Protein Engineering Strategies to Improve Efficiency ...

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CHAPTER 7

Protein Engineering Strategies to Improve Efficiency in Biomass Degradation Lucas F. Ribeiro1,*, Tina Xiong1, Pricila Hauk2,3 and Liliane F.C. Ribeiro4 1

Johns Hopkins University, Chemical and Biomolecular Engineering, Baltimore, USA; 2Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, USA; 3Fischell Department of Bioengineering, University of Maryland, College Park, USA and 4Department of Chemical, Biochemical and Environmental Engineering, University of Maryland, Baltimore County, Baltimore, USA Abstract: Protein engineering has become the most important tool for enabling industrial application of biocatalysts. Advances in structure-guided methods and novel techniques for directed evolution and high-throughput screening have facilitated the design of enzymes with improved properties such as functional expression, stability, and catalytic activity. The utilization of lignocellulosic biomass for biofuels production is attractive because it is environmentally sustainable. However, commercialization of biomass biofuels requires efficient bioconversion of cellulosic material to sugar, which is largely hampered by biomass recalcitrance. In this chapter, we present the recent application of protein engineering techniques for maximizing the efficiencies of biocatalysts for biofuel production.

Keywords: Protein engineering; enzyme adaptation; semi-rational design; library construction, site-directed mutagenesis; screening; biomass. UNDERSTANDING PROTEIN ENGINEERING Protein engineering is a multidisciplinary research field that aims to improve or adapt proteins to be perfectly suited for biotechnology applications. About 30 years ago, the early studies, sought to understand the relationship between the structure and function of proteins through the construction of proteins modified by site-directed mutagenesis [1]. Knowledge of protein folding from experimental and in silico methods, along with advances in gene manipulation techniques and the design and screening of protein libraries have aided in the development and identification of novel proteins with enhanced properties [2]. From the standpoint of biotech pharmaceutical companies, a biocatalytic process can be modified to be *Corresponding author Lucas F. Ribeiro: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore-MD, USA; Tel/Fax: +1 (410) 516-4146; E-mail: [email protected] Roberto Nascimento Silva (Ed) All rights reserved-© 2015 Bentham Science Publishers

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applicable for new commercial applications. Many different approaches exist for enzymatic development, though they are generally represented as shown in Fig. 1, where the property that will be modified in target protein is defined initially. The prior biochemical and/or structural knowledge of the parental protein (step 1) serves as a starting point to define which property will be improved to obtain a better adaptation within the process in which it will be applied. Next, genetic diversity must be generated (step 2) to obtain different "variants". Genetic diversity can be achieved through mutations, deletions, recombinations, duplications, and etc. [2], which can be performed in a rational or random manner, depending on the depth of knowledge of the structure/function relationship of the target protein. After this step, the different variants will be screened or selected for the desired property (step 3). In a screening the intention is to identify those organisms that display the desired phenotype, e.g. different activity in high temperature. In selection the goal is to find those organisms that grow under a specific set of conditions, in which organism carrying the parental protein do not grow, e.g. presence or absence of antibiotic resistance. Posteriorly, the best variant will be chosen for further characterization (step 1). Frequently, several rounds of modifications, screening/selection and characterization (steps 1-3) must be carried out before incorporating the protein into a biotechnological process (step 4).

Figure 1: Protein engineering cycle.

MAIN APPROACHES IN PROTEIN ENGINEERING Several protein engineering experiments involve a molecular analysis of a given structure in order to infer a particular function. These methods offer insight into the

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protein structure and crucial interactions that may facilitate the understanding of enzymatic mechanism for specific modification of key amino acid residues via site directed mutagenesis [3]. This method, also known as rational design, requires information pertaining to the structure of the protein or a homologous protein, sometimes in complex with its ligand. Homologous enzymes that bind to the same substrate tend to have conserved residues in the substrate binding site. Sequence alignment of those structures can be used to identify important residues that facilitate substrate binding in a substrate binding site Selected regions for changes usually are near the active site, flexible sites, sites identified by structure-based modeling and sites identified by sequence comparisons [4]. Computational tools such as molecular modeling and Statistical Coupling Analysis (SCA) programs have guided protein design [5-10]. The most classical rational design method is site-directed mutagenesis; this method is adopted to replace, add or even delete specific amino acids in the protein sequence. There are two different ways to make site-directed mutagenesis, Overlap extension and Whole plasmid single round Polymerase Chain Reaction (PCR). The details of these techniques can be seen in Figs. 2 and 3 below:

Figure 2: Overlapping PCR technique scheme. PCR#1, two PCRs are performed using the target gene and a combination of primers, non-mutated (dark arrow) and mismatched (star arrow) to generate fragments to be overlapped in PCR#2. For the A fragment, the PCR is performed using non-mutated primer forward and mismatched primer reverse. The B fragment is amplified using the mismatched primer forward and non-mutated primer reverse. The fragments (A and B) containing the complementary mutated region (star) are used as template for PCR#2. PCR#2 is performed using the same non-mutated pair primers that were used in PCR#1. In PCR#2 the complementary mutated regions created in PCR#1 (A and B fragment) overlap so that the highfidelity polymerase amplifies the entire target gene containing the desired mutation.

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Figure 3: Schematic whole plasmid single-round technique. (A) Gene target (light gray) cloned into the plasmid. For this PCR, an identical mismatched primer pair (star arrow) is used to amplify the entire plasmid harboring the gene to be mutated. (B) The PCR product must be treated with the DpnI restriction enzyme to digest possible parental plasmid contamination (template). (C) After DpnI treatment, the PCR is transformed into E. coli to repair the nicks, to be ligated and replicated.

While rational design can be a powerful technique for the development of industrialized proteins, knowledge of structures is often unavailable. New techniques have been developed for high throughput selection of proteins with characteristics of interests. Starting from a library of genetically modified proteins, subsequent selection or screening of proteins for properties that are critical for biotechnological applications could identify “hits” or proteins with desired characteristics. This strategy named directed evolution has developed quickly in the last decade, allowing us to change the amino acid sequences of macromolecules and modify enzymes with "evolved" properties [11, 12]. There are three basic requirements for the success of directed evolution: 1.

The property to be improved must be identifiable through in vitro or in vivo assays.

2.

A library with genetic diversity must be created.

3.

The screening must be efficient to identify variants with desired property from multifarious candidates without this property.

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Genetic diversity can be achieved through recombinant or non-recombinant DNA technology, UV and chemical exposures. Of the methods, error-prone of the polymerase chain reaction (epPCR) is the most commonly used method for random mutagenesis [13]. The most important parameter in epPCR experiments is the random distribution of mutations. This can be estimated based on the binomial distribution. For a sequence of length “n” which is mutated with an error rate ε, the probability of the introduction of k mutations is given by: , ,



! –

! !

1–

–

(1)

Besides and beyond the mutagenic method, the recombination of genes can increase library diversity and potentially combine phenotypes from homologous or nonhomologous parental genes. Well-known techniques for randomly altering DNA sequences also include DNA shuffling, Biased Clique DNA shuffling, and Staggered extension process (StEP) [14-17] (Table 1). A large number of techniques for generating genetic diversity in directed evolution have been developed [18], however, comparing these techniques is beyond the scope of this chapter. Table 1. Homologous recombination methods.

Classical DNA shuffling Biased Clique DNA shuffling Staggered extension process

Method for creating DNA fragments for crossover DNAse DNAse PCR (short extensions)

Application Equimolar amounts of parental genes; Better for generating smaller fragments for crossover. More of one parental gene is used; Better for generating smaller fragments for crossover; Library favors one gene. One sample process; Better for generating larger fragments for crossover.

A third approach shown here is known as semi-rational design. It is a combination between directed evolution and random design in which “hotspot” residues are selected using structural and/or functional data, and they are used as starting points for random mutagenesis. A semi-rational approach may be advantageous and an intelligently designed library can be constructed with higher functional content than a random library [19]. BIOFUELS AND PROTEIN ENGINEERING The main impediment of commercializing cellulosic biofuel is biomass recalcitrance [20]. Alcohol production from cellulosic material requires the degradation of lignin, cellulose, and hemicellulose. Crystalline cellulose

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microfibrils are susceptible to enzymatic degradation via hydrolysis. However, the microfibrils exist within a hemicellulose-lignin matrix that makes the cellulose inaccessible to water. Pretreatment of biomass helps disrupt the cellulosic matrix. Extreme physical conditions have been implemented, including steam treatment and chemical conditions such as alkaline or acid environment. However, the pretreatment process and maintenance is costly, slow, and must be paired with enzymatic reactions to efficiently produce simple sugars. Pretreatment conditions are highly incompatible with enzymes that are adapted to physiological conditions and usually milder environments. Industrial application of proteins outside of physiological conditions, such as extremely high temperature or low pH, would require large scale production of proteins with adapted features. Thus, these proteins need to be engineered to improve expression and functions based on rational, random or semi-rational approaches. Therefore, efficiency in biofuel production is closely related to advances in protein engineering. In the following paragraphs, we will present several examples of protein engineering applications that sought to increase the adaptability of these proteins in order to get the desired biotechnological effect. STRATEGIES TO IMPROVE FUNCTIONAL EXPRESSION Hundreds of microbial proteins are currently produced at commercial scale [2123]. However, the low production and secretion of many recombinant proteins remains as a bottleneck in many projects that study different enzymatic activities, their structure, and the application of directed evolution to improve or even to achieve a certain enzymatic characteristic [24-27]. Some bacterial strains are protease knockouts, which may induce higher protein accumulation. Thus, the choice of the expression host is vital. Moreover, eukaryotic proteins are not always successfully expressed in prokaryotic cells (e.g. inactive insoluble proteins or low expression). For biofuel applications, many fungal proteins are preferably expressed in yeasts or filamentous fungi, such as Saccharomyces cerevisiae and Aspergillus nidulans, respectively [28-30]. The increase in the functional expression of enzymes with many different applications would benefit with higher production efficiencies. Random mutagenesis and a good screening method have been shown to improve the expression and secretion of many different enzymes. Directed evolution techniques have been applied to increase the functional expression of proteins that may be used for biomass degradation. Laccase is one of the enzymes responsible for the delignification of biomass, and the bottleneck for the directed evolution of this protein is poor functional expression in the eukaryotic host,

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Saccharomyces cerevisiae [29]. In order to overcome this issue, the native signal sequence of laccase from P. cinnabarius (PcL) was replaced by the preproleader of the α-factor mating pheromone from S. cerevisiae and the whole construct (α-PcL) was subjected to six rounds of evolution: random mutagenesis and/or DNA recombination and a semi-rational approach called in vivo Overlap Extension (IVOE) [29]. In this case, five mutations in the preproleader sequence increased the secretion level of laccase 40-fold. However, a total of 14 mutations in the entire sequence enhanced the secretion by 584-fold [29]. In this method, mutagenized primers are used that amplify two separated fragments that overlap in the sequence region. After the two separate PCR amplifications, they are mixed together and another PCR reaction will setup the constructed gene containing the mutations (Fig. 4A) [31, 32]. This technique is often used in association with many rounds of error prone PCR and DNA shuffling in order to increase the variability. Another laccase, from Myceliophthora thermophile, was also evolved and expressed in S. cerevisiae and its secretion was even higher than the corresponding enzyme from P. cinnabarinus. After ten generations of error prone PCR, in vivo recombinations and a Staggered Extension Process (StEP), the expression of the most active laccase was about 8 times higher than the wild type [28]. StEP is also a recombination that uses full-length templates in very short time PCR reactions in sub-optimal extension conditions. StEP generates small fragments that will randomly anneal to different regions of the template sequence generating recombinant products (Fig. 4B) [16]. This technique was also used to increase the expression of Fusarium galactose oxidase in E. coli 18-fold. Authors speculated that the increased expression was probably due to a mutation in the Nterminal region of the protein that influenced gene transcription [33]. Another example of the use of StEP is for a versatile peroxidase (VP) from Pleurotus eryngii [34]. This enzyme is characterized by its promiscuous activity and attempts have been made to express it in different hosts, however, due to its structural complexity, its proper folding and functional expression at desired levels are affected. Even when Aspergillus nidulans was used as a host the expression level was as low as the homologous expression of VP in Pleurotus eryngii [35]. The attempt to express the protein in Escherichia coli was also not very successful. Inclusion bodies formed or, when soluble, the enzyme sometimes presented atypical properties related to improper folding [36]. For this case, the αfactor preproleader from S. cerevisiae was also used and, after four rounds of evolution (three error prone PCR, three in vivo shuffling and a StEP), eight

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mutations were introduced in the preproleader that led to a 51.6-fold improvement in functional expression [34].

Figure 4: Different methods used to improve protein secretion. A. IVOE – Amplification of the gene in two independent PCR reactions using mutagenized/degenerate primers. PCR products are recombined in vivo when cloned into S. cerevisiae with the linearized plasmid in a single transformation; B. StEP - Several parental genes are used as templates. This method generates random mutations during short annealing and extension cycles. The products are mixed with the linear plasmid and in vivo recombination occurs upon cloning into S. cerevisiae in a single transformation; C. MORPHING – Some segments of the gene are subjected to random mutagenesis (M1 to M3) and some are amplified with high fidelity (HF1 to HF4). After the PCRs, all of the segments are mixed with the linearized vector and it is used to transform S. cerevisiae. Each segment was amplified with an overlapping area of 50 bp flanking, which allow specific recombination events to occur between the segments. Stars represent single mutations.

A new method was developed and used to enhance the functional expression of an unspecific peroxygenase (UPO) in S. cerevisiae [37]. UPO is a new peroxidase that can be ligninolytic and is not readily expressed in heterologous hosts. In order to increase the expression and secretion of this enzyme, MORPHING (Mutagenic Organized Recombination Process by Homologous In vivo) was used to evolve

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the signal peptide. This method is a random domain mutagenesis/recombination for short pieces, introducing mutations in specific protein fragments by overlapping areas to favor in vivo recombination in yeast (Fig. 4C). The use of this method led to several independent mutations in the leader sequence. Each of these mutations enhanced secretion by about 20% compared to the parental type. Although it was possible to verify these beneficial mutations, S. cerevisiae machinery was unable to homologously recombine them. Therefore, a signal peptide containing the full set of these mutations was constructed and resulted in a 27-fold increase in the total secretion compared to the original UPO leader [37]. Different methods of directed evolution have been used to improve the production and secretion of fungal enzymes. An important sequence that has been a focus of these methods and showed great results is the α-factor preproleader from S. cerevisiae. These findings suggest that efforts to evolve it may yield a universal signal peptide for the heterologous expression of foreign proteins in yeasts [38]. STRATEGIES TO IMPROVE PROTEIN STABILITY The feasibility of an enzymatic process depends greatly on the stability of the protein. Protein stability determines the longevity of the application and the efficiency of the activity. Protein function is dependent on protein structure. Proteins are folded based on unique amino acid sequences and their conformations are driven mainly by the hydrophobic effect [39, 40]. The protein shape is defined by van der Waals interactions, hydrogen bonds, salt bridges, disulfide bonds, and all these interactions and bonds can suffer alterations due to changes in pH, temperature or solvent polarity. [41]. The following examples highlight some of the designs and strategies applied in cellulases, xylanases and laccases to improve their tolerance to operating conditions of saccharification, e.g., neutral pH levels and thermal tolerance to increase biomass degradation efficiency. pH Adaptation Changes in H+ or OH- concentrations can result in protein destabilization or denaturation. This is due of the intrinsic charge and pKas of the amino acids that give the corresponding protein a net charge. Backbone interactions, side chainside chain, and side chain- backbone interactions govern proper formation of the secondary, tertiary, and quaternary structure [42].

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Cellulases The high abundance of Endo-β-1,4-glucanases II (Cel5A) and III (Cel12A) make them great candidates for lignocellulosic degradation, but it operates optimally at a pH of 4.0 and 5.0, whereas saccharification processes require a neutral pH [43]. β-glucosidases with increased stability and activity have been explored, to increase the overall rate of sugar conversion [44]. Three mutagenesis methods were employed to increase tolerance of neutral pH levels of Cel5A from T. reesei [45]. Two libraries were created by introducing mutations with error-prone PCR (epPCR) at high and low mutation frequencies. About 60,000 candidates were screened from the low mutation frequency ep-PCR library and 80,000 from the high mutation frequency epPCR. From the low mutation library, variants were found with better than wild type activity at desirable pH levels. A third was made by saturation mutagenesis (by Strand Overlap Extension (SOE)) of asparagine at amino acid 342, because the group previously found that this residue affects tolerance of neutral pH [45, 46]. Variants with optimal activity at more neutral pH levels were subjected to two rounds of DNA shuffling. Three variants displayed 4.5-fold higher activity than the wild-type enzyme at pH 7.0 [45]. In addition to residue 342, mutagenesis studies have found that residue 321 can also increase the tolerance of neutral pH for Cel12A from T. reesei [46]. Wildtype endoglucanase is about 10% as active at pH 7 as its maximal activity at pH 4.8. A library of 200,000 transformants was screened for CarboxymethycelluloseNa (CMC) degradation using a colorimetric Congo Red Assay. Random mutagenesis revealed that a threonine mutation from asparagine at amino acid 321, N321T, was found to increase the optimal pH by 0.6 units [46]. Subsequent substitutions with aspartic acid and histidine at N321 confirmed that the position greatly affected the enzyme activity profile at different pH levels. At pH 7.0, N321T had at least 40% of its maximal activity. A non-conservative mutation to aspartic acid, a negatively charged residue, decreased the optimal pH to 4.0. The activities of the variants at their optimal pH levels are comparable to the wild type at its own pH optimum. Replacement with histidine, a positively charged residue, broadened the optimum pH range for Cel12A from 4.6 to 5.5. At pH 6.5, N321H retained 84% of its activity while wild type had 40% residual activity. Tips for Codon Optimization In the SOE method mentioned in the above example, two separate PCR reactions amplify two fragments that contain an overlapping sequence [47]. Each primer

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pair is synthesized with a random nucleotide sequence (such as-NNN-) encoding one of the 20 amino acids, flanked on both sides by nucleotides that specifically anneal to the target regions. However, synthesis of the 64 possible codons (NNN) for all 20 amino acids may lead to codon bias, since the genetic code is degenerate and there are, for example, six times more codons for Ser than for Trp or Met. Thus, codon optimization allows for small and more focused libraries, as showed in Table 2. Table 2. Analysis of codon usage for saturation mutagenesis. No. of codons

No. of amino acids

No. of stops

Amino acids encoded

Library sizea For 2 positions

Library sizea For 3 positions

NNN

64

20

3

20

995

25585

NNK

32

20

1

20

875

21051

DBK

18

12

0

ARCGILMFSTWV

279

3812

NDT

12

12

0

RNDCGHILFSYV

215

2587

NRT

8

8

0

RNDCGHSY

95

766

Codon type

a

To ensure a 0.95 probability of discovering at least one of the top two variants. The library sizes were calculated using an online tool [48] that can be found at the following website: http://stat.haifa.ac.il/~yuval/toplib/

Xylanases Xylanase with better pH adaptation have also been sought. Site-directed mutagenesis of Scytalidium acidophilum endo-xylanase XYL1p was performed at residues that were important to the acidophilic family of xylanases. Three conserved residues near the catalytic site (Y35W, D60N and E141A), were found among the 11 xylanases. [49]. All mutants increased the pH optimum by 0.5-1.5 pH units. In this study Al Balaa and colleagues (2009) found that the E141A mutation improved the specific activity with a small decrease in its pH-dependent thermal stability (the apparent melting temperature (Tm) was decreased by 2 ̊ C). The single substitution of E141 by Ala resulted in a shift of the pH by 0.8 units with a significant increase in specific activity of 50% [49]. Laccases Laccases are polyphenol oxidases and, in addition to lignocellulosic substrates, several xenobiotics families containing phenolic or aromatic amino groups are transformed by laccases. In order to improve xenobiotic degradation, a laccase from the fungus Trametes versicolor was modified. Mazdak and colleagues (2006) previously found the crystallized enzyme bonded xenobiotic 2,5-xylidine, which was used as a laccase inducer in the fungus culture. Structural analysis

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showed a strong interaction between the acidic Asp 206, present at the T1 active site of the enzyme, and the 2,5-xylidine [50, 51]. A single residue change Asp206Asn increased the optimal pH activity of the laccase (1.4 pH units) and improved its catalytic activity towards xenobiotics, substrates that are not normally oxidized by laccase [52]. Higher affinities for phenolic substrates, such as phenylurea derivatives, were observed with increasing pH [52]. To study the effect of protonation of the laccase, phenolic substrates were used. Since the protonation state of phenolic derivatives remains unchanged in this pH range, the degree of interaction between the substrate and the enzyme depends on the protonation state of substrateinteracting residue Asp206, more so than at others. Side chains interacting with the substrate, suggesting the involvement of an acidic amino acid, possibly Asp206 [52]. In this site-directed mutagenesis assay, the authors chose to replace Asp206 with Glu, Asn and Ala, based on multiple alignments with fungus and plant laccases in the vicinity of position 206. At site 206, glutamate is usually found among ascomycetes, whereas aspartate is found among basidiomycetes. Interestingly, Asp206 is conserved among plant laccases. Alanine is also a nonpolar amino acid and, for this reason, it was selected for replacement [52]. Thermal Stability Glycosyl hydrolase (GH) libraries from Biased Clique DNA shuffling (BCS) and family DNA shuffling were used to create thermostable Talaromyces emersonii Cel7 (Te Cel7A) [53]. Half of the parental DNA in BCS comprised of Te Cel7A gene, and the other half was split evenly among ten other homologous proteins. The eleven GH genes shared 68-85% DNA sequence similarity, allowing DNA crossover between genes and sufficient differences for library diversity. Only the catalytic domain was shuffled. Sequence analysis of six random sequences showed that diversity was greater when equimolar ratios of each gene were used. 70-110 mutations from the closest parent were found, whereas the variant with the greatest mutation rate in the BCS library was 29 mutations away from Te Cel7A. Despite the lower diversity in BCS library, there were more chimeras that were active (86%) and thermostable (11%). The presence of only one active chimera at 37 ˚C in the equimolar- parental gene library suggests that the chimeras diverged too far from the parental genes thereby losing functional similarity [53]. Enzyme stability was determined based on substrate hydrolysis. The breakdown of Avicel implied that enzymes were still active and thus stable. From the BCS library, variants with higher activitities at higher temperatures, 65 ˚C and 70 ˚C were found. Three variants (1G21, 2I13, and 2E10) had 2- to 4-fold higher activity at 65 ˚C than wild – type

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Cel7A. After one-day incubation at 65 ˚C, 1G21 and 2l13 had at least 20% residual activity, whereas Te Cel7A activity could not be measured [53]. Thermostable exocellulases Cel6A and Cel7A chimeras were developed using StEP in vitro recombination and SCHEMA. The approach increases the fraction of active variants in the library. SCHEMA identifies sequences of contiguous amino acid “blocks” that reduce the number of broken side chain interactions during shuffling based on sequence and structural information of the parental genes. The total number of blocks is the number of blocks x number of variants [54]. The effect on stability of Cel7A was screened with each block from each parental gene. Sixteen variants with different combinations of gene blocks were predicted to have increased stability relative to thermophilic Talaromyces emersonii [55]. All of the mutants were found to have higher thermal stability. Screens via hydrolysis of solid cellulose revealed stable Cel7A chimeras were active at 70 ˚C, whereas wild-type displayed no activity beyond 65 ˚C [54]. SCHEMA was also used to engineer a more stable Cel6A than its parents. Twenty-three of 48 chimeric sequences sampled in yeast were active, and five had greater stability than the most stable parent at 63 ˚C [55]. STRATEGIES TO IMPROVE ENZYMATIC ACTIVITY When proteins have low activity or undesirable substrate specificity or selectivity, amino acids close to the active site or interacting residues are the prime targets for mutations rather than surface residues[56]. These sites can be identified from three-dimensional structures, from homology modeling, or from directed evolution experiments. Using Computer Modeling to Improve Catalytic Efficiency The cellulase activity on the endo/exocellulase Cel9A (formerly called E4) from Thermobifida fusca was 40% improved through the integration of computer modeling created using the structure-based program CHARMM and site directedmutagenesis [9, 57]. In order to improve crystalline cellulose degradation by Cel9A, Escobar-Kousen and colleagues[9] modeled Cel9A using CHARMM and the PDB high-resolution structure of Cel9 to determine conserved residues that were essential for cellohexose binding in the cellulose binding domain [58]. The model showed that the cellohexose molecule was attached to the enzyme catalytic site, revealing interactions between the cellulose chain and the conserved CBM residues (F476, D513, R563, Y520, Q561) [9, 59]. Molecular graphics visualization programs such as QUANTA and energy minimization were used

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initially to extend the cellulose chain to 18 glucose residues (to mimic cellulose) spanning the catalytic domain (CD) and cellulose-binding module (CBM) [9]. The cellulose chain model (initially a cellohexose) was created using parameters specifically developed for carbohydrates [9]. The lengths of chemical bonds to hydrogen atoms were kept fixed using the constraint algorithm SHAKE to perform all calculations. The computational results indicated that conserved CBM residues (F476, D513, R563, Y520, Q561) [59] are well aligned and interact with the cellulose chain, with most of them forming hydrogen bonds or other interactions. Site-directed mutagenesis assays showed that the F476Y mutation improved binding, thereby increasing the activity of the mutated enzyme. The authors hypothesized that this improvement was achieved through the hydrogen bond formation of Y476 and the cellulose chain. According to the model from Kousen et al. the conserved aromatic residue F476 did not form a hydrogen bond with the cellodextrin chain. The authors hypothesized that the hydrogen bond between Y476 and cellulose further stabilized the binding interaction of the complex. Altogether, the experimental results corroborated with previous computational studies conducted on this residue [9]. Inhibition For the endoglucanase Cel5A from Acidothermicus cellulolyticus, with a published high-resolution structure [57], a single mutation Y245G was shown to increase the inhibition constant (KI) of cellobiose by 15-fold. Baker and colleagues [60] performed the point mutation based on the detailed description of substrate-enzyme interactions in the active site of Cel5A [57]. The authors performed a careful structural examination of crystal wild-type enzyme/substrate complex, which provided insights into the enzyme mechanism about Tyr245 acting as a key residue interacting with a leaving group. Furthermore, Barker and colleagues [60] were following the catalytic theory proposed by them, that end product inhibition could be relieved by a substitution of a non-aromatic residue at site 245, a mutant Y245G. This thesis was based on previous knowledge that the enzyme interacts with all four residues of cellotetraose by both hydrophobic contacts and hydrogen bonding. Moreover, it is known that polysaccharidebinding enzymes the hydrophobic face of each glucose unit interacts with an aromatic side chain at the active-site cleft [58, 61]. Additionally, Sakon and colleagues [57] had already demonstrated that Glc1, Glc2, Glc3, and Glc4 interact with Tyr245, Trp213, Trp319, and Phe29, respectively. For this reason, Baker and colleagues [60] proposed that the leaving group of the glycosylation half-reaction could bind to platform residues Tyr245 and Trp213.

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The designed mutation to replace Tyr245 with Gly245 was determined on theoretical binding-energy calculations using high-resolution X-ray crystallographic structures of Cel5A which indicated that a specific mutation (Tyr245 to Gly245) should reduce the affinity of the enzyme active site for the product, cellobiose. The Y245G mutation was able to increase the rate of release of soluble sugars from biomass cellulose by as much as 40% relative to the wildtype enzyme [60]. Tuning Substrate Specificity Small and intelligent libraries can be constructed with the same representation of a large and random library through a semi-rational approach [19, 62]. Thus the identification of variants with altered substrate specificity can circumvent large libraries. A combinatorial library with mutations focused on 12 residues directly responsible for binding β-glucans and xylans of a carbohydrate binding module (CBM) from the Rhodothermus marinus was created. Conservative mutations, with similar physical and chemical properties as the original site were implemented to avoid changes that destabilize the structure. The variants were screened using phage display. Variants that lost the ability to bind to xylan and gained the ability to bind other carbohydrates (Avicel and ivory nut mannan) and other drastically different ligand groups such as the protein portion of a human glycoprotein (a monoclonal IgG4 antibody) were selected [63]. These results show that applying an evolutionary approach enables greater exploration of protein plasticity, allowing for the creation of new functions for the same protein. In another relevant work, Bastian and Colleagues (2011) [64] proposed that the limited 2-methylpropan-1-ol (isobutanol) production under anaerobic conditions could be caused by a cofactor imbalance in the metabolic pathway. In order to overcome this imbalance between the cofactors NADPH / NADH, a ketol-acid reductoisomerase (IlvC E. coli) was engineered to change the cofactor dependence from NADPH to NADH. Initially, the structure was analyzed and the residues responsible for binding the cofactor were identified. These residues were subjected to site-saturation mutagenesis and the beneficial mutations were recombined by SOE-PCR to generate NADH-dependent variants, leading to production of isobutanol near the theoretical maximum [64]. Chiral compounds, especially single enantiomers, have many applications in the production of pharmaceutical drugs and agricultural chemicals. To improve the

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enantioselectivity of an epoxide hydrolase from Aspergillus niger (Candidate lignin degradation), this enzyme was “evolved” through iterative saturation mutagenesis (ISM) [65]. This method identifies key residues for catalysis based on proximity to catalytic residues. Residues within a radius of about 10Å, were selected to create a diagram, which could be divided into five regions, defined as A, B, C, D and E, having 2 or 3 residues each. Saturation mutagenesis was then performed at site A, and the libraries were screened for the catalytic property of interest and the best hit sequenced. Subsequently, the gene corresponding to the best variant was utilized as a template to create a saturation mutagenesis library at site B, and the best clone selected was utilized as a template to construct the next library (site C), and so on. This process mimics the potential evolutionary pathways that adapt a protein’s activity based on sites that impact catalytic activity. After five cycles of ISM and screening of only 20,000 clones, a variant with 25-fold higher enantioselectivity than wild type was identified. Multifunctional Enzymes A chimeric enzyme can be a good alternative to maximize the enzymatic efficiency in an industrial catalysis process and to reduce enzyme production cost [8]. Additionally, when enzymes are fused to form chimeras, the physical proximity of the catalytic domains can improve performance, compared to the activities of the separate parental enzymes [7, 8]. There are a significant number of challenges to create a multifunctional chimera. Before starting the experimental procedures, it is important to conduct a careful protein design project where available domain compatibility (pH, temperature, etc.), and high–resolution three-dimensional structures, computer modeling, and multiple alignment sequences are essential tools to get the desire protein. Peptide linker fusion is useful to develop a multifunctional enzyme by fusion of other proteins that possess different functionalities. One challenge is determining the appropriate peptide linker sequence length and amino acid composition to make the chimera have the expected activity. For this reason, semi-rational protein engineering is largely used to create chimeras through protein library construction because, in this way, it is possible to increase the peptide linker sequence combinations. Xylanase/-glucosidase and xylanase/arabinofuranosidase/xylosidase are examples of multifunctional enzymes created by peptide linker fusion [66, 67]. In the case of the bifunctional enzyme, an optimized flexible peptide linker was used

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as a bridge to connect the -glucanase of Bacillus amyloiquefaciens and the xylanase of B. subtilis. The addition of a flexible peptide linker substantially improved the catalytic efficiencies of the -glucanase and xylanase moieties compared to an end to end fusion of the same proteins [66, 68]. For the trifunctional enzyme, a multifunctional xylan-degrading enzyme was created by the fusion of a 23-residue linker (GGGGGADPAIGPMYNQVVYQYPN) to the xylanase domain of Clostridium thermocellum and a dual functional arabinofuranosidase/xylosidase (DeAFc) [67]. The multifunctional hemicellulose was comparable to the wild-type enzymes in protein and enzymatic properties and more active than the wild-type enzyme mixture in hydrolysis of natural xylans, including arabinoxylans and corn stover [67]. CONCLUDING REMARKS Biocatalysts have been improved through rational design and directed evolution technologies that presents a promising way to adapt enzymes to various bioprocesses. Here we have briefly presented different techniques and concepts applied mostly to fungal enzymes to solve problems such as stability, functional expression, selectivity, inhibition, catalytic efficiency and other enzyme properties. Continuing development in protein engineering will lead to broader applications of biocatalysis in chemical and energy industries. CONFLICT OF INTEREST The authors confirm that this chapter contents have no conflict of interest. ACKNOWLEDGMENTS Our thanks to Dr. Nathan Nicholes for critical reading of the chapter and comments. REFERENCES [1] [2] [3] [4] [5]

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