Improving Enzyme Performance Research Articles

Artificial Small Molecules as Cofactors and Biomacromolecular Building Blocks in Synthetic Biology: Design, Synthesis, Applications, and Challenges.

Enzymes are essential catalysts for various chemical reactions in biological systems and often rely on metal ions or cofactors to stabilize their structure or perform functions. Improving enzyme performance has always been an important direction of protein engineering. In recent years, various artificial small molecules have been successfully used in enzyme engineering. The types of enzymatic reactions and metabolic pathways in cells can be expanded by the incorporation of these artificial small molecules either as cofactors or as building blocks of proteins and nucleic acids, which greatly promotes the development and application of biotechnology. In this review, we summarized research on artificial small molecules including biological metal cluster mimics, coenzyme analogs (mNADs), designer cofactors, non-natural nucleotides (XNAs), and non-natural amino acids (nnAAs), focusing on their design, synthesis, and applications as well as the current challenges in synthetic biology.

Framework for Optimizing Polymeric Supports for Immobilized Biocatalysts by Computational Analysis of Enzyme Surface Hydrophobicity

Immobilization is a powerful strategy for improving enzyme usability and stability in various technologies that employ biocatalysis. However, the interactions leading to stabilization or destabilization remain poorly understood, and a support that may stabilize one enzyme may destabilize another. Employing chemically heterogeneous and complex random copolymer brushes as supports, we demonstrate a rational approach toward estimating the chemical composition of an optimally stabilizing enzyme immobilization support by computational analysis of enzyme surface hydrophobicity. This approach was tested by immobilizing a range of enzymes with diverse functions and hydrophobicity on tunable statistical random copolymer brush supports composed of poly(ethylene glycol) methacrylate (PEGMA) and sulfobetaine methacrylate (SBMA). Remarkably, we observed greatly improved enzyme performance as a function of brush composition with enhancements in the retention of catalytic activity at temperatures as high as 90 °C. Additionally, we observed an increase in activity at the optimal temperature by as much as 20-fold relative to the activity at the optimal temperature of the unimmobilized form of the enzyme. Most significantly, our results showed that the optimal composition of the brush support correlated with the overall hydrophobicity of the enzyme surface (ΔGsolv,total/area), which was determined from computational analysis. This correlation provides a framework for the choice of polymer brush supports based on enzyme structure and stabilizing enzymes using complex synthetic materials.

Engineered Imine Reductase for Larotrectinib Intermediate Manufacture

Imine reductases (IREDs) are increasingly identified and characterized for the reduction of imines and reductive amination of ketones. However, their practical application is still limited due to the low activity and poor stability of native enzymes. Herein, we developed an engineered IRED through three rounds of evolution from wild-type Streptomyces clavuligerus. The specific activity of the engineered enzyme, ScIRED-R3-V4, was increased >100-fold, and stability was increased >270-fold. Using the more active and stable ScIRED-R3-V4, 80 g L–1 cyclic imine 2-(2,5-difluorophenyl)-pyrroline was completely reduced, producing kilogram quantities of a key chiral intermediate of larotrectinib in 82.5% yield, with >99.5% enantiomeric excess and a space–time yield of 352 g L–1 day–1. In addition, crystal structures of enzyme–substrate complexes and molecular dynamics simulations were conducted to gain insight into the molecular mechanism for improved enzyme performance. The significantly improved process demonstrated the potential of the engineered IRED in industrial applications.

Protein-Glutaminase Engineering Based on Isothermal Compressibility Perturbation for Enhanced Modification of Soy Protein Isolate.

Protein-glutaminase plays a significant role in future food (e.g., plant-based meat) processing as a result of its ability to improve the solubility, foaming, emulsifying, and gel properties of plant-based proteins. However, poor stability, activity, high pressure, and high shear processing environments hinder its application. Therefore, we developed an application-oriented method isothermal compressibility perturbation engineering strategy to improve enzyme performance by simulating the high-pressure environment. The best variant with remarkable improvement in specific activity and half-time, N16M/Q21H/T113E, exhibited a 4.28-fold increase compared to the wild type in specific activity (117.18 units/mg) and a 1.23-fold increase in half-time (472 min), as one of the highest comprehensive performances ever reported. The solubility of the soy protein isolate deaminated by the N16M/Q21H/T113E mutant was 55.74% higher than that deaminated by the wild type, with a tinier particle size and coarser texture. Overall, this strategy has the potential to improve the functional performance of enzymes under complex food processing conditions.

Magnetic Halloysite Nanotube/α-Amylase Based Nanobiocatalytic Transformation of Food Processing Waste into an Active Fermentation Ingredient

The concept of “nanobiocatalysis” creates exciting opportunities for improving enzyme performance via immobilization onto nanomaterials. A nanobiocatalyst consisting of magnetic halloysite nanotube (MHNT)/α-amylase was evaluated to transform food processing waste into an active fermentation medium formulation for low-cost bioprocessing. A high loading of α-amylase (185.5 mg (g of support)−1) was achieved on the surface of MHNTs through polydopamine functionalization. We validated the establishment of an enzyme-support system retaining >89% catalytic activity (27332 IU (g of support)−1) with improved enzyme handling (>99.1% recovery) and reusability (>56% activity, 10 cycles). MHNTs remarkably improved the enzyme kinetics and thermodynamic characteristics along with operational and storage stabilities and mitigated the likely inhibitory effects of cellulose/metal ions as contaminants. In addition to facilitating a continuous production of reducing sugars from the extracted starch over 72 h, the nanobiocatalyst was equally effective in preparing a nutritive food waste hydrolysate as a fermentable medium substitute for batch culturing of E. coli and a single-cell protein (A. niger). The commercial relevance of waste hydrolysate was also investigated to promote calcite precipitation via Bacillus sp. induced biocementation. We evidenced that nanobiocatalyst-assisted “starch depolymerization” released more nutritional components into the hydrolysate suspension, easily accessible to growing microbial cultures.

Conjugation of a zwitterionic polymer with dimethyl chains to lipase significantly increases the enzyme activity and stability

Enzyme-polymer conjugates are complex molecules with great practical significance. This work was designed to develop a novel enzyme-polymer conjugate by covalently coupling a zwitterionic polymer with side dimethyl chains (pID) to Candida rugosa lipase (CRL) via the reaction between the anhydrides of polymer chains with the amino groups of the enzyme. The resulting two CRL-pID conjugates with different pID grafting densities were investigated in term of the catalytic activity, stability and structural changes. In comparison with native CRL, both the CRL conjugates displayed 2.2 times higher activity than the native enzyme, and showed an increase in the maximum reaction rate (Vmax) and a decrease in the Michaelis constant (Km), thus resulting in about three-fold increases in the catalytic efficiency (kcat/Km). These are mainly attributed to the activation of lipase by the hydrophobic alky side chains. Moreover, the thermostability and pH tolerance of the lipase conjugates were significantly enhanced due to the stabilizing effect of the zwitterion moieties. For instance, a five-fold increase of the enzyme half-life at 50 °C for the high-pID conjugated CRL was observed. Spectroscopic studies reveal that the pID conjugation protected the enzyme in the changes in its microenvironment and conformation, well correlating with enhanced activity and stability of lipase conjugates. The findings indicate that enzyme conjugation to the zwitterionic polymer is promising for improving enzyme performance and deserves further development.

Highly effective Candida rugosa lipase immobilization on renewable carriers: Integrated drying and immobilization process to improve enzyme performance

The overall stability of Candida rugosa lipase (CRL) was improved via immobilization onto low-cost eco-friendly carriers (agroindustrial wastes) followed by using spouted bed dryer or spray dryer. Lipase covalently immobilized on glutaraldehyde-activated rice husk proved the greatest performance, retaining 94.1% of the initial activity, followed by sugarcane bagasse (90.3%) and green coconut fiber (89.3%). On the other hand, the enzyme activity retention of CRL-lipase bioencapsulated in magnetic chitosan treated with glutaraldehyde or sodium tripolyphosphate ranged from 73.6% to 84.6%. The dried product moisture content, and water activities ranged from 4.1% and 6.5% and from 0.2 to 0.35, respectively. After storage for six months at 5 °C, the immobilized enzyme systems retained at least 70.0% of its initial activity (vs 18.3% retained activity for the free lipase). After ten reuse cycles, enzyme immobilized onto lignocellulosic carriers retained on average 72.7% of its initial activity. The thermostability of all immobilized derivatives was significantly improved and lipase immobilized onto rice husk showed a stabilization factor of 20.44 at 80 °C. The kinetic data showed that the CRL-lipase suffered alterations in catalytic behavior after the immobilization/drying, mainly a slight decrease in affinity to the substrate (↑KM). Among all the assessed immobilized lipase for aroma synthesis the rice husk immobilized lipase displayed the best result towards the production of isoamyl caprylate (62.40 g.L−1). In conclusion, this report reinforces the urgent need for the economic development of eco-sustainable immobilized biocatalysts to boost the use of enzymatic technology on industrial scale, and the feasibility of the association of immobilization and drying processes to improve the enzyme-quality features.

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization.

Enzyme immobilization has been developing since the 1960s and although many industrial biocatalytic processes use the technology to improve enzyme performance, still today we are far from full exploitation of the field. One clear reason is that many evaluate immobilization based on only a few experiments that are not always well-designed. In contrast to many other reviews on the subject, here we highlight the pitfalls of using incorrectly designed immobilization protocols and explain why in many cases sub-optimal results are obtained. We also describe solutions to overcome these challenges and come to the conclusion that recent developments in material science, bioprocess engineering and protein science continue to open new opportunities for the future. In this way, enzyme immobilization, far from being a mature discipline, remains as a subject of high interest and where intense research is still necessary to take full advantage of the possibilities.

Computation-aided design of the flexible region of zearalenone hydrolase improves its thermal stability

The zearalenone hydrolase (ZHD101) derived from Clonostachys rosea can effectively degrade the mycotoxin zearalenone (ZEN) present in grain by-products and feed. However, the low thermal stability of ZHD101 hampers its applications. High throughput screening of variants using spectrophotometer is challenging because the reaction of hydrolyzing ZEN does not change absorbance. In this study, we used ZHD101 as a model enzyme to perform computation-aided design followed by experimental verification. By comparing the molecular dynamics simulation trajectories of ZHD101 at different temperatures, 32 flexible sites were selected. 608 saturated mutations were introduced into the 32 flexible sites virtually, from which 12 virtual mutants were screened according to the position specific score and enzyme conformation free energy calculation. Three of the mutants N156F, S194T and T259F showed an increase in thermal melting temperature (ΔTm>4 °C), and their enzyme activities were similar to or even higher than that of the wild type (relative enzyme activity 95.8%, 131.6% and 169.0%, respectively). Molecular dynamics simulation analysis showed that the possible mechanisms leading to the improved thermal stability were NH-π force, salt bridge rearrangement, and hole filling on the molecular surface. The three mutants were combined iteratively, and the combination of N156F/S194T showed the highest thermal stability (ΔTm=6.7 °C). This work demonstrated the feasibility of engineering the flexible region to improve enzyme performance by combining virtual computational mutations with experimental verification.

Immobilization of lysozyme on layer-by-layer self-assembled electrospun nanofibers treated by post-covalent crosslinking

In this study, lysozyme and dextran sulfate sodium salt (DS) were alternately deposited on the surface of electrospun cellulose acetate (CA) nanofibers, which were then heated at 55 °C for 6 days to induce crosslinking via Maillard reaction. The non-heated self-assembled nanofibers presented the increased average fiber diameter from 397 to 501 nm with increasing number of layers, but an improved thermal stability was induced by Maillard reaction due to the formation of covalent bonds. After heating, the immobilized lysozyme lost less than 40% of its initial activity, but exhibited the enhanced temperature tolerance and storage stability, and retained over 55.6% of its original activity after 12 reuses. Moreover, the heated nanofibrous mats showed substantially higher antibacterial activities against Staphylococcus aureus and Escherichia coli, compared to the non-heated ones. These results suggested the promising applications of the post-covalent layer-by-layer (LBL) nanofibrous mats in improving enzyme performance in food, pharmaceutical or personal care products.

A thermophilic, pH-tolerant, and highly active GH10 xylanase from Aspergillus fumigatus boosted pre-treated sugarcane bagasse saccharification by cellulases

We have heterogously expressed a recombinant GH10 xylanase from Aspergillus fumigatus Af293 (Af-XYLA) in Pichia pastoris X-33 and investigated its biochemical and kinetic properties and its synergy with cellulases during lignocellulose sacchararification. Af-XYLA showed optimal activity at 80 °C and pH 5–7, and it withstood a temperature of 50 °C and pH 4–8 for 72 h with about 50 % and 80 % of its maximal activity, respectively, in the absence of substrate. Kinetic assays revealed that it presented the highest Vmax (35,756 U mg−1 at 50 °C and 88,082 U mg−1 at 80 °C) and kcat/KM (15,456 mL mg−1 s−1 at 50 °C and 15,081 mL mg−1 s−1 at 80 °C) constants among fungal GH10 xylanases. Af-XYLA was xylose- and glucose-tolerant and displayed >70 % activity at 10 % (v/v) ethanol. Few additives affected Af-XYLA negatively, and the surfactants Tween 20, Triton X-100, and SLS and the reducing agents DTT and β-mercaptoethanol improved enzyme performance by 28 %, 11 %, 8%, 44 %, and 32 %, respectively. We applied a 3-factor Central Composite Rotational Design (CCRD) to optimize important parameters for biomass saccharification and thus maximize the reducing sugars yields obtained from sugarcane exploded bagasse (SEB) and sugarcane delignified bagasse (SDB) hydrolyses. We carried out synergism assays under the previously determined optimal concentrations of Celluclast 1.5®L (1.8 FPU g−1), Af-XYLA (3.75 μg g−1), and Tween 20 (0.75 % v/v). Af-XYLA boosted SEB and SDB hydrolyses by cellulases by 155 % and 277 %, respectively, after 48 h. On the basis of the unique properties of Af-XYLA, it is a robust enzyme for application in biorefineries.

Characterization of polyphenol oxidase from purple sweet potato (Ipomoea batatas L. Lam) and its affinity towards acylated anthocyanins and caffeoylquinic acid derivatives

Biochemical characterization of polyphenol oxidase (PPO) present in purple sweet potato (PSP) is a key step in developing efficient methodologies to control oxidative damage caused by this enzyme to the valuable components of PSP, such as caffeoylquinic acid derivatives and acylated anthocyanins. Thus, this work focused on the assessment of the effects of pH, temperature, and chemical agents on the PPO activity as well as characterization of the PPO substrate specificity towards major phenolic compounds found in PSP. The optimum conditions of enzyme activity were pH 7 and a temperature range of 20–30 °C at which phenolic substrates were oxidized with 72.5–99.8% yield. Zn2+ ions remarkably reduced PPO activity while Cu2+ ions improved enzyme performance. The highest substrate preference was shown for 3,4,5-tri-caffeoylquinic and 3,5-di-caffeoylquinic acid, followed by 5-caffeoylquinic and caffeic acid, 3,4- and 4,5-di-caffeoylquinic acids, peonidin-3-caffeoyl-p-hydroxybenzoyl-sophoroside-5-glucoside. The highest Km values were found for 4,5-feruloyl-caffeoylquinic acid and catechol.

Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study.

Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.

Improving protein solubility and activity by introducing small peptide tags designed with machine learning models

Improving catalytic ability of enzymes is critical to the success of many metabolic engineering projects, but the search space of possible protein mutants is too large to explore exhaustively through experiments. To some extent, highly soluble enzymes tend to exhibit high activity due to their better folding quality. Here, we demonstrate that an optimization algorithm based on a regression model can effectively design short peptide tags to improve solubility of a few model enzymes. Based on the protein sequence information, a support vector regression model we recently developed was used to evaluate protein solubility after small peptide tags were introduced to a target protein. The optimization algorithm guided the sequences of the tags to evolve towards variants that had higher solubility. The optimization results were validated successfully by measuring solubility and activity of the model enzyme with and without the identified tags. The solubility of one protein (tyrosine ammonia lyase) was more than doubled and its activity was improved by 250%. This strategy successfully increased solubility of another two enzymes (aldehyde dehydrogenase and 1-deoxy-D-xylulose-5-phosphate synthase) we tested. The presented optimization methodology thus provides a valuable tool for improving enzyme performance for metabolic engineering and other biotechnology projects.

Computer-Assisted Recombination (CompassR) Teaches us How to Recombine Beneficial Substitutions from Directed Evolution Campaigns.

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 (ΔΔG fold). 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 ([BMIM][Cl]). 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.

Industrial enzymes market to attain revenue of $12.8 bn by 2025

The use of biological methods for industrial manufacturing is an excellent starting point to create a green process since biocatalysts naturally follows the Principles of Green Chemistry. Biocatalysts are highly biodegradable and are considered natural products. Nevertheless, they also have some drawbacks, since enzyme have high molecular complexity, high production costs and intrinsic fragility. Several strategies have been developed in order to stabilize enzymes under process conditions: chemical modification, enzyme immobilization, aggregation, modern techniques of protein engineering and so on. Enzyme improvement via immobilization is a useful technique of enzyme engineering, which allows not only the re-use of enzymes but also the enhancement of enzyme properties. Some reasons to engineer enzymes include better accommodate unnatural substrates, increase their stability under the reaction conditions and create new reactions or new biochemical pathways. Among enzymes, lipases appear as important tools in the application of the principles of Green Chemistry, offering a convenient way to prepare derivatives of natural compounds with great potential in food and pharmaceutical industries. With this in mind, some strategies to improve enzyme performance at industrial scale and ways of conducting processes will be discussed.

Directly covalent immobilization of Candida antarctica lipase B on oxidized aspen powder by introducing poly‑lysines: An economical approach to improve enzyme performance.

In our previous study, we could achieve high soluble expression of Candida antarctica lipase B (CalB) in E. coli by fusion poly‑amino acid tags on CalB (pCalB). Herein, we are surprised to find that pCalB can be easily and directly covalent binding on a simply oxidized aspen powder (OAP) by the aid of poly‑lysine tags. Under the optimal conditions, 72.9 ± 3.6% of the total protein could be immobilized, and the activity recovery of immobilized pCalB (pCalB-OAP) was 98.9 ± 3.8%. The analysis of scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) indicated that OAP was a suitable carrier for enzyme immobilization. The immobilized pCalB-OAP could exhibit excellent thermal stabilities, and it retained a residual activity of 58.4 ± 2.8% at 55 °C, whereas only 21.2 ± 2.2% of its initial activity for free pCalB was observed. And it could also display a nice tolerance for the changes of pH environment, compared with that of free pCalB. The results that pCalB-OAP could retained 73.6 ± 2.9% of their initial activity in (R, S)-NEMPAME hydrolysis after the tenth cycles, suggested that pCalB-OAP could be effectively recycled. The immobilization strategies established here were simple and inexpensive.

A Novel Bacterial Cellulose Aerogel Modified with PGMA via ARGET ATRP Method for Catalase Immobilization

Formation of Poly(Glycidyl Methacrylate) (PGMA) films on bacterial cellulose (BC) supports (aerogels) was achieved via activators regenerated by electron transfer for atom transfer radical polymerization (ARGET ATRP). Epoxy groups on PGMA were used for covalent coupling with catalase. Immobilized catalases on BC@PGMA were characterized by Fourier transform infrared spectroscopy (FTIR) and Typical scanning electron microscopy (SEM). The immobilized catalase amount reached a high value of 116 mg/g. Furthermore, the thermal, pH and storage stabilities of the immobilized catalase were improved significantly. After 10 use cycles, the BC@PGMA-catalase still retained approximately 63.5 % of the initial activity. This work demonstrates the potential of hierarchical nanomaterials for improving enzyme performance, leveraging the benefits of both nano- and macroscale supports. These results demonstrate that the BC@PGMA-catalase has high stability and good reusability, suggesting that the BC aerogel modified with PGMA may find applications in biotechnology and as biocatalyst.

Grafting through Method for Implanting of Lysozyme Enzyme in Molecular Brush for Improved Biocatalytic Activity and Thermal Stability

We report a “grafting through” conjugation strategy to improve lysozyme catalytic activity and thermal stability by the synthesis of a synthetic polymer–enzyme hybrid. The lysozyme was first conjugated with glycidyl methacrylate via ring-opening reaction between epoxy groups and lysine residues of the enzyme to synthesize the enzyme macromonomer. The conjugation was followed by free radical copolymerization of the macromonomer with poly(ethylene glycol) methyl ether acrylate (PEGMEA) (Mn = 5000 g/mol) and poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) (Mn = 500 g/mol). We demonstrated that implanting of a single lysozyme molecule in the molecular brush polymer chain resulted a significant improvement of the thermal stability up to 90 °C and 9-time extended half-life for this synthetic enzyme structure. The improved enzyme performance is explained by the crowding effect provided by molecular brush architecture of the synthetic hybrid.

Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1

The apparent bottleneck in the accumulation of oil during seed development in some oleaginous plant species is the formation of triacylglycerol (TAG) by the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol catalyzed by diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). Improving DGAT activity using protein engineering could lead to improvements in seed oil yield (e.g. in canola-type Brassica napus). Directed evolution of B. napus DGAT1 (BnaDGAT1) previously revealed that one of the regions where amino acid residue substitutions lead to higher performance in BnaDGAT1 is in the ninth predicted transmembrane domain (PTMD9). In this study, several BnaDGAT1 variants with amino acid residue substitutions in PTMD9 were characterized. Among these enzyme variants, the extent of yeast TAG production was affected by different mechanisms, including increased enzyme activity, increased polypeptide accumulation, and possibly reduced substrate inhibition. The kinetic properties of the BnaDGAT1 variants were affected by the amino acid residue substitutions, and a new kinetic model based on substrate inhibition and sigmoidicity was generated. Based on sequence alignment and further biochemical analysis, the amino acid residue substitutions that conferred increased TAG accumulation were shown to be present in the DGAT1-PTMD9 region of other higher plant species. When amino acid residue substitutions that increased BnaDGAT1 enzyme activity were introduced into recombinant Camelina sativa DGAT1, they also improved enzyme performance. Thus, the knowledge generated from directed evolution of DGAT1 in one plant species can be transferred to other plant species and has potentially broad applications in genetic engineering of oleaginous crops and microorganisms.