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International Journal of Biological Macromolecules 108 (2018) 893–901 Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Review 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, Shuqi Guo a, Hongbo Hu a,c,∗, Wei Wang a, Xuehong Zhang a 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 article info abstract Article history: Over the past years, technological and scientific advances have proven biocatalysis as a sustainable Received 10 September 2017 alternative than traditional chemical catalysis including organo- or metallocatalysis. In this context, Received in revised form 18 October 2017 immobilization based approaches represent simple but effective routes for engineering enzyme cata- Accepted 31 October 2017 lysts with higher activities than wild-type derivatives. Many enzymes including oxidoreductases have Available online 2 November 2017 been engineered by rational and directed evolution, to realize the catalytic activity, enantioselectiv- ity, and stability attributes which are essential for their biotechnological exploitation. Induce yet stable Keywords: activity in enzyme catalysis offer new insights and motivation to engineer efficient catalysts for prac- Protein engineering tical and commercial purposes. It has now become possible to envisage substrate accessibility to the Enzyme catalytic site of the enzyme by current computational capabilities that reduce the experimental work Biocatalysis related to the enzyme selection, screening, and engineering. Herein, state-of-the-art protein engineering Chemically modified enzyme approaches for improving enzymatic activities including chemical modification, directed evolution, and Catalytic activity rational design or their combination methods are discussed. The emphasis is also given to the applica- Rational design tions of the resulting tailored catalysts ranging from fine chemicals to novel pharmaceutical compounds Directed evolution that use biocatalysts as a vital step. © 2017 Elsevier B.V. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894 2. Biocatalysis – the big picture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894 3. Biocatalysis engineering – carrier-bound immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 894 4. CLEA and combi-CLEA – carrier-free immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 896 5. Protein engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897 5.1. Rational design and directed evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897 5.2. Combinatorial rational design and directed evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897 6. Examples of tailored enzymes in industrial biocatalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897 7. Engineering the oxidative enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 898 8. New trends in enzyme engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 899 9. Ongoing challenges in biocatalysis engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 899 10. Concluding remarks and prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900 ∗ Corresponding author at: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China. E-mail address: [email protected] (H. Hu). https://doi.org/10.1016/j.ijbiomac.2017.10.182 0141-8130/© 2017 Elsevier B.V. All rights reserved.

894 M. Bilal et al. / International Journal of Biological Macromolecules 108 (2018) 893–901 1. Introduction 2. Biocatalysis – the big picture Biotechnologists and microbiologists have long viewed enzy- Biocatalysis involves the application of microbes and biocat- matic catalysis with enormous potential in diverse fields for alysts (enzymes) in synthetic chemistry and exploits nature’s the manufacture of specialty chemicals, pharmaceuticals, biofuel catalysts for new purposes, which they have not been, devel- production, and food processing, etc. High catalytic potentiality, oped before [69,20]. Todays, the biocatalysis area, has realized its stability, and repeatability of enzymes are the characteristic fea- industrially established level through several phases of the biotech- tures mainly expected for industrial biocatalytic processes [3,48]. nological investigation, research, and inventions. More than 100 The strategies employed for designing biocatalysts with desirable years ago, researchers documented that the living cells components activity and stability for industrial applications can be categorized can be used for valuable biochemical transformations. Rosenthaler into 1) protein engineering, such as site-directed mutagenesis and [50] used a plant extract to synthesize (R)-mandelonitrile from ben- direct evolution, and 2) chemical approaches, including chemi- zaldehyde and hydrogen cyanide. Similarly, usage of proteases in cal modification and immobilization [14]. Also, the integration of laundry detergents [21], glucose conversion to fructose by glucose above-stated methods is useful for modifying the catalytic proper- isomerase [31] and penicillin G acylase to manufacture semisyn- ties of biocatalysts [71]. thetic antibiotics are the more recent examples [15]. The restricted stabilities of the enzymes were the significant technical chal- Protein engineering offers a straightforward way to upgrade lenges for these applications, and such inadequacies were primarily enzymatic activities by altering the structure of amino acid residues encountered by enzyme immobilization, which also facilitated the at enzyme catalytic site by fusion protein technologies, directed repeatability of the biocatalysts (First wave of biocatalysis) (Fig. 3). evolution and site-targeted mutagenesis. Thanks to the key sci- entific advancements in biotechnology and protein engineering, a In the second biocatalysis wave (during the 1980s and 1990s), plenty of achievements have been accomplished in the last twenty initial protein engineering strategies, particularly structure-guided years. The directed evolution also played a noticeable role in the technologies, broadened the enzyme ranges towards the unusual successful re-engineering of several enzymes in particular oxyge- substrate, thus allowing the manufacture of non-natural synthetic nases for biocatalysis [17]. intermediates. This wave of engineering enabled the use of biocat- alysts for the biosynthesis of value-added pharmaceutical and fine On the other hand, enzyme immobilization, firstly commer- chemicals. Notable examples are the lipase-driven development of cialized in the 1960s [64], has been developed as a unique a blood pressure drug (diltiazem), hydroxy nitrile-lyase-mediated chemical-based engineering technique that facilitates the reusabil- biosynthesis of herbicides [27], synthesis of cholesterol-lowering ity and recovery of enzymes. Importantly, high stabilization of an statin drugs by carbonyl-reductase-catalyzed reaction [29], and enzyme is often achieved through immobilization, which can trim- nitrile-hydratase-catalyzed hydration of acrylonitrile to acry- down the cost of enzyme-based industrial catalysis. In the last lamide for polymers [41]. In parallel to stabilizing features, the several years, continuous attempts in this dimension have achieved challenges now involved optimization of the biocatalyst towards several immobilization approaches, such as attachment on solid unnatural substrates. carriers, conjugation with (bio) polymers, encapsulation in gels, or porous/hollow materials, and development of cross-linked enzyme In the present, so-called third biocatalysis wave, Pim Stem- aggregates or crystals [71,7–10]. Among various immobilization mer and Frances Arnold introduced directed evolution methods strategies, enzyme attachment on solid surfaces either through that can extensively modify biocatalysts in a short time. Prelim- physical adsorption or covalent linkages establishes one of the inary, this technology encompasses recurrent cycles of random best practical techniques. The multiple covalent bonds between amino-acid changes, which were then selected and screened for carrier supports and protein molecules can considerably modify finding variants with better catalytic stability, substrate specificity, the protein conformation to optimally retaining the enzymatic and selectivity. Subsequently, scientists focused were revived on activity and stability [18]. Immobilization significantly improves enhancing the directed evolution efficiency to construct ‘smarter’ the enzymatic properties such as pH and thermal stability, tol- libraries. The metabolic pathways were optimized, in some cases, erance to organic solvents, activity, and selectivity, to encounter for instance, integrating pertinent genes from various natural hosts the growing demands for green and sustainable industrial per- to biosynthesize 1,3-propanediol in a novel candidate rendering spectives [58,6,10]. An increasing number of recent reports have it promising to shift from glycerol to glucose as the more useful demonstrated that immobilized enzymes can remarkably display feedstocks [42]. enhanced activities than their native counterparts in aqueous solu- tion [2,11–13]. Fig. 1 illustrates basic methods and sub-methods 3. Biocatalysis engineering – carrier-bound immobilization of enzyme immobilization strategies, whereas, various advan- tages and disadvantages of enzyme immobilization technology are A biocatalytic process consists of many variables, wherein the shown in Fig. 2 [2,10]. enzyme catalyst is merely one part. After selecting an enzyme for the targeted-oriented biocatalytic application, and optimiz- Keeping in mind the enzymatic engineering potentialities of ing its properties, the enzyme should be expressed in a microbial biological macromolecules, an effort has been made to highlight host with a Generally Regarded as Safe (GRAS) status for pro- state-of-the-art protein engineering approaches and their applied ducing in large quantities at relatively low cost. Since enzyme perspectives. The first part of the review describes biocatalysis catalysts are typically water soluble and challenging to recover engineering with special reference to the carrier-bound immobi- them from aqueous solutions; therefore, many enzymes can only lization and carrier free immobilization of enzymes. The second be exploited on a single use, throw-away basis. It has been demon- part is focused on the protein engineering and rational design. strated that the enzyme expenses per kg of target product can be Some examples of tailored enzymes in industrial biocatalysis have significantly diminished by immobilization that develops an easily also been introduced. Towards the end, information is given on the recoverable and reusable heterogeneous catalyst [58]. This biocat- new trends in enzyme engineering, current challenges in biocatal- alytic engineering consequences substantial process simplification ysis engineering and the section is wrapped up with concluding accompanied by a greater product quality and negligible environ- remarks and futuristic perspectives. mental viewpoints. Enzyme immobilization led to an augmented

M. Bilal et al. / International Journal of Biological Macromolecules 108 (2018) 893–901 895 Fig. 1. Basic methods and sub-methods of enzyme immobilization. (Reproduced from [10], with permission from Elsevier) Fig. 2. Advantages vs. disadvantages of enzyme immobilization technology. (Reproduced from [10], with permission from Elsevier) stabilization, by overwhelming the unfolding and, therefore, deac- Truppo et al. [65] investigated and compared several polymer- tivation of the catalyst [47,1]. based resins immobilized transaminase (TA) enzyme with the

896 M. Bilal et al. / International Journal of Biological Macromolecules 108 (2018) 893–901 Fig. 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. corresponding lyophilized free counterpart. The enzyme adsorbed 4. CLEA and combi-CLEA – carrier-free immobilization on a highly hydrophobic octadecyl functionalized polymethacry- late resin exhibited the best results with 4% loading and 45% activity Inevitably, the use of an immobilization carrier leads to dilution recovery. The immobilized TA derivative was surprisingly more vig- of activity which results in reduced space-time yields and produc- orous in dry isopropyl acetate at 50 ◦C, with a slight deactivation tions. Enhancing the catalytic loading, however, does not overcome rate over 6 days. No notable deactivation experimented over the this issue, because some of the enzyme molecules become inacces- same period following the use of water-saturated isopropyl acetate, sible due to multiple carrier layers or located deeply within the and 10 successive cycles were achieved with no apparent activ- carrier pores. There is an escalating interest in carrier-free immo- ity loss for 200 h. The native TA, on the other hand, was entirely bilized enzymes, such as cross-linked enzyme aggregates (CLEAs) deactivated and presented no activity in the organic solvent. The which apparently presents the advantages of high yields, higher utilization of organic solvents represents significant advantages stability and lower processing costs presumably due to the elimi- than that of aqueous suspensions which necessitate the addition nation of an additional costly supporting carrier [59]. of buffer solution and constant pH regulation throughout the reac- tion. The organic solvent is subsequently employed for product CLEAs are developed by simple precipitation of the enzyme extraction followed by mixture filtration to eliminate the dena- from a solution by adding salt, (such as ammonium sulfate), or tured enzyme. The residual aqueous solution generates a solvent a water-miscible organic solvent, followed by cross-linking with polluted waste stream. In contrary, an immobilizate application in a bifunctional reagent (Fig. 4). Since the selective precipitation organic solvents precludes the requirement for the buffer, pH con- is often employed to purify enzymes from an aqueous medium; trol and labor-intensive separation of the residual denatured waste. therefore, the CLEA approach merges purification and immobi- This reduces the work up and markedly diminishes the process- lization into a single step operation that essentially obviates a ing duration and the waste expenses. Biocatalysts could be reused highly purified enzyme preparation. The CLEA technology has been again and again in a commercially more attractive way [57]. extensively attempted to a wide-range of hydrolases, lyases, oxi- doreductases, and transferases. Magnetic CLEAs have been reported The methods for enzyme immobilization can be categorized into for the biotransformation of lignocelluloses to second-generation solid support attachment, entrapment and cross-linking. Attach- biofuels [5]. ment to a pre-designed carrier support can be physical, ionic, or covalent bonding. Physical binding is considered too feeble to Interestingly, Ning et al. [43] demonstrated that two or more retain the enzyme fixed to the supporting matrix in harsh environ- enzymes could be co-immobilized to form a so-called combi-CLEA ments of elevated reactant and product concentrations and high by co-precipitating the enzymes followed by cross-linking of the ionic strength. Ionic binding is durable, whereas the covalent link- resulting aggregates. They developed a combi-CLEA of two enzymes ages prevent the enzyme leaching from the surface but sometimes containing ketoreductase (KRED) and glutamate dehydrogenase presents the disadvantage of irreversible enzyme deactivation, and (GDH) and repeatedly employed for synthesizing the essential ator- both the biocatalyst and the (often expensive) carrier materials vastatin intermediates. It appeared to be an excellent candidate are rendered impracticable. Entrapment implicates enclosure of a displaying elevated pH and thermal stability accompanied by high biocatalyst in organic or inorganic polymeric materials or mem- substrate resistance and durable, functional stability. These novel brane devices (such as hollow fibers) or microcapsules. Physical combi-CLEAs have found potential utilities in the food and bever- attachments are too weak; therefore, to preclude complete enzyme ages processing. In another study, Sojitra et al. [60] synthesized escaping an extra covalent linkage is often needed. a tri-enzyme magnetic combi-CLEA of cellulase, a-amylase, and pectinase and subsequently employed for the clarification of dif- ferent fruit juices.

M. Bilal et al. / International Journal of Biological Macromolecules 108 (2018) 893–901 897 Fig. 4. A schematic illustration of cross-linked enzyme aggregates (CLEAs) and COMBI-CLEAs development in the presence of cross-linker and Fe2O3 particle, respectively. 5. Protein engineering any suitable vector and screened. This process can be reiterated until the achievement of enzyme variant with the desired traits. 5.1. Rational design and directed evolution Though pioneer reports regarding the directed evolution were primarily concentrated on enhancing the enzymes stability pro- The native soluble enzymes are often not efficient for catalyzing files, the scientist’s in particular organic chemists rekindled their a reaction with unnatural substrates particularly under unfavor- interest to improving another noteworthy enzyme property, so- able industrial environments, which consequence low activities, called stereoselectivity. Reetz et al. [46] reported a groundbreaking, selectivity, and volumetric throughputs. In this juncture, they proof-of-concept for improving enantioselectivity of biocatalysts need to be redesigned/re-engineered to furnish high productivities by directed evolution. Later on, DNA shuffling, and evolutionary and selectivity at elevated substrate level and minimum cata- approaches have widely been attempted aimed at improving the lyst loadings. This can be accomplished by generating libraries of existing properties [63] and evolving new yet unexplored activi- mutant enzymes and the screened for desired characteristics, using ties of biocatalysts [49]. It is now possible to modify the biocatalyst directed evolution or in-vitro evolution. Alternatively, space-time e.g. dihydroxyacetone (DHA) kinase properties according to the yields can be enhanced using higher catalyst quantities but, this predefined designs by directed evolution approaches (Fig. 5) [53]. gives rise to complications in downstream processing probably due to the emulsions formation which is difficult to separate [57]. 5.2. Combinatorial rational design and directed evolution Rational design by site-directed mutagenesis (SDM) is a novel Even though the rational or in-vitro evolution has identified genetic engineering approach. This strategy implicates the con- any specific amino acid that should be exchanged to enhance bio- struction of point mutations, whereby a particular amino acid at a catalyst potentiality, it is unclear whether the ideal amino acid specific location is substituted by any of the standard amino acids. substitution has previously been identified. Consequently, site- A major dilemma is that meticulous information concerning the directed saturation mutagenesis, substituting a certain amino acid three-dimensional structure and mechanism of the target enzyme by all naturally-occurring amino acids, may be carried out for is required for SDM. In contrary, random mutagenesis (RM) neces- catalytic improvement [33]. The substrate recognition or enantios- sitates no structural information, and an error-prone polymerase electivity can be improved by combinatorial active-site saturation chain reaction (ep-PCR) was used to generate mutants’ libraries in testing (CASTing) using the structural information on the active-site the early 1990s. cavity of the target enzymes. It is demonstrated that the mutations adjacent to the catalytic site more efficiently improve the catalytic In the ideal situation, the target biocatalyst should be purified, properties of enzymes [40,45]. Novel enzyme mutants are created crystallized, and investigated by X-ray crystallography. Apart from either by merely random mutagenesis or by including a rational the catalytic residues in a protein structure, the pertinent amino component, the influential factor for efficacious enzyme evolution acids defining the active-site cavity are of considerable importance is a stable, functional enzyme assay and the working environments since they dictate whether an unusual substrate can get accessi- under which the enzyme is projected to be applied. bility to the biocatalytic site. This is very crucial if the non-natural substrate is larger compared to the natural one (s) [62]. Based on 6. Examples of tailored enzymes in industrial biocatalysis crystal structures, some amino acids hindering substrate approach to the catalytic site can be substituted, thereby improving the cat- In the last decade, a continuous regeneration of cofactors and alyst performance [16,30]. Remarkably, rationally exchanging only a variety of enzymes have been reported as predicted by Schmid two amino acids can modify an esterase enzyme to a hydroxy nitrile et al.[56]. The exploitation of non-metabolizing cells has demon- lyase [44]. Guided by structural information of the enzyme; ratio- strated to be more challenging for biocatalytic purposes than nal design might be regarded as the easiest and straightforward anticipated and inclination has instead revived towards tailored enzyme tailoring approach, but the chances of getting the desired catalysts used either in crude and semi-purified form. The exploita- outcomes are often still too low that reflects our lack of appropriate tion of isolated enzymes presents notable advantages that they are understandings regarding enzyme functions. easier to remove, resist harsher conditions, eliminate diffusional restrictions and are easier to ship, around the globe. Ketoreductases A milestone in the progress of directed evolution was the rapid (KRED)-based catalytic bioprocesses have entirely substituted the development of a protein by DNA shuffling report published by whole-cell reductions and metal–ligand-assisted chemo catalysis, Stemmer [61]. In this development technique, a set of differ- over the past few years [39,62]. ent 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

898 M. Bilal et al. / International Journal of Biological Macromolecules 108 (2018) 893–901 Fig. 5. Directed evolution approach applied to modify the phosphoryl donor specificity of the DHAK from C. freundii. (Reproduced from [53], an open access article distributed under the Creative Commons Attribution License) KREDs and many other enzymes have been extensively stud- 7. Engineering the oxidative enzymes ied for producing chiral pharmaceutical intermediates such as atorvastatin – a cholesterol-lowering drug with global sales of Rational design and directed molecular evolution, as well as US$11,900,000.000 in 2010. In parallel to highly efficient (bio) cat- combinations of both techniques, have been attempted for engi- alyst, a low-cost bioprocess also necessitates easily-available raw neering oxidoreductases focusing on the whole protein or any feedstock materials and facile isolation of purified target compound targeted domains [37]. All the protein engineering technologies in elevated yields. One contemporary biocatalytic process employs prerequisites the availability of a suitable expression system to three enzymatic steps: first, the combination of KRED and glu- produce variants with improved features. The heterologous expres- cose dehydrogenase; second, this combination with a halohydrin sion of oxidoreductase genes in Escherichia coli, Saccharomyces dehalogenase to make the ethyl (R)-4-cyano-3- hydroxybutyrate cerevisiae, or other systems was standardized for aryl-alcohol intermediate; and, third, the enzyme-assisted reduction for the oxidase (AAO), dye-decolorizing peroxidase (DyP), ligninolytic per- manufacturing of advanced diol intermediate [36]. oxidases, unspecific peroxygenases (UPO), and vanillyl-alcohol oxidase (VAO) enabling their subsequent engineering [25,67,28]. In Recently, it has been reported that biocatalysis engineering many cases, a considerable increase in the gene expression of oxi- broadened the substrate range of transaminases to ketones with doreductase was obtained after many cycles of directed molecular two bulky substituents [55]. Notably, the catalytic re-engineering evolution, which were followed by additional rounds to increase started with a smaller ketone substrate, generated more space in the desired catalytic traits further. the catalytic site and used progressively larger ketones. Several rounds of directed evolution successfully constructed an engi- Notably, versatile peroxidase (VP) has been used as a model per- neered amine transaminase with 40,000-folds amplified catalytic oxidase in systematic engineering studies, and oxidative, as well as activity. The engineered catalyst can substitute the transition- alkaline inactivation, has been examined to get variants with bet- metal-based hydrogenation catalyst for the manufacturing of ter industrial applicability. In this context, two different strategies sitagliptin [14]. were merged to enhance the oxidative stability of VP towards H2O2 [51]. A different strategy based on i) selection of a naturally-stable The enzyme mutants obtained from optimization studies are peroxidase by genome screening and heterologous expression; ii) an inimitable source of initiating points for future programs. For identification of the structural determinants for this stability, such example, tailoring KREDs to make (R)-3-hydroxy-thiolane (R3HT) as H-bonding patterns, salt bridges and basic residues and iii) intro- led to several enzyme variants with excellent stability including ducing them into the target enzyme by directed mutagenesis, was some which were incompatible due to insignificant enantiose- effectively employed for rational improvement of alkaline stabil- lectivity. Nevertheless, one of these unsuitable variants was the ity [52]. Rational design engineerings have also been applied to preliminary enzyme in engineering a KRED for (S)-1-(2,6-dichloro- develop a ligninolytic VP with potential capability to function par- 3-fluorophenyl)-ethanol (DCFPE) [34]. Likewise, the transaminases ticularly at highly acidic pH (pH 3), which in turn increases the obtained during the pro sitagliptin evolution can give rise many redox potential of the heme iron [23]. In another study, Linde other amines and might function as starting points for several dif- et al.[35] reported that rational design of the active site of DyP ferent engineered biocatalysts for amine biosynthesis [19]. resulted in efficient stereoselective sulfoxidation reactions.

M. Bilal et al. / International Journal of Biological Macromolecules 108 (2018) 893–901 899 In parallel to rational designing stated-above, molecular evo- The directed evolution of the halohydrin dehalogenase substituted lution and combinatorial approaches have also been envisioned around 35 of the 254 amino acids for the manufacturing of atorvas- to improve the H2O2 and alkaline steadiness of VP. In case of tatin [24]. directed evolution, the best variant harbored eight mutations resulting in improvement of VP half-life from 3 to 35 min [26]. A second state-of-the protein engineering approach explored González-Pérez et al. (2016) achieved a VP variant that efficiently in the last decade is the creation of new, so-called non-natural, oxidizes substrates at alkaline pH both the Mn2+ site and the heme catalytic activities. The base for these new engineered activities channel, whereas the catalytic amino acid tryptophan was not is usually a catalytically promiscuous reaction. Promiscuity is the functioning under these conditions. Likewise, UPO has been tai- capability of one active site to catalyzing more than one reac- lored by directed evolution to magnify its mono(per)oxygenase tion type, which may be additional side reactions in combination activity and to reduce competing one-electron oxidation. The with one normal reaction. Interestingly, this new reaction type is resulting evolved variants were employed in oxy functionalizations not merely a substituent addition to the existing substrate, but of biotechnological importance, such as the naphthalene oxygena- implicates different transition states and thereby generates vary- tion to 1-naphthol. In the past decade, the rational and evolutionary ing types of chemical linkages. The pyruvate decarboxylase (PDC) design has also been applied for laccase engineering. Nevertheless, natively catalyzes pyruvate to acetaldehyde and CO2, but, a promis- directed evolution of the whole genome and mutagenesis-based cuity activity of pyruvate decarboxylase led to the coupling of this evolution have better upgraded the ligninolytic laccase character- acetaldehyde to another aldehyde in an acyloin condensation. Such istics for targeted functions, such as the oxidation degradation of an un-natural PDC-driven acetaldehyde condensation with ben- phenolic compounds [66]. zaldehyde was the initiating point to manufacture a precursor of the drug Ephedrine [38]. In contrast to the normal reaction, the promis- 8. New trends in enzyme engineering cuous reaction does not require a proton transfer, and substitution of a single amino-acid eliminating proton donor deactivated the Intensive modifications in enzyme catalytic properties usually natural enzyme activity but enhanced the fivefold promiscuity involve multiple amino-acid replacements because they largely activity. alter the protein structure. Nonetheless, concurrent substitutions of several amino-acid can generate exponentially more vari- The restricting unsolicited or competing for metabolic pathways ants/mutants. Most of these variants are inactive and require to shift divert the flux towards the target product entirely is further testing to find the improved variants followed by incremental developed by the third wave advance so-called metabolic pathway improvements by several rounds of directed evolution. Efficient engineering. This engineering enables transferring of secondary screening of the variants is the simplest solution to overcome metabolism-related more complex pathways into new organisms this issue. Any notable changes in substrate specificity might be and creating entirely new biochemical pathways to manufacture scrutinized by high-throughput methods, such as fluorescence- industrially pertinent pharmaceutical compounds, fine chemicals, activated cell sorting, which can screen millions of variants in a and biofuels. Using these techniques, the normal metabolisms of short time [4,22]. Whittle and Shanklin [70] carried out six simulta- amino acids, fatty acids and terpenes have been re-engineered to neous amino-acid replacements in the catalytic site of a desaturase make alcohols, hydrocarbons, and polyesters for use as fuels, bulk enzyme and found that only the variants with modified substrate chemicals, and plastics. specificities were able to grow. 9. Ongoing challenges in biocatalysis engineering Currently, the best technique to generating multiple-site muta- tions is to add them concurrently but to limit the choices Notwithstanding the significant advances, several major chal- using bioinformatics or statistical approaches. Codexis researchers lenges remain to harness the entire advantages of biocatalysis. Now used one statistical correlation approach namely ProSAR (protein a day, enzyme engineering is much faster as ten years ago, but sub- structure-activity relationship) algorithm to improve the 4000- stituting amino acids and screening lots of variants entails a large fold reaction rate of a halohydrin dehalogenase [24]. Several research group. Many protein engineering approaches will gen- other scientists conducted random amino-acid replacements in the erate improved candidates, but some will produce variants with dehalogenase and monitored the catalytic rate by the variants. Sta- better attributes, however, which ones are the superior strategies tistical approaches identified that the variants containing a Phe is still uncertain. Directly comparing different strategies and test- 186 Tyr substitution were found to be the better than those with ing the assumptions behind these strategies would help to identify no substitutions. Though some mutants with such a substitution the most efficient variants [14]. were not beneficial possibly due to the detrimental effects of other mutations, on average, the statistical analysis concluded that Phe The first assumption is that protein engineering can fulfill the 186 Tyr is an advantageous mutation. The ultimately improved objective of biocatalysis. The reactions occurring with unnatural enzyme variant consists of 35 amino-acid substitutions among it’s substrates may be thermodynamically less favorable than that 254 amino acids [14]. of reactions involving natural substrates, and achieving certain enzyme activities might be impossible. In this context, a closer inte- Jochens and Bornscheuer introduced a novel approach, in which gration of biocatalytic process and thermodynamics is extremely the changes are restricted to the catalytic site, to improve the desirable in developing new methods. Secondly, protein engi- enantioselectivity of an esterase enzyme from Pseudomonas flu- neering often relies on good knowledge regarding the quaternary orescence. There were 160,000 ways simultaneously to change the structure of the enzyme, and our understanding of protein dynam- four amino acids adjoining to the substrate in the active site. The ics is still very narrow that makes predictions challenging. Third, amino-acid sequences of 2800 related enzymes were aligned by although most of the mutations are non-interactive, many coopera- the researchers to identify most common amino acids at these tive mutations are highly beneficial but hard to study. One possible positions. This analysis restricted the possibilities to several hun- way for the identification of cooperative effects includes statisti- dred variants, which were then investigated to discover double and cal analysis using the ProSAR algorithm [68], but more up-to-date triple mutants with the desired enantioselectivities [32]. techniques are required to predict at an early stage of protein engineering efficiently. Fourth, computer-based designing of new The magnitude of beneficial variations occurred during the evo- enzyme activities is not appropriate and usually creates an enzyme lution proteins have increased considerably over the past decade. with low catalytic activity, requiring further substantial engineer-

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