Critical Reviews in Biotechnology

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Biocatalysts: application and engineering for industrial purposes Sonia Jemli, Dorra Ayadi-Zouari, Hajer Ben Hlima & Samir Bejar To cite this article: Sonia Jemli, Dorra Ayadi-Zouari, Hajer Ben Hlima & Samir Bejar (2016) Biocatalysts: application and engineering for industrial purposes, Critical Reviews in Biotechnology, 36:2, 246-258, DOI: 10.3109/07388551.2014.950550 To link to this article: http://dx.doi.org/10.3109/07388551.2014.950550

Published online: 06 Nov 2014.

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Date: 15 November 2016, At: 02:52

http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, 2016; 36(2): 246–258 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.950550

REVIEW ARTICLE

Biocatalysts: application and engineering for industrial purposes Sonia Jemli, Dorra Ayadi-Zouari, Hajer Ben Hlima, and Samir Bejar Laboratoire de Microorganismes et de Biomole´cules, Centre de Biotechnologie de Sfax, Universite´ de Sfax, Tunisie

Abstract

Keywords

Enzymes are widely applied in various industrial applications and processes, including the food and beverage, animal feed, textile, detergent and medical industries. Enzymes screened from natural origins are often engineered before entering the market place because their native forms do not meet the requirements for industrial application. Protein engineering is concerned with the design and construction of novel enzymes with tailored functional properties, including stability, catalytic activity, reaction product inhibition and substrate specificity. Two broad approaches have been used for enzyme engineering, namely, rational design and directed evolution. The powerful and revolutionary techniques so far developed for protein engineering provide excellent opportunities for the design of industrial enzymes with specific properties and production of high-value products at lower production costs. The present review seeks to highlight the major fields of enzyme application and to provide an updated overview on previous protein engineering studies wherein natural enzymes were modified to meet the operational conditions required for industrial application.

Bioengineering, directed evolution, industrial requirements, protein design, rational design, semi-rational design, tailor-made enzyme, white biotechnology

Introduction Biocatalysts are considered an important alternative to traditional chemical catalysts since they offer attractive advantages, including their availability from renewable resources, biodegradability, ability to operate under relatively mild conditions of pH and temperature, and selectivity in both substrate and product stereochemistry. Enzymes have the potential to perform most of the reactions required for the production of chirally pure and complex molecules with attractive properties (Fendri et al., 2012; Karagu¨ler et al., 2007; Nakano et al., 2011). Accordingly, enzymes have become important tools in various fields, including medicine, chemical industry, food processing, agriculture and energy (Kirk et al., 2002; Tao & Xu, 2009). Although the advantages of using biocatalysts as substitutes to chemical processes are often compelling from an environmental perspective, their industrial applications requires some degree of cost-effectiveness to justify their cost competiveness with conventional chemical catalysts from an economic perspective (Jaeger, 2004; Liszka et al., 2012). For a given biocatalytic process, however, the native enzyme does not often meet the requirements for large-scale application, and its physico-chemical properties need to be modulated. In fact, industrial processes often operate under

Address for correspondence: Sonia Jemli, Laboratoire de Microorganismes et de Biomole´cules, Centre de Biotechnologie de Sfax, Universite´ de Sfax, Route de Sidi Mansour Km 6, BP 1177, 3038, Tunisie. Tel/Fax: 00 216 74 870 451. E-mail: [email protected]

History Received 26 February 2014 Revised 16 May 2014 Accepted 30 June 2014 Published online 3 November 2014

harsh conditions that can inactivate the enzyme, including elevated pressure and temperature, extreme pH and nonaqueous solutions, and oxidative conditions (Hilde´n et al., 2009; Luetz et al., 2008). The successful implementation of an enzyme as an industrial biocatalyst would, therefore, require the availability of an economical and suitable enzyme with high activity, specificity and stability that can improve the performance and cost-effectiveness of the process under the required operational conditions. Several protein engineering strategies have been developed to provide tailor-made enzymes with new properties adapted to specific industrial applications, increase protein solubility and hence reduce enzyme production cost. Large-scale fermentation and recombinant DNA technologies have also been widely used to make the over-expression of enzymes economically feasible. The optimization of enzyme activities and specificities (such as pH tolerance, solvent stability, enantioselectivity, etc.) has been extensively investigated using recent advances in protein engineering techniques. The latter consist of two main strategies, namely, rational design and directed evolution, or a combination of those methods in a semi-rational design (Table 1). Rational engineering, introducing controllable changes to proteins on the basis of their structure and related biochemical properties, has been a valuable tool for enzyme engineering since the 1980s (Kirk et al., 2002). Controlled changes are important to determine the effect of individual residue changes on protein structure, folding, stability and function. The generation of random mutations has become feasible with the advent of directed evolution, thus allowing for the construction and screening of variant libraries to identify mutants with improved properties (Ben Mabrouk

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Table 1. Comparison of protein engineering approaches.

Structural and/or functional data High-throughput screening technology Occurrence of Mutations

Rational design

Semi-rational design

Directed evolution

Both required Not required Several point mutations in some regions

Whichever is sufficient Advantageous but not essential Simultaneous saturation and synergistic mutations in a local region

Not required Required Random mutations distributed over the gene

et al., 2013; Cherry & Fidantsef, 2003). Semi-rational design uses information on protein sequence, structure and function, as well as computational predictive algorithms to target multiple or specific residues and create ‘‘smart’’ libraries that are more likely to yield positive results. The combination of directed evolution methods with elements of rational design successfully circumvents some of the limitations associated with both approaches and offers promising opportunities for the generation of desired functional properties (Bloom et al., 2005; Chica et al., 2005). In fact, while the rational approach entails an in-depth understanding of the structure–function relationship, which is not always available, the directed evolution approach requires the development of a highthroughput screening methodology, which is not always easily implemented. The three protein engineering methods described above have been extensively employed to adapt industrial enzymes. The method of choice needs to be selected on a case-by-case basis depending on several factors, including knowledge on the desired property, availability of 3D structure and function of the protein, and feasibility of a high-throughput screening system. The first part of this review provides an overview on the current status of industrial enzymes, with particular focus on technical applications, food processing and feed industries. The second part highlights previous studies in the literature where protein engineering was applied to enhance the biochemical properties of industrially significant enzymes.

Industrial applications of enzymes Natural enzymes have been used in the production of food and beverage products, including cheese, sourdough, beer, potable spirits and vinegar, and the manufacture of commodities, such as leather and linen, since ancient times. During the past few decades, the application of enzymes has expanded to include other industrial applications and processes ranging from chemo-enzymatic synthesis to the generation of novel biofuels from renewable biomass (Kim & Park, 2013; Wen et al., 2009). The enzyme industry, in its current form, is the result of a rapid development of modern biotechnology, including recombinant DNA, high throughput screening, and protein engineering technologies. The recent advances in recombinant gene technology have further improved manufacturing processes and allowed for the commercialization of enzymes that were not previously amenable to production in sufficient quantities. Furthermore, recent breakthroughs in high throughput and protein engineering technologies have revolutionized the development of tailor-made enzymes that can function under operational conditions, thus further expanding the scope of their industrial application (Kumar & Singh, 2013; Lutz, 2010).

The global market of industrial enzymes was estimated to exceed 3.3 billion USD in 2010 and is expected to reach 4.5 to 5 billion USD by 2015 (DiCosimo et al., 2013). Commercially available enzymes are derived from animals, plants and microorganisms. The greater part of industrial enzymes (more than 50%) is, however, obtained from microorganisms, especially bacteria and fungi, which is particularly due to their ease of growth, minor nutritional requirements, and broad biochemical diversity. Hydrolases, such as proteases, amylases, lipases and cellulases, cover more than 75% of commercially available enzymes (Prakash et al., 2013), and the demand for these enzymes is continuously increasing. Industrial enzymes are typically divided into three major types, namely, technical, food and animal feed enzymes (Cherry & Fidantsef, 2003). Technical enzymes are applied in various industries, including the detergent, textile, paper, fuel, and alcohol industries, and account for the major fraction of commercialized industrial enzymes. Food enzymes, the second largest group, includes enzymes employed in the dairy, brewing, wine, juice, fat and oil, and baking industries. The third class of industrial enzymes is used in the animal feed industry (Table 2). Over the past few years, the use of biocatalysts for organic synthesis has also received increasing interest due to their superior quality and efficiency with regard to the safety and environmental aspects of major industrial transformations. Chiral molecules, such as amino acids, amino alcohols, amines, and epoxides, have also been in growing demand by the chemical and pharmaceutical industries (Simon et al., 2013; Zheng & Xu, 2011). The enzyme’s capacity to display regio-, chemo- and stereoselectivity is particularly attractive and valuable because this high selectivity eliminates subsequent separation steps of undesired isomers. Many biocatalysts, mostly hydrolases, transaminases, reductases and oxidases (Table 2), are currently applied for the synthesis of a wide range of organic compounds (Huisman & Collier, 2013; Nestl et al., 2014). During recent years, biorefinery processes have also attracted a great deal of attention among researchers interested in the production of biofuels from renewable biomass (Cavka et al., 2011; Chandel et al., 2012). Furthermore, synthetic biology allows the synthesis of natural and unnatural biopolymers, including polyhydroxyalkanoates and poly(lactic acid) (Park et al., 2012; Yang et al., 2013), as a potential solution to environmental problems. With the aim of achieving efficient and cost-effective strategies for producing chemicals, synthetic pathways usually require engineering to improve enzyme performance, modify cofactor usage for redox balance, tune protein expression, and create scaffold multi-enzyme complexes for metabolite flux control (Li, 2012).

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Table 2. The major industrial enzyme applications [Aguilar et al., 2007; Araujo et al., 2008; Ba et al., 2013; Brady et al., 2004; Casas-Godoy et al., 2012; Choct, 2006; Demuner et al., 2011; Fendri et al., 2012; Kirk et al., 2002; Huisman & Collier, 2013; Jemli et al., 2007; Kalantzi et al., 2008; Khan, 2013; Rhimi et al., 2009; Sanchez & Demain, 2011; Meyer, 2010). Industry 1. Technical industry 1.1. Laundry detergents

1.2. Textile industry

1.3. Pulp and paper industry

1.4. Biorefinery

Enzyme Proteases Lipases Amylases Xylanases Cellulases Cellulases Amylases Laccases Peroxidases Pectate lyases Amylases Cellulases Lipases Xylanases Laccases Cellulases, hemicellulases Lipases

2. Food industry 2.1. Dairy industry

2.2. Starch industry

2.3. Baking industry

2.4. Juice industry

2.5. Brewing industry

2.6. Fat and oil industry 2.7 Functional food industry

3. Animal feed industry

4. Organic synthesis industry

Lipases Proteases b-galactosidases ´ rases L-arabinose isome a-amylases and amyloglucosidases, Glucose isomerases Cyclodextrin glycosyltransferases a-amylases Lipases Glucose oxidases Lipoxygenases Proteases Xylanases Pectinases Amylases Naringinases and limoninases Laccases a-amylases Pullulanases b-glucanases Proteases Pentosanases and xylanases Acetolactate decarboxylases Tannases Lipases Phospholipases Phytases Rhamnosidases Pectinases Phytases Xylanases b-glucanases Tannases Lipases Nitrilases, nitrile hydratases and amidases Laccases Ketoreductases, and alcohol dehydrogenases Monooxygenases Transaminases

Applications Protein-based stain removal Lipid stain removal Removal of resistant starch residues Plant-based stain removal Removal of cotton ‘‘fuzz’’ that accumulates with excessive washing Denim bleaching, textile softener Starch removal from woven fabrics Decolorization and detoxification of effluents from textile Dye excess removal Scouring Reducing viscosity in starch coatings, drainage Making fibers flexible Control pitch in pulping processes Enhancing pulp-bleaching process efficiency Improving brightness and removal of lignin from wood and non-wood fibres Degrading efficiently the lignocellulotic materials for ethanol production Biodiesel production by transesterification Cheese flavour Milk clotting, flavor Avoiding lactose intolerance Producing low caloric milk products containing D-tagatose Conversion of starch to glucose syrup Production of high fructose syrup Cyclodextrins production and carbohydrate glycosylation Controlling the volume and crumb structure of bread Improving stability of gas cells in dough Dough strengthening Dough strengthening, bread whitening Biscuits Improving dough handling as well as specific volume, color and crumb structure of bread Increasing juice production yield Clarifying cloudy juice Decreasing bitterness in citrus juices Phenol derivative removal Decreasing viscosity and rising maltose and glucose content Securing maximum wort fermentability Decreasing viscosity and improving wort separation Improving yeast growth Improving extraction and beer filtration Improving beer taste Clarifying agent Flavor production Lyso-lecithin production Dephosphorylation of phytic acid to enhance iron human absorption Enhancing bioavailability of the citrus flavonoid hesperetin Obtaining solubilized dietary fiber via treatment of potato pulp material Releasing phosphorus and increasing availability of cations Degrading fiber in viscous diets and increasing nutritive value of feed Increasing animal feed digestibility Hydrolysis of tannins and gallic acid esters Resolution of chiral compounds Synthesis of enantiopure compounds for pharmaceutical industry Oxidation of phenol derivatives for synthetic applications Reduction of the carbonyl functionality of esters and aromatic ketones Synthesis of optically pure epoxides Resolution of racemic amines and direct chiral synthesis

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Enzymes in technical industries Technical enzymes are typically applied as bulk enzymes in various industrial applications and processes, including the detergent, textile and leather, pharmaceutical, cosmetic, biofuel, and pulp and paper industries. The global sales of technical enzymes in 2011 were estimated at 1.2 billion USD (Prakash et al., 2013) and are expected to reach 1.5 billion USB by 2015 (Dewan, 2011). The most important sector for the application of technical enzymes in terms of volume and value is the detergent industry. The application of proteases as pancreatic extracts in detergents is old and dates back to 1913. The use of enzymes in detergent formulations has, however, been introduced only in the past five decades, particularly after the availability of enzymes from microbial origins in the 1960s (Maurer, 2004). Enzymes have been used in laundry detergents to remove soil and difficult stains at washing temperatures. Proteases represent the major fraction of technical enzymes used in detergents, but other hydrolases (lipases, amylases and cellulases) are also employed to achieve various desired results (Table 2). All of the proteases used in the detergent industry were obtained by subtilisins from Bacillus species. A recent innovation in this area is the use of psychrophilic enzymes able to operate efficiently at low temperatures, which offers important opportunities for environmental and economic benefits through energy savings (Cavicchioli et al., 2002; Mikhailova et al., 2014). Textile processing is a growing industry that has traditionally used a lot of water, energy and harsh chemicals. The use of enzymes in the textile industry is a good example of white biotechnology, which allows the application of environmentally friendly technologies and low energy consumption in almost all the steps of fiber processing. Several enzymes have been used in this area since the middle of the last century, including a-amylases, cellulases, laccases, peroxidases, lipases, and pectate lyases (Araujo et al., 2008). Among these enzymes, cellulases have commonly been used to improve the appearance and feel of garments made from a variety of cellulosics such as cotton, linen and viscose. Pectate lyases have also been successfully introduced as substitutes to the conventional chemical processes used in the preparation of cotton fabrics (Kalantzi et al., 2008). Enzymes, particularly proteases, have also been employed in several processes in the leather industry, including the processing of skins and hides to remove proteins associated with collagen and to replace toxic chemicals (Khan, 2013). The extent of removing non-collagenous constituents is a critical factor in controlling the quality of final leather, including its durability and softness. The substitution of chemicals with proteases has also been often reported to offer the only way to limit the pollution caused by the leather industry. The use of enzymes in the pulp and paper processes has grown rapidly since the mid 1980s (Demuner et al., 2011). Cellulases, xylanases, laccases and lipases are the most important enzymes used in this industry. While xylanases and laccases have been mainly applied in pulp production, cellulases and lipases have been widely used in paper manufacturing. Laccases and xylanases have been applied for fiber delignification and pulp bleach boosting in pulp

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production. Likewise, cellulases and lipases have found extensive application for fiber modification and pitch control, respectively. For efficiency, environmental and economic purposes, the search for enzymes that are active at lower temperatures has attracted growing interest in the pulp and paper industry (van den Burg, 2003). With the rising scarcity and cost of fossil fuels, the future of energy supply lies in the development of renewable energy sources and modern approaches for their use. Accordingly, the bioconversion of agricultural sources and abundant lignocellulosic biomass as a feedstock for biofuels and biobased chemicals has recently received growing attention (Yamada et al., 2013). Lignocellulose-degrading enzymes, including cellulases and hemicellulases, have received an increasing interest in biofuel production owing to their powerful ability to reduce the high cost associated with lignocellulose conversion by overcoming biomass recalcitrance (Himmel et al., 2007; Wen et al., 2009). Amylases and glucoamylases have also gained momentum in the conversion of starchy materials to glucose which is subsequently fermented by yeast to produce ethanol (Kim et al., 2011). Enzymes in food industries Biocatalysts have a long history of use in food processing. The earliest applications go back to 6000 BC with beer brewing, bread baking and cheese making (Vasic-Racki, 2006). The ever-increasing consumer awareness and demand for high quality and convenient food products has recently triggered a continuous search for natural enzymes for application as substitutes to the conventional synthetic chemicals used in food processing. Nowadays, enzymes are commonly used in a wide range of food industries, including the bread baking, fruit juice, cheese, starch, wine, and fat and oil industries (Table 2). Enzymes can improve the food texture, appearance, and nutritional value of food products, and may generate desirable flavors and aromas. The global sales of enzymes in the food and beverage industry are expected to reach about 1.3 billion USD by 2015 (Figure 1). The highest sales reported for the enzymes used in this area in 2009 were recorded in the milk and dairy market, with 401.8 million USD (Dewan, 2011).

Figure 1. Expected global enzyme sales in 2015 (Dewan, 2011; Frost & Sullivian, 2011).

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Pectinases were used for juice clarification in the 1930s (Vasic-Racki, 2006). However, the large application of food enzymes did not become truly established before the 1960s, with the use of amylases and amyloglucosidases as substitutes the traditional acid hydrolysis of starch (Fernandes, 2010). Some years later, the starch conversion approach also included the use of glucose isomerase to produce high fructose syrup. By 2009, the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) issued a list of about 200 enzymes that were manufactured for use in the food industry, at least 57 of which were produced from genetically modified microorganisms (AMFEP, 2009). The key criterion in evaluating food enzyme safety is the toxigenic potential of the production microbe. Only few microorganisms from a relatively small number of bacterial and fungal species, including Aspergillus oryzae, A. niger, Saccharomyces cerevisiae, Streptomyces rubiginosus, Actinoplanes mussousriensis, Bacillus subtilis, and B. licheniformis which are recognized as Generally Recognized As Safe (GRAS), are used in enzyme preparations for food applications. One of the most important factors that determine the safety of enzyme preparations is the nontoxic and nonpathogenic potential of the microorganisms used. Characterized microbial strains with a history of safe use in food enzyme processing are also considered as potent candidates for the generation of a safe strain lineage through which improved strains may be derived via genetic modification (Pariza & Johnson, 2001). The authors have reported that the key elements required for the development of a safe strain lineage include the systematic characterization of the host organism, the determination of the safety of all new DNA introduced to it, and the confirmation that the procedure(s) used in the modification of the host organism are appropriate for food use. Olempska-Beer et al. (Olempska-Beer et al., 2006) reported on the engineering of microbial strains used for food enzyme production by reducing or eliminating their potential production of toxic secondary metabolites. Koushki et al. (Koushki et al., 2011) reported on the genetic manipulation of fungal strains, including protease-deficient and sporulation-deficient, to improve enzyme production yield. Enzymes in the animal feed industry The use of enzymes as supplements in animal feed started in the late 1980s and has, since then, considerably evolved particularly in the pig and poultry feed markets (Ravindran & Son, 2011). The global sales of feed enzymes in 2007 were estimated at around 344 USD million and are expected to reach 727 USD million in 2015 (Frost & Sullivian, 2011). In fact, exogenous enzymes offer numerous advantages for diet formulations. They improve the digestion of dietary components, which enhances feed conversion and, hence, growth. They also help overcome the negative effects of anti-nutritional factors (e.g. NSPs: non-starch polysaccharides), thus reducing the negative impacts on the environment through lowering phosphorus and nitrogen excretion and reducing the amount of manure discharge. Cereal grains (such as barley, rye and wheat) and their by-products are used in animal diets in many countries. These materials have a high content of insoluble

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NSPs. The development of highly sophisticated enzymes that target NSPs, in a precise manner, would make these polymers a large source of potential energy and a promising class of prebiotics (Choct, 2006). The highest portion of enzymes applied as feed additives is in poultry feed and is followed by swine feed. Aquaculture and ruminant feed have recently received attention and become emerging markets for exogenous feed enzymes (Ravindran & Son, 2011). The enzymes currently used in animal feed are those that have the ability to break down NSPs, proteins, starch, and phytate. Accordingly, the typically commercialized feed enzymes are b-glucanases, xylanases, pectinases, a-galactosidases, proteases, a-amylases and phytases. Of these, phytases have attracted special attention in animal feed research and industry (Farhat-Khemakhem et al., 2012; Selle & Ravindran, 2008). They are particularly valued for their ability to provide phosphate and other phytate-bound nutriments by the degradation of phytic acid, a potent antinutritional factor (Yi et al., 1996), and for their promising potential to reduce phosphate pollution. Because the formulation of commercial animal feed involves high temperatures, the use of feed enzymes in wide scale applications has often been constrained by their limited thermostability inactivating protein at processing temperatures (Katrolia et al., 2013). The search for and development of heat-stable enzymes has, therefore, been imperative in this continuously growing market. This search should also target enzymes that are active during the transit in the digestive tract which entails activity and stability at wide range of pH.

Enzymes engineering for tailoring fundamental properties Despite the growing interest in enzyme application, the performance of natural enzymes has often been reported to be not suitable for most industrial processes without further tailoring or redesign of the enzyme traits (Nestl et al., 2011; Schoemaker et al., 2003). This has triggered the need for designing novel biocatalysts with ‘‘ideal’’ specificities and efficiencies under reaction conditions. The first engineered subtilisins were marketed in the beginning of the 1990s (Maurer, 2004). This marked the beginning of a revolutionary era in protein engineering where several enzymes were designed with desired properties. Nowadays, numerous examples of enzyme property optimizations are available (as developed in the following sections) demonstrating that biocatalysts can be engineered to meet the requirements of industrial applications, including enhanced activity, higher thermostability, better organic solvent tolerance, and increased or modified substrate specificity. Several strategies have been applied to modify the native sequence of enzymes and engineer proteins with better performance. Previously, two broad approaches, namely, directed evolution and rational design, were considered unique and exclusive (Bornscheuer & Pohl, 2001). Directed evolution techniques are based upon the principle of natural evolution processes such as random mutagenesis (Chen & Arnold, 1993) and genetic recombination (Coco et al., 2001). Following the creation of a large mutant library (reaching 106–109 mutants), the sequence diversity is explored by high-throughput screening to identify

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and select mutations that produce the desired enzyme phenotype (Labrou, 2010). High-throughput screening using solid selective media has been successfully used to discover and characterize novel activities from metagenomic libraries at a throughput of 200 000 clones per week (Cecchini et al., 2013; Tasse et al., 2010). Automated liquid-handling systems using chromogenic or fluorescent substrate transformations have also been developed as efficient methods (Nyysso¨nen et al., 2013). Ultra-high screening techniques combining high sensitivity and high throughput have also emerged with fluorescence activated cell sorting (FACS) (Yoo et al., 2012) or microfluidic platforms (Beneyton et al., 2014). The rational design, on the other hand, requires data about enzyme structure–function relationships to allow the modification of one or more amino acids that are predicted to elicit the desired enhancements to enzyme properties. A semi-rational design, which combines directed evolution and rational design, has recently been advanced as a powerful approach to accelerate the engineering of biocatalysts in a less labor-intensive and more time-efficient way through creating a much smaller library (102–103 mutants) with a higher proportion of beneficial mutations (Zhao et al., 2002). The development of powerful computer algorithms simplifies the decision making process and allows the screening of libraries in silico for the identification of beneficial mutations (Fox et al., 2007). More recently, broad advances in the field of computational biology have enabled the emergence of de novo design of enzyme catalyzing a chemical reaction for which a natural biocatalyst does not exist (Barrozo et al., 2012; Bommarius et al., 2011). The remarkable success that the applied computational algorithms and models have achieved in designing novel activities toward unnatural substrates takes into consideration several structural features, including the backbone flexibility, structural remodeling (Eiben et al., 2012), and molecular dynamics of protein structures (Ruscio et al., 2009). By far, computational design has obviously improved the possibility of finding active enzymes. It needs, however, to be coupled with optimized experimental protocols in order to obtain the efficient biocatalysts. To date, several approaches have been proposed to improve enzyme properties or even design novel biocatalysts with new catalytic properties. However, each strategy presents some limitations, and the choice of the appropriate experimental approach for enzyme engineering is, therefore, based on the availability of experimental tools, computer algorithms, and in-depth knowledge of structure–function relationships (Figure 2). On the other hand, demands are increasingly placed on enzymes to cover new synthetic reactions that are not catalyzed by existing biocatalysts. Hence, the success in finding useful proteins is principally a matter of persistent trial and consistent optimization of strategies. Stability As industrial conditions are often different from the natural environment of enzymes, considerable attention has been paid to the improvement of stability of most engineered proteins (Eijsink et al., 2004; Eijsink et al., 2005). The stability of an enzyme can be altered by several deactivating factors,

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including temperature, solvent, pH and presence of oxidants. The design of more thermostable enzymes is receiving growing attention and remains a challenge in the field of biotechnology given that industrial applications often operate at elevated temperatures, which partly or completely inactivate the biocatalyst (Ebrahimi et al., 2011; Yeoman et al., 2010). Using a range of protein engineering techniques, several studies have successfully enhanced enzymes thermostability to meet the needs of biotechnological applications. Efforts have, for instance, been made to design thermostable xylanases of significant interest for the paper and pulp industry and other industries involving the bioconversion of lignocellulosic material into fermentable sugars. The thermostability of xylanases has been improved through the introduction of classic factors that are widely applied to increase stability, including additional hydrophobic interactions (Georis et al., 2000), optimization of protein electrostatic surface (Torrez et al., 2003), introduction of disulfide bonds (Ruller et al., 2008), and the enrichment of hydrogen bonds (Vieira & Degre`ve, 2009). Recently, Fonseca-Maldonado et al. (Fonseca-Maldonado et al., 2013) have studied the effects of glycan content and glycosylation position on the thermostability of the bacterial xylanase through the combinatorial mutagenesis of all potential N-glycosylation sites. They reported that the glycosylated enzymes expressed in Pichia pastoris showed enhanced thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. It needs to be emphasized that the sitedirected mutagenesis of the glycosylation sites significantly alters the glycosylation pattern and modulates the proteinglycan interface, which increases the thermostability of the engineered xylanase. Owing to the great value and economic importance of enzymes in laundry detergent applications, several active and stable enzymes have been successfully engineered in the literature. An impressive example of the combination of sitedirected mutagenesis, error-prone PCR (ep-PCR) and DNA shuffling was reported, by Novo Nordisk (Denmark) researchers, with the aim of designing a fungal peroxidase that is more efficient as a dye-transfer inhibitor in laundry detergent (Cherry et al., 1999). After multiple mutagenesis rounds, the researchers obtained a mutant displaying 174 times the thermal stability and 100 times the oxidative stability of the wild-type peroxidase under conditions mimicking those in a washing machine (pH 10.5, 50  C, 5–10 mM peroxide). In a more recent study, the thermostability of a variant of the alpha-amylase from Geobacillus stearothermophilus sp. US100 has been improved by the deletion of a small extra loop (Ben Ali et al., 2006). The deletion increased the enzyme half-life time from 15 min to 70 min at 100  C and from 3 min to 13 min at 110  C. With the purpose of making the enzyme more suitable for detergent applications, the stability towards chemical oxidation was also enhanced by the selective replacement of Met197 with Ala (Khemakhem et al., 2009). This enzyme engineering study showed that the obtained variant displayed higher resistance to oxidation compared to the well-known commercial amylase used in detergent, Termamyl300. The serine protease subtilisin is an important industrial enzyme widely used as a model system for protein

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Figure 2. Selection of the preferred approach for enzyme engineering. aas: amino acids; Ep-PCR: Error-prone PCR; HTS: High-Throughput Screening.

engineering since the 1980s (Bryan, 2000). By 1985, the stability of subtilisin towards hydrogen peroxide, generated in the cleaning process of bleach-containing products, has become a textbook example of site-directed mutagenesis of

a methionine residue adjacent to the catalytic Ser221 (Estell et al., 1985). In 1990, the thermostability of subtilisin E, a cysteine-free protease, was tremendously increased by the introduction of a disulfide bond (Takagi et al., 1990).

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The directed evolution approach through the DNA shuffling of 26 subtilisin genes was successfully applied to design chimeras possessing all the combinations of attractive parental properties. The library (out of 10 000 in total) was screened for variants with considerably improved thermostability, organic solvent tolerance, and activity at broad pH range (Ness et al., 1999). Jaouadi et al. (Jaouadi et al., 2010) combined site-directed mutagenesis and 3D molecular modeling approaches to engineer the thermostability enhancement of the Bacillus pumilus CBS serine alkaline protease previously noted to exhibit excellent laundry detergent compatibility and high dehairing ability in the leather and poultry processing industries (Jaouadi et al., 2009). The authors reported on the substitution of five amino acids and the construction of 12 single, double and triple mutants. Of these, four mutants were particularly noted to exhibit enhanced thermostability and improved stability at alkaline pH compared to the wild-type enzyme. According to the authors, the latter mutant enzymes could be considered as potential strong candidates for future industrial application in laundry detergents. Using site-directed mutagenesis, sequence alignment, and structural studies, Ben Hlima et al. (Ben Hlima et al., 2012; Ben Hlima et al., 2013) showed the role of two amino acid residues adjacent to the catalytic histidines in the thermostability of the Streptomyces sp. SK glucose (xylose) isomerase (SKGI) and in the resistance to calcium inhibition. Due to its high thermostability and acid tolerance, SKGI represents an attractive enzyme for the production of high-fructose corn syrup (HFCS) from corn starch (Borgi et al., 2004; Srih-Belghith et al., 2002). The mutations F53L (Ben Hlima et al., 2012) and G219A (Ben Hlima et al., 2013) were reported to significantly enhance the thermostability of the evolved mutants by 1.5- and 2-fold compared to the wildtype enzyme, respectively. In order to further improve the thermal stability of the SKGI, the double mutant F53L/G219A was designed and noted to display a half-life time of about 150 min at 85  C as compared to 50 min for the wild-type SKGI (Ben Hlima et al., 2013). This double mutant enzyme could, therefore, be potentially considered as a promising candidate for application in the HFCS production process. In a recent work, Fei et al. (Fei et al., 2013) used a computational design combining three common structural features, namely protein flexibility, protein surface, and salt bridges, to engineer phytase with enhanced thermostability. The authors constructed single, double and triple mutants, and showed that a double mutant exhibited about 2-fold higher thermostability at 80  C than the wild-type enzyme. These multi-factors rational design strategy revealed positive and negative synergies of some mutations and can be practically applied as a thermostabilization strategy instead of the conventional single-factor approach. Likewise, directed evolution was used in combination with site-directed mutagenesis to enhance the thermostability of A. niger glucoamylase by creating a multiply-mutated enzyme which is substantially more thermostable than the wild-type enzyme at 80  C, with a 5.1 kJ/mol increase in the free energy of thermo-inactivation, making it the most thermostable A. niger glucoamylase mutant ever reported (Wang et al., 2006).

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Thermostable glucoamylases are required to ovoid, or at least to minimize, the cooling step during starch processing. With the aim of creating active enzymes at lower temperatures but conserving high thermostability, random mutagenesis was applied to adapt a thermophilic 3-isopropylmalate dehydrogenase to low temperatures (Suzuki et al., 2001). The 3-isopropylmalate dehydrogenase is one of the leucine biosynthetic pathway enzymes. Four variants were isolated with enhanced specific activities at a moderate temperature (up to 4.1-fold at 40  C). Two of these variants were reported to retain thermostability levels comparable to that of the wild-type enzyme, thereby indicating the possibility of obtaining cold-adapted mutant enzymes with high thermal stability. The use of organic solvents in biocatalytic reactions could offer several attractive advantages, including high solubility of hydrophobic substrates, drastic changes in enzyme specificity, suppression of water-dependent side reactions, easy product recovery, and diminution of microbial contamination risk (Castro & Knubovets, 2003; Khmelnitsky & Rich, 1999). The prospective application of enzymes in industrial process would be dramatically expanded if biocatalysts could act in non-aqueous environments. Several studies have noted that the majority of naturally available enzymes are usually not optimally suitable for catalysis in organic solvents (such as acetone, DMF, DMSO) and sought to develop better enzymes for synthetic applications. Directed evolution methods have been widely employed to engineer proteins for non-aqueous environments (Hao & Berry, 2004; Kawata & Ogino, 2009; Zuma´rraga et al., 2007), mainly because the structural basis of resistance towards organic solvents is not well known (Eijsink et al., 2005). Through rational engineering, Martinez et al. (Martinez et al., 1992) showed that the substitution of surfacecharged residues is a generally useful mechanism for the stabilization of enzymes in organic media. This stabilization was observed only at high concentrations of organic solvent and not at low organic acid concentrations (40% DMF). A double substitution showed, however, an additive effect and provided a mutant that was 3.4 times more stable than the wild-type in 80% DMF. Specificity The engineering of enzymes with desired specificities is a challenging task that has attracted the attention of the research community, particularly in the fields of chemo-enzymatic and synthetic biotechnology. Using a semi-rational design and in attempt to promote the detoxification role of epoxide hydrolases, Rui et al. (Rui et al., 2004) have targeted the vicinity of the active site of an epoxide hydrolase from Agrobacterium radiobacter AD1 to successfully expand the substrate specificity and accept toxic chlorinated epoxyethanes. The enzyme was tuned to recognize cis1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues. A generated triple mutant F108L-I219L-C248L was noted to enhance the degradation of cis-1,2-dichloroethylene up to 10-folds as compared to the wild-type enzyme at high substrate concentrations.

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Similarly, using a cyclical random/targeted approach, Reetz et al, (Reetz et al., 2001; Reetz, 2004) considerably increased the enantioselectivity (E) of Pseudomonas aeruginosa lipase toward a p-nitrophenyl ester in favor of the (S)-2 acid. Through this semi-rational method, the authors progressively improved the enantioselectivity of the enzyme from E ¼ 1.1 (wild type) to E451, thereby providing the most highly enantioselective lipase variant described to date. May et al. (May et al., 2000) successfully inverted the enantioselectivity of a hydantoinase from D-selectivity (with enantiomeric excess (ee) of 40%) to moderate L-preference (20% ee at 30% conversion) by a combination of ep-PCR and saturation mutagenesis. A single amino acid substitution was found to be sufficient to invert enantioselectivity. Despite the low L-selectivity obtained, this example of inverting enantioselectivity of a hydantoinase is particularly remarkable because it addresses a problem with high impact on an industrial application. Another equally impressive example lies in the creation of novel enzyme substrate specificities through the directed evolution of two highly homologous triazine hydrolases different by only nine residues (Raillard et al., 2001). The library created by DNA shuffling contained many chimeric enzymes with higher activity (up to 150-fold) than either parents. More importantly, it contained a set of variants with novel substrate specificity allowing the hydrolysis of five triazines that were not substrates for the two wild-type enzymes. A recent study (Chen et al., 2012) has reported on a successful redesign towards dual substrate specificity in glycoside hydrolase family 5 through the engineering of a conserved active site motif. In the same context, the exchange of active site motifs allowed for switching the substrate specificity between O- and C-glycosyltransferase (Gutmann & Nidetzky, 2012). The enhancement of the minor hydrolytic side reaction of cyclodextrin glycosyltransferase (CGTase) may have useful applications in the bread baking industry (Jemli et al., 2012). In an attempt to improve the anti-staling properties of the CGTase from alkalophilic Bacillus sp. I-5, a combination of ep-PCR and site directed mutagenesis was used (Shim et al., 2004). A triple mutant was obtained with a 10-fold decrease in cyclization activity and 15-fold increase in hydrolytic activity. Interestingly, the addition of this CGTase mutant reduced the retrogradation rate of bread in the same way attained with the commercial anti-staling enzyme Novamyl during 7 d of storage at 4  C. Recently, and using a semi-rational approach, Champion and collaborators (Champion et al., 2012) have reported on an unprecedented specificity enhancement for an amylosucrase (up to 400-fold toward a non-natural acceptor, the allyl 2-acetamido-2-deoxy-a-D-glucopyranoside). Actually, they obtained three double mutants with high levels of catalytic efficiency toward both the sucrose (up to 8-fold) and acceptor substrate (up to 400-fold). Irague et al. (Irague et al., 2012) have also reported on the successful synthesis of seven original types of biopolymers that could be safely used in analytical and therapeutic applications. These biopolymers were obtained in vitro by mutants evolved through the combinatorial engineering of the most efficient glucansucrase applied to date, dextransucrase

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DSR-S from Leuconostoc mesenteroides NRRL B-512F. It is worth mentioning that the considerable success of combinatorial engineering to tailor-made biocatalysts and identify the key residues involved in the enzyme specificity cannot be achievable without the development of powerful screening protocols that allow for the rapid and efficient identification of specific functions. Hence, a quantitative and sensitive NMR-based method was, for instance, developed for the screening of a library of 3.6  104 clones at a throughput of 480 enzyme mutants per day (Irague et al., 2011). This screening method proved efficient as a tool to mine mutant libraries for novel enzyme specificities (Irague et al., 2013). Activity The activities of naturally available enzymes are often not amenable to several industrial processes. The enhancement of the activity of the industrial biocatalyst is usually needed to overcome this limitation. The pH-activity profile of the phytase from Aspergillus niger used as a feed supplement was, for instance, enhanced by combining several individual mutations that allowed the mutants to be quite active at pH 3.5. The efficient hydrolysis of phytate, which occurs generally at a pH around 3.5 in the stomach of animals and which was otherwise ineffective for the parent phytase, has, therefore, become feasible. Moreover, the hydrolytic activity of the mutants at pH 3.5 was enhanced by approximately 1.5-fold as compared to that of the wild-type at its optimum pH (pH 5.5). The work showed that the strategic and cumulative improvements in pH activity and thermostability could be achieved by assembling mutations derived from rational design and random mutagenesis (Zhang & Lei, 2008). Gene shuffling has also been used to engineer another phytase from Aspergillus niger 113 with enhanced catalytic properties (Tian et al., 2010). Over 50 000 mutants were screened and two mutants were retained. None of the substitutions (K41E and E121F) in the two mutants was in a position known to be important for catalysis or substrate binding. The substitutions were, however, noted to give rise to 2.5- and 3.1-fold increased specific activity and 1.4- and 1.6-fold enhanced catalytic efficiency. The use of starch modifying enzymes in industrial applications usually requires the adaptation of the enzyme to a specific pH (Kelly et al., 2009). Hence, with the aim of adapting the a-amylase from Bacillus amyloliquefaciens (BAA) to detergency, the alkali-activity of the enzyme was engineered by ep-PCR and DNA shuffling (Bessler et al., 2003). By screening the library, the superior mutant (BAA 42) obtained exhibited an optimal activity at pH7, corresponding to a shift of one pH unit as compared to the wild-type enzyme. BAA 42 was active over a broader pH-range than the wild-type enzyme, resulting in a 5-fold higher activity at pH 10. Furthermore, the activity in periplasmic extracts and the specific activity increased 4 and 1.5-fold, respectively. A further mutant (BAA 29) showed a wild-type-like pH profile but displayed a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity.

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DOI: 10.3109/07388551.2014.950550

Another example of specific pH adjustment for enzymes is the adaptation of the maltogenic amylase (Novamyl) to breads made at low pH recipes (Jones et al., 2008). Novamyl is widely used in the baking industry as an anti-staling agent in breads baked at neutral or near neutral pH. The enzyme is, however, rapidly inactivated during the process of baking low pH bread (such as sourdough and rye). In an attempt to improve the applicability of Novamyl to low pH, two ep-PCR libraries were constructed and screened for variants with improved thermal stability at 80  C and activity at pH 4.3 for 25 min. Variants exhibiting improved performance were iteratively recombined using gene shuffling to create two generations of libraries. Relative to wild-type Novamyl, a number of the resulting variants exhibited more than 10  C increase in thermal stability at pH 4.5. Of these, a triple mutant significantly outperformed the anti-staling activity of the wild-type enzyme in bread made at low pH.

Concluding remarks Enzymes are economically and environmentally attractive biocatalysts for various industrial processes, including technical, food, beverage and animal feed industries. Accordingly, the global demand for enzymes is expected to increase in the coming years. However, in several cases, the use of natural biocatalysts has often been hampered by their low stability, lack of specificity, and low catalytic efficiency under operational industrial conditions. To overcome these inadequacies and meet the growing demands for robust, high turnover, and economical enzymes, efforts have largely been devoted to engineer enzymes at gene and protein levels with the aim of developing industrial biocatalysts with improved or new catalytic functions. Over the last decades, considerable success in enzyme engineering has been achieved thanks to the significant advances in structural biology, screening protocols, and bioinformatic tools. These advances have also been paralleled with an ever increasing need for novel biocatalysts to cover a wide field of chemo-enzymatic synthesis. Nevertheless, it is worth noting that the currently known natural biocatalysts represent only about 1% of existing enzymes. Hence, the functional screening of metagenomic libraries could provide a powerful tool to discover promising novel enzymes from uncultured microbial communities. By far, the progress in technology and protocols can provide immense momentum in the future with regard to the design or discovery of powerful biocatalysts. In the present review, we have first provided a survey of representative industrial applications of enzymes. We have then presented some illustrative examples of enzymes engineered to overcome the limitations associated with parent wild-type enzymes, including stability, specificity, and pH or solvent tolerance. The survey and illustrative examples clearly show that no definite universal approach is currently available for the design of ideal catalysts for industrial application.

Acknowledgements The authors are thankful to Mr. Anouar Smaoui and Mrs. Hanen Ben Salem for their valuable language polishing services.

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Declaration of interest Due to the large flow of studies on the subject under investigation, the authors would like to apologize for any work that they were not able to cite. Related projects in our laboratory are supported by Grants-in-Aid for Scientific Research from the Ministry of High Education and Scientific Research of Tunisia. The authors report no declaration of interest.

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