Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several ...cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production.
The functionalization of polymer surfaces by polymer-binding peptides offers tremendous opportunities for directed immobilization of enzymes, bioactive peptides, and antigens. The application of ...polymer-binding peptides as adhesion promoters requires reliable and stable binding under process conditions. Molecular modes of interactions between material surfaces, peptides, and solvent are often not understood to an extent that enables (semi-) rational design of polymer-binding peptides, hindering the full exploitation of their potential. Knowledge-gaining directed evolution (KnowVolution) is an efficient protein engineering strategy that facilitates tailoring protein properties to application demands through a combination of directed evolution and computational guided protein design. A single round of KnowVolution was performed to gain molecular insights into liquid chromatography peak I peptide, 47 aa (LCI)-binding to polypropylene (PP) in the presence of the competing surfactant Triton X-100. KnowVolution yielded a total of 8 key positions (D19, S27, Y29, D31, G35, I40, E42, and D45), which govern PP-binding in the presence of Triton X-100. The recombination of two of the identified amino acid substitutions (Y29R and G35R; variant KR-2) yielded a 5.4 ± 0.5-fold stronger PP-binding peptide compared to LCI WT in the presence of Triton X-100 (1 mM). The LCI variant KR-2 shows a maximum binding capacity of 8.8 ± 0.1 pmol/cm² on PP in the presence of Triton X-100 (up to 1 mM). The KnowVolution approach enables the development of polymer-binding peptides, which efficiently coat and functionalize PP surfaces and withstand surfactant concentrations that are commonly used, such as in household detergents.
Cytochromes P450 have gained much interest for their broad substrate scope in the catalysis of oxidation reactions for pharmaceuticals, plastics, and hormones. However, achieving high coupling ...efficiency by the engineering of P450s is still a big challenge. The presence of extra water around the active site is deemed to be related to uncoupling. In this study, the access tunnels of P450 BM3 from Bacillus megaterium are engineered to control water access from bulk solvent to the active site. Nine residues located in tunnels are investigated by site‐saturation mutagenesis to reduce water diffusion, thereby improving the coupling efficiency. The recombined variant N319L/T411V/T436A shows improved coupling efficiency (from 31.2 % to 52.6 %). Tunnel polarity analysis and molecular dynamics simulation further indicate that reduced water molecules around the active site lead to higher coupling efficiency. Overall, this study provides valuable insight on improving coupling efficiency by controlling water diffusion through tunnel engineering.
Hydrophobic tunnels: Hydrophobic residues are introduced to the access tunnels of P450 BM3 from Bacillus megaterium, reducing extra water entering the active site. The access tunnels with increased hydrophobicity positively affect the catalytic activity and coupling efficiency of P450 BM3.
A main remaining challenge in protein engineering is how to recombine beneficial substitutions. Systematic recombination studies show that poorly performing variants are usually obtained after ...recombination of 3 to 4 beneficial substitutions. This limits researchers in exploiting nature's potential in generating better enzymes. The Computer‐assisted Recombination (CompassR) strategy provides a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding (ΔΔGfold). The performance of CompassR was evaluated by analysis of 84 recombinants located on 13 positions of Bacillus subtilis lipase A. The finally obtained variant F17S/V54K/D64N/D91E had a 2.7‐fold improved specific activity in 18.3 % (v/v) 1‐butyl‐3‐methylimidazolium chloride (BMIMCl). In essence, the deducted CompassR rule allows recombination of beneficial substitutions in an iterative manner and empowers researchers to generate better enzymes in a time‐efficient manner.
True North: The Computer‐assisted Recombination (CompassR) strategy provides a selection guide for beneficial substitutions that can be recombined to gradually improve enzyme performance by analysis of the relative free energy of folding (ΔΔGfold).
Cytochrome P450s are heme-containing enzymes capable of the oxidative transformation of a wide range of organic substrates. A protein scaffold that coordinates the heme iron, and the catalytic pocket ...residues, together, determine the reaction selectivity and regio- and stereo-selectivity of the P450 enzymes. Different substrates also affect the properties of P450s by binding to its catalytic pocket. Modulating the redox potential of the heme by substituting iron-coordinating residues changes the chemical reaction, the type of cofactor requirement, and the stereoselectivity of P450s. Around hundreds of P450s are experimentally characterized, therefore, a mechanistic understanding of the factors affecting their catalysis is increasingly vital in the age of synthetic biology and biotechnology. Engineering P450s can enable them to catalyze a variety of chemical reactions viz. oxygenation, peroxygenation, cyclopropanation, epoxidation, nitration, etc., to synthesize high-value chiral organic molecules with exceptionally high stereo- and regioselectivity and catalytic efficiency. This review will focus on recent studies of the mechanistic understandings of the modulation of heme redox potential in the engineered P450 variants, and the effect of small decoy molecules, dual function small molecules, and substrate mimetics on the type of chemical reaction and the catalytic cycle of the P450 enzymes.
Different from other laccases, copper efflux oxidase (CueO) from Escherichia coli possesses an additional methionine-rich segment (MetRich), which is generally considered to be detrimental to its ...oxidase activity. Herein, we reveal that MetRich plays an important role in rapid immobilization of CueO on electrodes by studying the adsorption and bioelectrocatalysis behaviors of CueO, a truncated CueO (ΔMetRich CueO), and a serine-rich substituted CueO (SerRich CueO). Atomic molecular dynamics (MD) simulations demonstrate that the synergistic effect of π–π stacking and hydrophobic interactions contribute to the high affinity of MetRich to carbon nanotubes. Considering that the location of the electron acceptor (i.e., T1 Cu active site) in the family of laccases is close to the C-terminus, MetRich fused to another bacterial laccase, spore coat protein A (CotA) from Bacillus licheniformics, is found to endow CotA with the properties of rapid and oriented adsorption at the electrode surface. The finding and validation make MetRich a valuable binding motif for the rapid immobilization and high performance of laccases and perhaps other oxidoreductases in bioelectrocatalytic applications.
Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low ...thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from
. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15⁻21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52⁻58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15⁻22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.
The CompassR rule enables to identify the beneficial substitutions, which can be recombined in directed evolution with gradually improving the enzymatic properties. However, the question of how to ...efficiently explore the protein sequence space when ten or more beneficial substitutions are identified has not yet been addressed. Two recombination strategies 2GenReP and InSiReP employing CompassR are systematically investigated to minimize experimental efforts and maximize possible improvements. Here we describe the details of the 2GenReP and InSiReP procedure with an example of recombining 15 substitutions and discuss some important practical issues that should be considered for the application of 2GenReP and InSiReP, such as placing the substitutions into subsets. The core part of the protocol (Step1 to Step5) is transferable to other enzymes and any recombination of potential substitutions.
Biocatalysis for the synthesis of fine chemicals is highly attractive but usually requires organic (co‐)solvents (OSs). However, native enzymes often have low activity and resistance in OSs and at ...elevated temperatures. Herein, we report a smart salt bridge design strategy for simultaneously improving OS resistance and thermostability of the model enzyme, Bacillus subtilits Lipase A (BSLA). We combined comprehensive experimental studies of 3450 BSLA variants and molecular dynamics simulations of 36 systems. Iterative recombination of four beneficial substitutions yielded superior resistant variants with up to 7.6‐fold (D64K/D144K) improved resistance toward three OSs while exhibiting significant thermostability (thermal resistance up to 137‐fold, and half‐life up to 3.3‐fold). Molecular dynamics simulations revealed that locally refined flexibility and strengthened hydration jointly govern the highly increased resistance in OSs and at 50–100 °C. The salt bridge redesign provides protein engineers with a powerful and likely general approach to design OSs‐ and/or thermal‐resistant lipases and other α/β‐hydrolases.
By removing unfavorable surface salt bridges, the organic solvent and thermal resistance of enzymes can be significantly improved. This design strategy results in locally refined flexibility and strengthened hydration.
Understanding the mechanisms of modulators' action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regulated mainly by ...a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM17's broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New 'exosite' (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modulators as therapeutic drugs. By combining experimental and in silico methods (structural modeling and docking), we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibition mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity.
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