www.sciencedirect.com
Industrial applications of extremozymes Elleuche et al. 121
and 378C are still being discovered. These candidates are suitable for cold washing purposes [36]. Additionally, heat-active enzymes with optimal activity at high temperatures, such as a protease from the thermophilic Coprothermobacter proteolyticus, are also in demand for use under rough process conditions [37]. Lipolytic enzymes can be used for the synthesis or hydrolysis of fatty acids. Esterases prefer short chain acyl esters (10 carbon atoms). Multiple industrial applications favour the screening and identification of novel lipid-modifying enzymes from extreme environments. Because of their unique attributes including their enantioselectivity and regioselectivity, these enzymes are applied in the synthesis of drugs and other valuable chemical components [38]. Cold-active lipolytic enzymes, such as an esterase from Pseudoalteromas arctica, have been shown to exhibit residual activity even near 08C [39]. Moreover, a heatactive lipase (Topt: 708C) from Thermoanaerobacter thermohydrosulfuricus has recently been modified by incorporating non-canonical amino acids to widen the range of industrial applications by enhancing enzyme activation, activity, and substrate tolerance [40]. The identification of heat-active phytases from extremophiles is crucial for ensuring rich animal nutrition preparations and optimal ecology-minded monogastric stockbreeding. Phytate is found in grains, legumes, and oil seeds and consists of six phosphate molecules that can be enzymatically released stepwise to give inositol. The cyclic acid molecule can be utilized by ruminants but not by other animals. Hence, phosphorolytic enzymes, the so called phytases, are used for swine, poultry, and also fish diets to increase the phytate phosphorus uptake without external phosphate supplementation, thereby simultaneously reducing the faecal phosphorus excretion. Therefore, these hydrolases contribute on the one hand to reduce animal feed costs and on the other hand to environmental protection by minimizing animal waste pollution. Heat-active phytases are needed for various processes such as feed pelleting at high temperatures [41]. So far, no applicable phytases with high temperature optima were discovered, opening an up-to-date research field to handle the prospective demand of these enzymes [42].
challenges to be met, including the production of extremozymes in large-scale. Moreover, the establishment of efficient heterologous production systems based on extremophilic hosts is still at the beginning, but the advancing availability will pave the way for easier and faster production of extremozymes in the future. Additionally, a lot of work that has been done in the past 50 years using traditional laboratory workhorses such as E. coli and unicellular yeasts can be easily transferred to research on extremophiles. Moreover, the rapid development in the biological ‘-omics’ fields of (meta-) genomics, proteomics, and metabolomics and of directed evolution, multienzyme complexes and synthetic biology will soon provide tailor-made biocatalysts with desired properties leading to the development of a sustainable biobased industry.
Acknowledgements Mazen Rizk is thanked for critically reading the manuscript. Research at the institute is funded by BMBF (German Federal Ministry for Education and Research) through clusters Biokatalyse2021 (0315171) and Biorefinery2021 (0315559A).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Woodley JM: Protein engineering of enzymes for process applications. Curr Opin Chem Biol 2013, 17:310-316.
2.
Adams MW, Perler FB, Kelly RM: Extremozymes: expanding the limits of biocatalysis. Biotechnology (N Y) 1995, 13:662-668.
3.
Davids T, Schmidt M, Bottcher D, Bornscheuer UT: Strategies for the discovery and engineering of enzymes for biocatalysis. Curr Opin Chem Biol 2013, 17:215-220.
4.
Rizk M, Antranikian G, Elleuche S: End-to-end gene fusions and their impact on the production of multifunctional biomass degrading enzymes. Biochem Biophys Res Commun 2012, 428:1-5.
5.
Nisha M, Satyanarayana T: Recombinant bacterial amylopullulanases: developments and perspectives. Bioengineered 2013, 4. This review provides an excellent overview about GH13-family and GH57family amylopullulanases considering their distribution in microorganisms, catalytic functionality, and industrial applications. 6.
Albers SV, Jonuscheit M, Dinkelaker S, Urich T, Kletzin A, Tampe R, Driessen AJ, Schleper C: Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus. Appl Environ Microbiol 2006, 72:102-111.
7.
Leis B, Angelov A, Liebl W: Screening and expression of genes from metagenomes. Adv Appl Microbiol 2013, 83:1-68.
Conclusion and future prospects
8.
Because of economical awareness towards environmental protection and the need of sustainable biofuel alternatives, processes catalysed by enzymes are gradually replacing chemical routes in industry. So far, the majority of biocatalysts that are used in industry are derived from mesophilic fungi or bacteria, but the number of industrial relevant proteins of extremophilic origin is continuously increasing. Nevertheless, there are some essential
9.
www.sciencedirect.com
Lin L, Xu J: Dissecting and engineering metabolic and regulatory networks of thermophilic bacteria for biofuel production. Biotechnol Adv 2013, 31:827-837. A research review demonstrating genetic engineering techniques of thermophiles in detail. The authors give an excellent overview about carbohydrate utilization for biofuel production and take different stress responses into account. Sunna A, Moracci M, Rossi M, Antranikian G: Glycosyl hydrolases from hyperthermophiles. Extremophiles 1997, 1:2-13.
10. Kumar P, Satyanarayana T: Microbial glucoamylases: characteristics and applications. Crit Rev Biotechnol 2009, 29:225-255. Current Opinion in Biotechnology 2014, 29:116–123
122 Cell and pathway engineering
11. Bertoldo C, Antranikian G: Starch-hydrolyzing enzymes from thermophilic archaea and bacteria. Curr Opin Chem Biol 2002, 6:151-160. 12. Elleuche S, Antranikian G: Starch-hydrolyzing enzymes from thermophiles. In Thermophilic Microbes in Environmental and Industrial Biotechnology: Biotechnology of Thermophiles. Edited by Satyanarayana T, Littlechild J, Kawarabayasi Y. Springer; 2013:509-533. A comprehensive and up-to-date review, that offers deep insights into heat-stable amylolytic enzymes alongside with the production in appropriate hosts and possible biotechnological applications. 13. Antranikian G, Egorova K: Extremophiles: a unique source of biocatalysts for industrial biotechnology. In In Physiology and Biochemistry of Extremophiles. Edited by Gerday C, Glansdorff N. ASM Press; 2007:361-406. 14. Koch R, Zablowski P, Spreinat A, Antranikian G: Extremely thermostable amylolytic enzyme from the archaebacterium Pyrococcus furiosus. FEMS Microbiol Lett 1990, 71:21-26. 15. Sharma A, Satyanarayana T: Cloning and expression of acidstable, high maltose-forming, Ca2+-independent alphaamylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis. Extremophiles 2012, 16:515-522. 16. Han T, Zeng F, Li Z, Liu L, Wei M, Guan Q, Liang X, Peng Z, Liu M, Qin J et al.: Biochemical characterization of a recombinant pullulanase from Thermococcus kodakarensis KOD1. Lett Appl Microbiol 2013, 57:336-343. 17. Kim JW, Flowers LO, Whiteley M, Peeples TL: Biochemical confirmation and characterization of the family-57-like alphaamylase of Methanococcus jannaschii. Folia Microbiol (Praha) 2001, 46:467-473. 18. Jabbour D, Sorger A, Sahm K, Antranikian G: A highly thermoactive and salt-tolerant alpha-amylase isolated from a pilot-plant biogas reactor. Appl Microbiol Biotechnol 2013, 97:2971-2978. An amylase that tolerates elevated temperatures in combination with high concentrations of salt offers the potential to be applicable for starch degradation under harsh conditions. 19. Clark JH, Luque R, Matharu AS: Green chemistry, biofuels, and biorefinery. Annu Rev Chem Biomol Eng 2012, 3:183-207. 20. Alvira P, Tomas-Pejo E, Ballesteros M, Negro MJ: Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 2010, 101:4851-4861. 21. Thomsen MH, Thygesen A, Jorgensen H, Larsen J, Christensen BH, Thomsen AB: Preliminary results on optimization of pilot scale pretreatment of wheat straw used in coproduction of bioethanol and electricity. Appl Biochem Biotechnol 2006, 129–132:448-460. 22. Klippel B, Antranikian G: Lignocellulose converting enzymes from thermophiles. In Extremophiles Handbook. Edited by Horikoshi K, Antranikian , Bull AT, Robb FT, Stetter KO. Springer; 2011:443-474. 23. Martinez AT, Speranza M, Ruiz-Duenas FJ, Ferreira P, Camarero S, Guillen F, Martinez MJ, Gutierrez A, del Rio JC: Biodegradation of lignocellulosics: microbial, chemical, and enzymatic aspects of the fungal attack of lignin. Int Microbiol 2005, 8:195-204. 24. Kirsch C, Zetzl C, Smirnova I: Development of an integrated thermal and enzymatic hydrolysis for lignocellulosic biomass in fixed-bed reactors. Holzforschung 2011, 65:483-489. 25. Bhalla A, Bansal N, Kumar S, Bischoff KM, Sani RK: Improved lignocellulose conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresour Technol 2013, 128:751-759. 26. Shi R, Li Z, Ye Q, Xu J, Liu Y: Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum. Bioresour Technol 2013, 142:338-344. A highly thermostable bacterial cellulase with a half-life of two weeks at high temperatures distinguishes this enzyme from other cellulolytic biocatalysts. Since the enzymatic supply is often the most cost-effective position, processes may run for more than two weeks using the same load of enzyme, thus, contributing to an enormous cost reduction. Current Opinion in Biotechnology 2014, 29:116–123
27. Graham JE, Clark ME, Nadler DC, Huffer S, Chokhawala HA, Rowland SE, Blanch HW, Clark DS, Robb FT: Identification and characterization of a multidomain hyperthermophilic cellulase from an archaeal enrichment. Nat Commun 2011, 2:375. An endoglucanase displays optimal activity at 1098C and exhibits an outstanding half-life of 5 hours at 1008C. This research emphasizes Archaea to become focus of research for prospective metagenomic studies on thermostable cellulases. 28. Barabote RD, Parales JV, Guo YY, Labavitch JM, Parales RE, Berry AM: Xyn10A, a thermostable endoxylanase from Acidothermus cellulolyticus 11B. Appl Environ Microbiol 2010, 76:7363-7366. 29. Shi H, Zhang Y, Li X, Huang Y, Wang L, Wang Y, Ding H, Wang F: A novel highly thermostable xylanase stimulated by Ca2+ from Thermotoga thermarum: cloning, expression and characterization. Biotechnol Biofuels 2013, 6:26. 30. Staufenberger T, Imhoff JF, Labes A: First crenarchaeal chitinase found in Sulfolobus tokodaii. Microbiol Res 2012, 167:262-269. 31. Kuzu SB, Guvenmez HK, Denizci AA: Production of a thermostable and alkaline chitinase by Bacillus thuringiensis subsp. kurstaki strain HBK-51. Biotechnol Res Int 2012, 2012:1354-1398. 32. Hamid R, Khan MA, Ahmad M, Ahmad MM, Abdin MZ, Musarrat J, Javed S: Chitinases: an update. J Pharm Bioallied Sci 2013, 5:21-29. 33. Garcia-Fraga B, da Silva AF, Lopez-Seijas J, Sieiro C: Functional expression and characterization of a chitinase from the marine archaeon Halobacterium salinarum CECT 395 in Escherichia coli. Appl Microbiol Biotechnol 2013, 98:2133-2143. 34. Cabeza MS, Baca FL, Mun˜oz Puntes E, Loto F, Baigorı´ MD, Morata VI: Selection of psychrotolerant microorganisms producing cold-active pectinases for biotechnological processes at low temperature. Food Technol Biotechnol 2011, 49:187-195. 35. Li Q, Yi L, Marek P, Iverson BL: Commercial proteases: present and future. FEBS Lett 2013, 587:1155-1163. 36. Saba I, Qazi PH, Rather SA, Dar RA, Qadri QA, Ahmad N, Johri S, Taneja SC, Shawl S: Purification and characterization of a cold active alkaline protease from Stenotrophomonas sp., isolated from Kashmir, India. World J Microbiol Biotechnol 2012, 28:1071-1079. 37. Toplak A, Wu B, Fusetti F, Quaedflieg PJ, Janssen DB: Proteolysin, a novel highly thermostable and cosolventcompatible protease from the thermophilic bacterium Coprothermobacter proteolyticus. Appl Environ Microbiol 2013, 79:5625-5632. 38. Sharma R, Thakur V, Sharma M, Birkeland N-K: Biocatalysis through thermostable lipases: adding flavor to chemistry. In Thermophilic Microbes in Environmental and Industrial Biotechnology: Biotechnology of Thermophiles. Edited by Satyanarayana T, Littlechild J, Kawarabayasi Y. Springer; 2013:905-927. 39. Al Khudary R, Venkatachalam R, Katzer M, Elleuche S, Antranikian G: A cold-adapted esterase of a novel marine isolate, Pseudoalteromonas arctica: gene cloning, enzyme purification and characterization. Extremophiles 2010, 14: 273-285. 40. Hoesl MG, Acevedo-Rocha CG, Nehring S, Royter M, Wolschner C, Wiltschi B, Budisa N, Antranikian G: Lipase congeners designed by genetic code engineering. ChemCatChem 2011, 3:213-221. 41. Yao MZ, Zhang YH, Lu WL, Hu MQ, Wang W, Liang AH: Phytases: crystal structures, protein engineering and potential biotechnological applications. J Appl Microbiol 2012, 112:1-14. 42. Lei XG, Weaver JD, Mullaney E, Ullah AH, Azain MJ: Phytase, a new life for an ‘‘old’’ enzyme. Annu. Rev. Anim. Biosci. 2013, 1:283-309. This review summarizes structural information about phytases, possibilities of application, new approaches of industry and academia as well as related issues and future directions. An overview covering all interests is given and recommended as greatly readable. www.sciencedirect.com
Industrial applications of extremozymes Elleuche et al. 123
43. Moshfegh M, Shahverdi AR, Zarrini G, Faramarzi MA: Biochemical characterization of an extracellular polyextremophilic alpha-amylase from the halophilic archaeon Halorubrum xinjiangense. Extremophiles 2013, 17:677-687. 44. Kang J, Park KM, Choi KH, Park CS, Kim GE, Kim D, Cha J: Molecular cloning and biochemical characterization of a heatstable type I pullulanase from Thermotoga neapolitana. Enzyme Microb Technol 2011, 48:260-266. 45. Cihan AC, Benli M, Cokmus C: Purification and characterization of intracellular and extracellular alpha-glucosidases from Geobacillus toebii strain E134. Cell Biochem Funct 2011, 30:6981. 46. Li X, Yu HY: Purification and characterization of novel organicsolvent-tolerant beta-amylase and serine protease from a newly isolated Salimicrobium halophilum strain LY20. FEMS Microbiol Lett 2012, 329:204-211. 47. Zhao L, Xie J, Zhang X, Cao F, Pei J: Overexpression and characterization of a glucose-tolerant b-glucosidase from Thermotoga thermarum DSM 5069T with high catalytic efficiency of ginsenoside Rb1 to Rd. J Mol Catal B Enzym 2013, 95:62-69. 48. Schro¨der C, Elleuche S, Blank S, Antranikian G: Characterization of a heat-active archael beta-glucosidase from a hydrothermal spring metagenome. Enzyme Microb Technol 2014, 57:48-54.
www.sciencedirect.com
49. ul Haq I, Hussain Z, Khan MA, Muneer B, Afzal S, Majeed S, Akram F: Kinetic and thermodynamic study of cloned thermostable endo-1,4-beta-xylanase from Thermotoga petrophila in mesophilic host. Mol Biol Rep 2012, 39:7251-7261. 50. Royter M, Schmidt M, Elend C, Hobenreich H, Schafer T, Bornscheuer UT, Antranikian G: Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis. Extremophiles 2009, 13:769-783. 51. Chow J, Kovacic F, Dall Antonia Y, Krauss U, Fersini F, Schmeisser C, Lauinger B, Bongen P, Pietruszka J, Schmidt M et al.: The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases. PLoS ONE 2012, 7:4766-4775. 52. Stetter KO: History of discovery of extremophiles. In Extremophiles Handbook. Edited by Horikoshi K, Antranikian G, Bull AT, Robb FT, Stetter KO. Springer; 2011:403-425. 53. Zaparty M, Siebers B: Physiology, metabolism and enzymology of thermoacidophiles. In Extremophiles Handbook. Edited by Horikoshi K, Antranikian G, Bull AT, Robb FT, Stetter KO. Springer; 2011:601-639. 54. Luo H, Robb FT: Thermophilic protein folding systems. In Extremophiles Handbook. Edited by Horikoshi K, Antranikian G, Bull AT, Robb FT, Stetter KO. Springer; 2011:583-599.
Current Opinion in Biotechnology 2014, 29:116–123
