State-of-the-art protein engineering approaches using biological macromolecules: A review from immobilization to implementation view point

Accepted Manuscript Title: State-of-the-art protein engineering approaches using biological macromolecules: a review from immobilization to implementation view point Authors: Muhammad Bilal, Hafiz M.N. Iqbal, Guo Shuqi, Hongbo Hu, Wei Wang, Xuehong Zhang PII: DOI: Reference:

S0141-8130(17)33484-0 https://doi.org/10.1016/j.ijbiomac.2017.10.182 BIOMAC 8477

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

10-9-2017 18-10-2017 31-10-2017

Please cite this article as: Muhammad Bilal, Hafiz M.N.Iqbal, Guo Shuqi, Hongbo Hu, Wei Wang, Xuehong Zhang, State-of-the-art protein engineering approaches using biological macromolecules: a review from immobilization to implementation view point, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.10.182 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

State-of-the-art protein engineering approaches using biological macromolecules: a review from immobilization to implementation view point Muhammad Bilal,a Hafiz M. N. Iqbal,b Guo Shuqi a, Hongbo Hua,c*, Wei Wang,a and Xuehong Zhanga a

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology,

Shanghai Jiao Tong University, Shanghai, 200240, China b

Tecnologico de Monterrey, School of Engineering and Sciences, Campus Monterrey, Ave.

Eugenio Garza Sada 2501, Monterrey, N.L., CP 64849, Mexico c

National Experimental Teaching Center for Life Sciences and Biotechnology, Shanghai Jiao

Tong University, Shanghai, 200240, China *Corresponding author e-mail: [email protected] (H. Hu) Graphical abstract

Highlights

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State-of-the-art protein engineering approaches are reviewed.

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Protein engineering offers a straightforward way to upgrade enzymatic activities.

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Tailored enzymes and challenges in biocatalysis engineering are discussed.

Abstract Over the past years, technological and scientific advances have proven biocatalysis as a sustainable alternative than traditional chemical catalysis including organo- or metallocatalysis. In this context, immobilization based approaches represent simple but effective routes for engineering enzyme catalysts with higher activities than wild-type derivatives. Many enzymes including oxidoreductases have been engineered by rational and directed evolution, to realize the catalytic activity, enantioselectivity, and stability attributes which are essential for their biotechnological exploitation. Induce yet stable activity in enzyme catalysis offer new insights and motivation to engineer efficient catalysts for practical and commercial purposes. It has now become possible to envisage substrate accessibility to the catalytic site of the enzyme by current computational capabilities that reduce the experimental work related to the enzyme selection, screening, and engineering. Herein, state-of-the-art protein engineering approaches for improving enzymatic activities including chemical modification, directed evolution, and rational design or their combination methods are discussed. The emphasis is also given to the applications of the resulting tailored catalysts ranging from fine chemicals to novel pharmaceutical compounds that use biocatalysts as a vital step.

Keywords: Protein engineering; Enzyme; Biocatalysis; Chemically modified enzyme; Catalytic activity; Rational design; Directed evolution

1. Introduction Biotechnologists and microbiologists have long viewed enzymatic catalysis with enormous potential in diverse fields for the manufacture of specialty chemicals, pharmaceuticals, biofuel production, and food processing, etc. High catalytic potentiality, stability, and repeatability of enzymes are the characteristic features mainly expected for industrial biocatalytic processes (Asgher et al., 2017; Rehman et al., 2017). The strategies employed for designing biocatalysts with desirable activity and stability for industrial applications can be categorized into 1) protein engineering, such as site-directed mutagenesis and direct evolution, and 2) chemical approaches, including chemical modification and immobilization (Bornscheuer et al., 2012). Also, the integration of above-stated methods is useful for modifying the catalytic properties of biocatalysts (Zhang et al., 2015). Protein engineering offers a straightforward way to upgrade enzymatic activities by altering the structure of amino acid residues at enzyme catalytic site by fusion protein technologies, directed evolution and site-targeted mutagenesis. Thanks to the key scientific advancements in biotechnology and protein engineering, a plenty of achievements have been accomplished in the last twenty years. The directed evolution also played a noticeable role in the successful reengineering of several enzymes in particular oxygenases for biocatalysis (Cirino and Arnold, 2002). On the other hand, enzyme immobilization, firstly commercialized in the 1960s (Tosa et al., 1967), has been developed as a unique chemical-based engineering technique that facilitates the reusability and recovery of enzymes. Importantly, high stabilization of an enzyme is often

achieved through immobilization, which can trim-down the cost of enzyme-based industrial catalysis. In the last several years, continuous attempts in this dimension have achieved several immobilization approaches, such as attachment on solid carriers, conjugation with (bio) polymers, encapsulation in gels, or porous/hollow materials, and development of cross-linked enzyme aggregates or crystals (Zhang et al., 2015; Bilal et al., 2017a; Bilal et al., 2017b; Bilal et al., 2017c; Bilal et al., 2017d). Among various immobilization strategies, enzyme attachment on solid surfaces either through physical adsorption or covalent linkages establishes one of the best practical techniques. The multiple covalent bonds between carrier supports and protein molecules can considerably modify the protein conformation to optimally retaining the enzymatic activity and stability (Cowan and Fernandez-Lafuente, 2011). Immobilization significantly improves the enzymatic properties such as pH and thermal stability, tolerance to organic solvents, activity, and selectivity, to encounter the growing demands for green and sustainable industrial perspectives (Sheldon and van Pelt, 2013; Bilal et al., 2016; Bilal et al., 2017d). An increasing number of recent reports have demonstrated that immobilized enzymes can remarkably display enhanced activities than their native counterparts in aqueous solution (Asgher et al., 2014; Bilal et al., 2017e; Bilal et al., 2017f; Bilal et al., 2017g). Figure 1 illustrates basic methods and sub-methods of enzyme immobilization strategies, whereas, various advantages and disadvantages of enzyme immobilization technology are shown in Figure 2 (Asgher et al., 2014; Bilal et al., 2017d). Keeping in mind the enzymatic engineering potentialities of biological macromolecules, an effort has been made to highlight state-of-the-art protein engineering approaches and their applied perspectives. The first part of the review describes biocatalysis engineering with special reference to the carrier-bound immobilization and carrier free immobilization of enzymes. The

second part is focused on the protein engineering and rational design. Some examples of tailored enzymes in industrial biocatalysis have also been introduced. Towards the end, information is given on the new trends in enzyme engineering, current challenges in biocatalysis engineering and the section is wrapped up with concluding remarks and futuristic perspectives. 2. Biocatalysis — the big picture Biocatalysis involves the application of microbes and biocatalysts (enzymes) in synthetic chemistry and exploits nature’s catalysts for new purposes, which they have not been, developed before (Wenda et al., 2011; Drauz et al., 2012). Todays, the biocatalysis area, has realized its industrially established level through several phases of the biotechnological investigation, research, and inventions. More than 100 years ago, researchers documented that the living cells components can be used for valuable biochemical transformations. Rosenthaler, (1908) used a plant extract to synthesize (R)-mandelonitrile from benzaldehyde and hydrogen cyanide. Similarly, usage of proteases in laundry detergents (Estell et al., 1985), glucose conversion to fructose by glucose isomerase (Jensen and Rugh, 1987) and penicillin G acylase to manufacture semisynthetic antibiotics are the more recent examples (Bruggink et al., 1998). The restricted stabilities of the enzymes were the significant technical challenges for these applications, and such inadequacies were primarily encountered by enzyme immobilization, which also facilitated the repeatability of the biocatalysts (First wave of biocatalysis) (Figure 3). In the second biocatalysis wave (during the 1980s and 1990s), initial protein engineering strategies, particularly structure-guided technologies, broadened the enzyme ranges towards the unusual substrate, thus allowing the manufacture of non-natural synthetic intermediates. This wave of engineering enabled the use of biocatalysts for the biosynthesis of value-added pharmaceutical and fine chemicals. Notable examples are the lipase-driven development of a

blood pressure drug (diltiazem), hydroxy nitrile-lyase-mediated biosynthesis of herbicides (Griengl et al., 2000), synthesis of cholesterol-lowering statin drugs by carbonyl-reductasecatalyzed reaction (Hills, 2003), and nitrile-hydratase-catalyzed hydration of acrylonitrile to acrylamide for polymers (Nagasawa et al., 1990). In parallel to stabilizing features, the challenges now involved optimization of the biocatalyst towards unnatural substrates. In the present, so-called third biocatalysis wave, Pim Stemmer and Frances Arnold introduced directed evolution methods that can extensively modify biocatalysts in a short time. Preliminary, this technology encompasses recurrent cycles of random amino-acid changes, which were then selected and screened for finding variants with better catalytic stability, substrate specificity, and selectivity. Subsequently, scientists focused were revived on enhancing the directed evolution efficiency to construct ‘smarter’ libraries. The metabolic pathways were optimized, in some cases, for instance, integrating pertinent genes from various natural hosts to biosynthesize 1,3propanediol in a novel candidate rendering it promising to shift from glycerol to glucose as the more useful feedstocks (Nakamura and Whited, 2003). 3. Biocatalysis engineering — Carrier-bound immobilization A biocatalytic process consists of many variables, wherein the enzyme catalyst is merely one part. After selecting an enzyme for the targeted-oriented biocatalytic application, and optimizing its properties, the enzyme should be expressed in a microbial host with a Generally Regarded as Safe (GRAS) status for producing in large quantities at relatively low cost. Since enzyme catalysts are typically water soluble and challenging to recover them from aqueous solutions; therefore, many enzymes can only be exploited on a single use, throw-away basis. It has been demonstrated that the enzyme expenses per kg of target product can be significantly diminished by immobilization that develops an easily recoverable and reusable heterogeneous catalyst

(Sheldon and van Pelt, 2013). This biocatalytic engineering consequences substantial process simplification accompanied by a greater product quality and negligible environmental viewpoints. Enzyme immobilization led to an augmented stabilization, by overwhelming the unfolding and, therefore, deactivation of the catalyst (Rehman et al., 2016; Amin et al., 2017). Truppo et al. (2012) investigated and compared several polymer-based resins immobilized transaminase (TA) enzyme with the corresponding lyophilized free counterpart. The enzyme adsorbed on a highly hydrophobic octadecyl functionalized polymethacrylate resin exhibited the best results with 4% loading and 45% activity recovery. The immobilized TA derivative was surprisingly more vigorous in dry isopropyl acetate at 50 °C, with a slight deactivation rate over 6 days. No notable deactivation experimented over the same period following the use of watersaturated isopropyl acetate, and 10 successive cycles were achieved with no apparent activity loss for 200 h. The native TA, on the other hand, was entirely deactivated and presented no activity in the organic solvent. The utilization of organic solvents represents significant advantages than that of aqueous suspensions which necessitate the addition of buffer solution and constant pH regulation throughout the reaction. The organic solvent is subsequently employed for product extraction followed by mixture filtration to eliminate the denatured enzyme. The residual aqueous solution generates a solvent polluted waste stream. In contrary, an immobilizate application in organic solvents precludes the requirement for the buffer, pH control and labor-intensive separation of the residual denatured waste. This reduces the work up and markedly diminishes the processing duration and the waste expenses. Biocatalysts could be reused again and again in a commercially more attractive way (Sheldon and Pereira, 2017). The methods for enzyme immobilization can be categorized into solid support attachment, entrapment and cross-linking. Attachment to a pre-designed carrier support can be physical,

ionic, or covalent bonding. Physical binding is considered too feeble to retain the enzyme fixed to the supporting matrix in harsh environments of elevated reactant and product concentrations and high ionic strength. Ionic binding is durable, whereas the covalent linkages prevent the enzyme leaching from the surface but sometimes presents the disadvantage of irreversible enzyme deactivation, and both the biocatalyst and the (often expensive) carrier materials are rendered impracticable. Entrapment implicates enclosure of a biocatalyst in organic or inorganic polymeric materials or membrane devices (such as hollow fibers) or microcapsules. Physical attachments are too weak; therefore, to preclude complete enzyme escaping an extra covalent linkage is often needed. 4. CLEA and combi-CLEA – Carrier-free immobilization Inevitably, the use of an immobilization carrier leads to dilution of activity which results in reduced space-time yields and productions. Enhancing the catalytic loading, however, does not overcome this issue, because some of the enzyme molecules become inaccessible due to multiple carrier layers or located deeply within the carrier pores. There is an escalating interest in carrierfree immobilized enzymes, such as cross-linked enzyme aggregates (CLEAs) which apparently presents the advantages of high yields, higher stability and lower processing costs presumably due to the elimination of an additional costly supporting carrier (Sheldon et al., 2013). CLEAs are developed by simple precipitation of the enzyme from a solution by adding salt, (such as ammonium sulfate), or a water-miscible organic solvent, followed by cross-linking with a bifunctional reagent (Figure 4). Since the selective precipitation is often employed to purify enzymes from an aqueous medium; therefore, the CLEA approach merges purification and immobilization into a single step operation that essentially obviates a highly purified enzyme preparation. The CLEA technology has been extensively attempted to a wide-range of

hydrolases, lyases, oxidoreductases, and transferases. Magnetic CLEAs have been reported for the biotransformation of lignocelluloses to second-generation biofuels (Bhattacharya and Pleschke, 2015). Interestingly, Ning et al., (2014) demonstrated that two or more enzymes could be coimmobilized to form a so-called combi-CLEA by co-precipitating the enzymes followed by cross-linking of the resulting aggregates. They developed a combi-CLEA of two enzymes containing ketoreductase (KRED) and glutamate dehydrogenase (GDH) and repeatedly employed for synthesizing the essential atorvastatin intermediates. It appeared to be an excellent candidate displaying elevated pH and thermal stability accompanied by high substrate resistance and durable, functional stability. These novel combi-CLEAs have found potential utilities in the food and beverages processing. In another study, Sojitra et al., (2016) synthesized a tri-enzyme magnetic combi-CLEA of cellulase, a-amylase, and pectinase and subsequently employed for the clarification of different fruit juices. 5. Protein engineering 5.1 Rational design and directed evolution The native soluble enzymes are often not efficient for catalyzing a reaction with unnatural substrates particularly under unfavorable industrial environments, which consequence low activities, selectivity, and volumetric throughputs. In this juncture, they need to be redesigned/reengineered to furnish high productivities and selectivity at elevated substrate level and minimum catalyst loadings. This can be accomplished by generating libraries of mutant enzymes and the screened for desired characteristics, using directed evolution or in-vitro evolution. Alternatively, space-time yields can be enhanced using higher catalyst quantities but, this gives rise to

complications in downstream processing probably due to the emulsions formation which is difficult to separate (Sheldon and Pereira, 2017). Rational design by site-directed mutagenesis (SDM) is a novel genetic engineering approach. This strategy implicates the construction of point mutations, whereby a particular amino acid at a specific location is substituted by any of the standard amino acids. A major dilemma is that meticulous information concerning the three-dimensional structure and mechanism of the target enzyme is required for SDM. In contrary, random mutagenesis (RM) necessitates no structural information, and an error-prone polymerase chain reaction (ep-PCR) was used to generate mutants’ libraries in the early 1990s. In the ideal situation, the target biocatalyst should be purified, crystallized, and investigated by X-ray crystallography. Apart from the catalytic residues in a protein structure, the pertinent amino acids defining the active-site cavity are of considerable importance since they dictate whether an unusual substrate can get accessibility to the biocatalytic site. This is very crucial if the non-natural substrate is larger compared to the natural one (s) (Strohmeier et al., 2011). Based on crystal structures, some amino acids hindering substrate approach to the catalytic site can be substituted, thereby improving the catalyst performance (Cedrone et al., 2000; Hult and Berglund, 2003). Remarkably, rationally exchanging only two amino acids can modify an esterase enzyme to a hydroxy nitrile lyase (Padhi et al., 2010). Guided by structural information of the enzyme; rational design might be regarded as the easiest and straightforward enzyme tailoring approach, but the chances of getting the desired outcomes are often still too low that reflects our lack of appropriate understandings regarding enzyme functions. A milestone in the progress of directed evolution was the rapid development of a protein by DNA shuffling report published by Stemmer, (1994). In this development technique, a set of

different parent genes were fragmented by DNase treatment followed by ligating these fragments into new chimeras using a primer-free PCR step. The resultant chimeras can then be expressed into any suitable vector and screened. This process can be reiterated until the achievement of enzyme variant with the desired traits. Though pioneer reports regarding the directed evolution were primarily concentrated on enhancing the enzymes stability profiles, the scientist’s in particular organic chemists rekindled their interest to improving another noteworthy enzyme property, so-called stereoselectivity. Reetz et al. (1997) reported a groundbreaking, proof-ofconcept for improving enantioselectivity of biocatalysts by directed evolution. Later on, DNA shuffling, and evolutionary approaches have widely been attempted aimed at improving the existing properties (Sun et al., 2016) and evolving new yet unexplored activities of biocatalysts (Renata et al., 2015). It is now possible to modify the biocatalyst e.g. dihydroxyacetone (DHA) kinase properties according to the predefined designs by directed evolution approaches (Figure 5) (Sánchez-Moreno et al., 2015). 5.2 Combinatorial rational design and directed evolution Even though the rational or in-vitro evolution has identified any specific amino acid that should be exchanged to enhance biocatalyst potentiality, it is unclear whether the ideal amino acid substitution has previously been identified. Consequently, site-directed saturation mutagenesis, substituting a certain amino acid by all naturally-occurring amino acids, may be carried out for catalytic improvement (Kretz et al., 2004). The substrate recognition or enantioselectivity can be improved by combinatorial active-site saturation testing (CASTing) using the structural information on the active-site cavity of the target enzymes. It is demonstrated that the mutations adjacent to the catalytic site more efficiently improve the catalytic properties of enzymes (Morley and Kazlauskas, 2005; Reetz et al., 2006). Novel enzyme mutants are created either by

merely random mutagenesis or by including a rational component, the influential factor for efficacious enzyme evolution is a stable, functional enzyme assay and the working environments under which the enzyme is projected to be applied. 6. Examples of tailored enzymes in industrial biocatalysis In the last decade, a continuous regeneration of cofactors and a variety of enzymes have been reported as predicted by Schmid and co-workers, (2001). The exploitation of non-metabolizing cells has demonstrated to be more challenging for biocatalytic purposes than anticipated and inclination has instead revived towards tailored catalysts used either in crude and semi-purified form. The exploitation of isolated enzymes presents notable advantages that they are easier to remove, resist harsher conditions, eliminate diffusional restrictions and are easier to ship, around the globe. Ketoreductases (KRED)-based catalytic bioprocesses have entirely substituted the whole-cell reductions and metal–ligand-assisted chemo catalysis, over the past few years (Moore et al., 2007; Strohmeier et al., 2011). KREDs and many other enzymes have been extensively studied for producing chiral pharmaceutical intermediates such as atorvastatin – a cholesterol-lowering drug with global sales of US$11,900,000,000 in 2010. In parallel to highly efficient (bio) catalyst, a low-cost bioprocess also necessitates easily-available raw feedstock materials and facile isolation of purified target compound in elevated yields. One contemporary biocatalytic process employs three enzymatic steps: first, the combination of KRED and glucose dehydrogenase; second, this combination with a halohydrin dehalogenase to make the ethyl (R)-4-cyano-3- hydroxybutyrate intermediate; and, third, the enzyme-assisted reduction for the manufacturing of advanced diol intermediate (Ma et al., 2010).

Recently, it has been reported that biocatalysis engineering broadened the substrate range of transaminases to ketones with two bulky substituents (Savile et al., 2010). Notably, the catalytic re-engineering started with a smaller ketone substrate, generated more space in the catalytic site and used progressively larger ketones. Several rounds of directed evolution successfully constructed an engineered amine transaminase with 40,000-folds amplified catalytic activity. The engineered catalyst can substitute the transition-metal-based hydrogenation catalyst for the manufacturing of sitagliptin (Bornscheuer et al., 2012). The enzyme mutants obtained from optimization studies are an inimitable source of initiating points for future programs. For example, tailoring KREDs to make (R)-3-hydroxy-thiolane (R3HT) led to several enzyme variants with excellent stability including some which were incompatible due to insignificant enantioselectivity. Nevertheless, one of these unsuitable variants was the preliminary enzyme in engineering a KRED for (S)-1-(2,6-dichloro-3fluorophenyl)-ethanol (DCFPE) (Liese et al., 2006). Likewise, the transaminases obtained during the pro sitagliptin evolution can give rise many other amines and might function as starting points for several different engineered biocatalysts for amine biosynthesis (Desai, 2011). 7. Engineering the oxidative enzymes Rational design and directed molecular evolution, as well as combinations of both techniques, have been attempted for engineering oxidoreductases focusing on the whole protein or any targeted domains (Mate et al., 2016). All the protein engineering technologies prerequisites the availability of a suitable expression system to produce variants with improved features. The heterologous expression of oxidoreductase genes in Escherichia coli, Saccharomyces cerevisiae, or other systems was standardized for aryl-alcohol oxidase (AAO), dye-decolorizing peroxidase (DyP), ligninolytic peroxidases, unspecific peroxygenases (UPO), and vanillyl-alcohol oxidase

(VAO) enabling their subsequent engineering (Garcia-Ruiz et al., 2014; Viña-Gonzalez et al., 2015; Gygli and van Berkel, 2017). In many cases, a considerable increase in the gene expression of oxidoreductase was obtained after many cycles of directed molecular evolution, which were followed by additional rounds to increase the desired catalytic traits further. Notably, versatile peroxidase (VP) has been used as a model peroxidase in systematic engineering studies, and oxidative, as well as alkaline inactivation, has been examined to get variants with better industrial applicability. In this context, two different strategies were merged to enhance the oxidative stability of VP towards H2O2 (Sáez-Jiménez et al., 2015a). A different strategy based on i) selection of a naturally-stable peroxidase by genome screening and heterologous expression; ii) identification of the structural determinants for this stability, such as H-bonding patterns, salt bridges and basic residues and iii) introducing them into the target enzyme by directed mutagenesis, was effectively employed for rational improvement of alkaline stability (Sáez-Jiménez et al., 2015b). Rational design engineerings have also been applied to develop a ligninolytic VP with potential capability to function particularly at highly acidic pH (pH 3), which in turn increases the redox potential of the heme iron (Fernández-Fueyo et al., 2014). In another study, Linde et al. (2016) reported that rational design of the active site of DyP resulted in efficient stereoselective sulfoxidation reactions. In parallel to rational designing stated-above, molecular evolution and combinatorial approaches have also been envisioned to improve the H2O2 and alkaline steadiness of VP. In case of directed evolution, the best variant harbored eight mutations resulting in improvement of VP half-life from 3 to 35 min (González-Pérez et al., 2014). González-Pérez et al. (2016) achieved a VP variant that efficiently oxidizes substrates at alkaline pH both the Mn2+ site and the heme channel, whereas the catalytic amino acid tryptophan was not functioning under these conditions.

Likewise, UPO has been tailored by directed evolution to magnify its mono(per)oxygenase activity and to reduce competing one-electron oxidation. The resulting evolved variants were employed in oxy functionalizations of biotechnological importance, such as the naphthalene oxygenation to 1-naphthol. In the past decade, the rational and evolutionary design has also been applied for laccase engineering. Nevertheless, directed evolution of the whole genome and mutagenesis-based evolution have better upgraded the ligninolytic laccase characteristics for targeted functions, such as the oxidation degradation of phenolic compounds (Vicente et al., 2016). 8. New trends in enzyme engineering Intensive modifications in enzyme catalytic properties usually involve multiple amino-acid replacements because they largely alter the protein structure. Nonetheless, concurrent substitutions of several amino-acid can generate exponentially more variants/mutants. Most of these variants are inactive and require testing to find the improved variants followed by incremental improvements by several rounds of directed evolution. Efficient screening of the variants is the simplest solution to overcome this issue. Any notable changes in substrate specificity might be scrutinized by high-throughput methods, such as fluorescence-activated cell sorting, which can screen millions of variants in a short time (Becker et al., 2008; Fernández‐ Álvaro et al., 2011). Whittle and Shanklin, (2001) carried out six simultaneous amino-acid replacements in the catalytic site of a desaturase enzyme and found that only the variants with modified substrate specificities were able to grow. Currently, the best technique to generating multiple-site mutations is to add them concurrently but to limit the choices using bioinformatics or statistical approaches. Codexis researchers used one statistical correlation approach namely ProSAR (protein structure-activity relationship)

algorithm to improve the 4,000-fold reaction rate of a halohydrin dehalogenase (Fox et al., 2007). Several other scientists conducted random amino-acid replacements in the dehalogenase and monitored the catalytic rate by the variants. Statistical approaches identified that the variants containing a Phe 186 Tyr substitution were found to be the better than those with no substitutions. Though some mutants with such a substitution were not beneficial possibly due to the detrimental effects of other mutations, on average, the statistical analysis concluded that Phe 186 Tyr is an advantageous mutation. The ultimately improved enzyme variant consists of 35 amino-acid substitutions among it's 254 amino acids (Bornscheuer et al., 2012). Jochens and Bornscheuer introduced a novel approach, in which the changes are restricted to the catalytic site, to improve the enantioselectivity of an esterase enzyme from Pseudomonas fluorescence. There were 160,000 ways simultaneously to change the four amino acids adjoining to the substrate in the active site. The amino-acid sequences of 2,800 related enzymes were aligned by the researchers to identify most common amino acids at these positions. This analysis restricted the possibilities to several hundred variants, which were then investigated to discover double and triple mutants with the desired enantioselectivities (Jochens and Bornscheuer, 2010). The magnitude of beneficial variations occurred during the evolution proteins have increased considerably over the past decade. The directed evolution of the halohydrin dehalogenase substituted around 35 of the 254 amino acids for the manufacturing of atorvastatin (Fox et al., 2007). A second state-of-the protein engineering approach explored in the last decade is the creation of new, so-called non-natural, catalytic activities. The base for these new engineered activities is usually a catalytically promiscuous reaction. Promiscuity is the capability of one active site to catalyzing more than one reaction type, which may be additional side reactions in combination

with one normal reaction. Interestingly, this new reaction type is not merely a substituent addition to the existing substrate, but implicates different transition states and thereby generates varying types of chemical linkages. The pyruvate decarboxylase (PDC) natively catalyzes pyruvate to acetaldehyde and CO2, but, a promiscuity activity of pyruvate decarboxylase led to the coupling of this acetaldehyde to another aldehyde in an acyloin condensation. Such an unnatural PDC-driven acetaldehyde condensation with benzaldehyde was the initiating point to manufacture a precursor of the drug Ephedrine (Meyer et al., 2011). In contrast to the normal reaction, the promiscuous reaction does not require a proton transfer, and substitution of a single amino-acid eliminating proton donor deactivated the natural enzyme activity but enhanced the fivefold promiscuity activity. The restricting unsolicited or competing for metabolic pathways to shift divert the flux towards the target product entirely is further developed by the third wave advance so-called metabolic pathway engineering. This engineering enables transferring of secondary metabolism-related more complex pathways into new organisms and creating entirely new biochemical pathways to manufacture industrially pertinent pharmaceutical compounds, fine chemicals, and biofuels. Using these techniques, the normal metabolisms of amino acids, fatty acids and terpenes have been re-engineered to make alcohols, hydrocarbons, and polyesters for use as fuels, bulk chemicals, and plastics. 9. Ongoing challenges in biocatalysis engineering Notwithstanding the significant advances, several major challenges remain to harness the entire advantages of biocatalysis. Now a day, enzyme engineering is much faster as ten years ago, but substituting amino acids and screening lots of variants entails a large research group. Many protein engineering approaches will generate improved candidates, but some will produce

variants with better attributes, however, which ones are the superior strategies is still uncertain. Directly comparing different strategies and testing the assumptions behind these strategies would help to identify the most efficient variants (Bornscheuer et al., 2012). The first assumption is that protein engineering can fulfill the objective of biocatalysis. The reactions occurring with unnatural substrates may be thermodynamically less favorable than that of reactions involving natural substrates, and achieving certain enzyme activities might be impossible. In this context, a closer integration of biocatalytic process and thermodynamics is extremely desirable in developing new methods. Secondly, protein engineering often relies on good knowledge regarding the quaternary structure of the enzyme, and our understanding of protein dynamics is still very narrow that makes predictions challenging. Third, although most of the mutations are non-interactive, many cooperative mutations are highly beneficial but hard to study. One possible way for the identification of cooperative effects includes statistical analysis using the ProSAR algorithm (Wells, 1990), but more up-to-date techniques are required to predict at an early stage of protein engineering efficiently. Fourth, computer-based designing of new enzyme activities is not appropriate and usually creates an enzyme with low catalytic activity, requiring further substantial engineering for better activity. Therefore, the better understanding of the structural, dynamic and mechanistic aspects of enzymatic catalysis is needed. Enzyme engineering deciphered the previous shortcomings of biocatalysts such as low activity and stability for non-natural reaction substrates. Highly active enzymes with a longer shelf-life and stability profiles in organic solvents should support biocatalysis to spread into industrial laboratories. The properties of the catalytically engineered enzymes have improved by thousands to millions of times, and these enzymes now can function in unusually harsh industrial environments (Savile and Lalonde, 2011).

10. Concluding remarks and prospects Recent advances in biotechnology and molecular biology have greatly benefitted the enzyme catalysis that now has emerged as an outstanding technology for the sustainable synthesis of specialty chemicals, and pharmaceutical intermediates and use in food-beverage processing. Also, biocatalysis applications are predicted to exponentially increase in the future as a consequence of the transition from fossil fuel-based economy to a biobased and environmentallyfriendlier economy. Therefore, there is a dire need to design new enzymes with excellent biotransformation capabilities under economically and environmentally-justified viewpoint. We believe that the recent biocatalysis engineering approaches would play a noteworthy role in satisfying the socio-industrial demand for bio-based processes and products, and the impending interest in enhancing the catalytic features of enzymes by immobilization and protein engineering will continue unabated in the upcoming days. Given the long-term striving for ecofriendlier processes, and mild reaction environments, the development and use of immobilized green catalysts are likely to remain the subject of intense future investigations. Conflict of interest We do not have any conflicting, competing and financial interests in any capacity.

Acknowledgements The literature facilities provided by Shanghai Jiao Tong University, Shanghai 200240, China and Tecnologico de Monterrey, Campus Monterrey, Mexico are thankfully acknowledged.

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Figures

Figure 1 Basic methods and sub-methods of enzyme immobilization (Reproduced from Bilal et al., 2017d, with permission from Elsevier).

Figure 2 Advantages vs. disadvantages of enzyme immobilization technology (Reproduced from Bilal et al., 2017d, with permission from Elsevier).

Figure 3 Biocatalytic evaluation and improvement strategies. A complete overview from biocatalyst (enzyme) production via fermentation and/or engineering to catalytic pathway. The low substrate conversion can be significantly induced following enzyme engineering.

Figure 4 A schematic illustration of cross-linked enzyme aggregates (CLEAs) and COMBI-CLEAs development in the presence of cross-linker and Fe2O3 particle, respectively.

Figure 5 Directed evolution approach applied to modify the phosphoryl donor specificity of the DHAK from C. freundii (Reproduced from Sánchez-Moreno et al., 2015, an open access article distributed under the Creative Commons Attribution License).