Enzyme Engineering

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Creative Enzymes offers a comprehensive suite of enzyme engineering services designed to enhance or redesign enzyme properties for industrial, research, and therapeutic applications. Leveraging advanced technologies such as directed evolution, rational and de novo design, site-directed and random mutagenesis, phage and mRNA display, and the incorporation of unnatural amino acids, we tailor enzyme performance to meet specific project goals.

Our services enable improvements in catalytic activity, substrate specificity, stability, solubility, and enantioselectivity, as well as the creation of entirely novel catalytic functions. With a multidisciplinary team skilled in molecular biology, structural biology, bioinformatics, and biophysics, Creative Enzymes provides one-stop, scalable solutions—from in silico modeling and mutagenesis design to experimental validation and large-scale enzyme production.

Enzyme Engineering: Background and Overview

Enzyme engineering lies at the intersection of biotechnology, structural biology, and computational design, transforming how enzymes are used in biocatalysis, biopharmaceutical production, and sustainable chemical synthesis.

Natural enzymes often fall short under industrial or therapeutic conditions—limited by narrow substrate specificity, low thermostability, or poor solvent tolerance. Through modern enzyme engineering, these challenges are systematically overcome by modifying enzyme sequences and structures to achieve desired functions with predictable outcomes.

Historically, enzyme engineering began with rational attempts to modify known active sites. The field advanced dramatically following the development of directed evolution—recognized by the 2018 Nobel Prize in Chemistry—which mimics natural evolution in the laboratory to generate optimized enzyme variants. Today, enzyme engineering integrates computational modeling, machine learning, quantum chemistry, and high-throughput screening, offering unprecedented control over enzyme properties.

Diagram of enzyme engineering strategies for enhancing enzyme performance Figure 1. Multiple strategies of protein engineering have significantly enhanced the performance of enzymes. (Grigorakis et al., 2025)

At Creative Enzymes, our integrated enzyme engineering platform combines rational design precision with directed evolution diversity, enabling customers to develop optimized biocatalysts faster, with higher success rates, and reduced cost.

Enzyme Engineering: What We Offer

Creative Enzymes provides end-to-end enzyme engineering services that cover the full range of current scientific and industrial needs. Whether your project involves improving an existing enzyme or designing a completely new catalytic system, our flexible service packages ensure reliable outcomes supported by data-driven design.

Service	Description	Applications	Price
Enzyme Engineering by Directed Evolution	We employ error-prone PCR, DNA shuffling, and site-saturation mutagenesis to generate large mutant libraries containing diverse enzyme variants. Through high-throughput screening and iterative selection, we identify mutants with superior activity, selectivity, or stability. Our automated workflows allow for the creation and testing of libraries exceeding 10¹² variants, drastically accelerating optimization cycles.	Improving catalytic efficiency, thermostability, substrate scope, and solvent tolerance.	Inquiry
Enzyme Engineering by Rational Design	Our rational design service leverages structural biology, bioinformatics, and molecular simulation to introduce precise amino acid substitutions based on mechanistic understanding. Using multiple sequence alignment, molecular dynamics, and energy minimization, we predict stabilizing or activity-enhancing mutations that can be introduced with minimal experimental screening.	Altering substrate specificity, enhancing thermostability, and fine-tuning enzyme kinetics.
Enzyme Engineering by De Novo Design	Through de novo design, Creative Enzymes constructs entirely new enzymes with custom catalytic functions or folds that do not exist in nature. Combining computational protein design, energy optimization algorithms, and machine learning models, we design active sites, folding scaffolds, and binding pockets from scratch. Experimental validation ensures that predicted enzymes fold correctly and perform the intended reactions.	Designing enzymes for novel reactions, non-natural substrates, or artificial metabolic pathways.
Enzyme Engineering by Site-Directed Mutagenesis	Using precise genetic editing, our site-directed mutagenesis service introduces defined mutations at specific residues to investigate structure–function relationships or enhance enzyme performance. We employ QuikChange-style PCR, cassette mutagenesis, and recombineering approaches for single-site or multi-site mutations.	Targeted improvement of catalytic residues, active site tuning, or mechanistic elucidation.
Enzyme Engineering by Random Mutagenesis and DNA Shuffling	When sequence–structure relationships are not well characterized, random mutagenesis offers a powerful exploration strategy. We introduce mutations stochastically throughout the gene and recombine fragments via DNA shuffling to create highly diverse chimeric libraries. Subsequent screening identifies novel variants with emergent, beneficial properties.	Discovering entirely new catalytic functions and improving poorly characterized enzymes.
Phage Display and mRNA Display for Enzyme Engineering	These in vitro display systems enable the selection of enzyme variants with desired binding or catalytic traits from vast libraries. Phage display links phenotype to genotype on the surface of bacteriophages, while mRNA display allows for screening libraries up to 10¹³ variants in a cell-free environment. These methods are particularly useful for affinity optimization and enzyme–substrate interaction studies.	Improving substrate affinity, evolving binding proteins, and screening enzyme scaffolds for biocatalysis.
Incorporation of Unnatural Amino Acids	By expanding the genetic code, Creative Enzymes enables the site-specific incorporation of unnatural amino acids into enzymes. This allows the introduction of non-canonical functional groups, reactive moieties, or spectroscopic probes for mechanistic analysis and enhanced stability.	Introducing catalytic metal centers, improving enzyme rigidity, or enabling photo-switchable or chemically responsive activity.

Service Workflow

Creative Enzymes' enzyme engineering service workflow illustration

Service Details

Creative Enzymes offers fully customized solutions, from single-site mutation studies to large-scale directed evolution projects. Our services include:

Computational Enzyme Design: Homology modeling, QM/MM simulations, and AI-driven structure prediction.
Mutagenesis Platforms: Site-directed, random, or combinatorial mutagenesis with high fidelity.
Library Construction & Screening: Up to 10¹² variants screened per project using fluorescence, absorbance, or activity-based assays.
Expression Systems: Expertise in E. coli, yeast, insect, and mammalian systems, with scalable expression and purification.
Characterization & Validation: Structural determination (X-ray, cryo-EM), kinetic assays, and stability tests under industrial conditions.
Reporting & Technology Transfer: Full documentation, data analysis, and IP-respecting project handover.

Our process is designed for flexibility and scalability, ensuring compatibility with both early-stage discovery and industrial production needs.

Contact Our Team

Why Partner with Creative Enzymes

Comprehensive Technical Expertise

Our multidisciplinary team combines expertise in structural biology, enzymology, and computational biochemistry, ensuring rationally guided, scientifically sound enzyme designs.

Integrated Technology Platform

By integrating computational modeling, high-throughput screening, and experimental evolution, we offer end-to-end solutions from concept to optimized enzyme.

Customization & Flexibility

Each project is customized to meet the client's goals, with adaptable workflows for specific enzymes, substrates, or process conditions.

High Success Rate & Efficiency

Our combined use of rational and evolutionary methods drastically reduces trial-and-error, accelerating project timelines and enhancing success rates.

Scalable & Industry-Ready Solutions

From lab-scale validation to industrial-scale enzyme production, our solutions are designed with scalability and regulatory compliance in mind.

Proven Track Record

Trusted by global pharmaceutical, biotech, and chemical companies, Creative Enzymes has successfully delivered hundreds of engineered enzymes with documented performance improvements.

Real-World Examples of Enzyme Engineering

Case 1: Rational Engineering of β-Amino Acid Dehydrogenase for Efficient Asymmetric Synthesis

This study reports the structural and mechanistic elucidation of L-erythro-3,5-diaminohexanoate dehydrogenase, the only known β-amino acid dehydrogenase (β-AADH). Through crystal structure determination, site-directed mutagenesis, and quantum chemical analysis, researchers uncovered key differences between β- and α-AADHs in substrate binding and catalysis. Guided by these insights, several rationally engineered variants were developed, exhibiting 110–800-fold activity improvements toward various β-amino acids without sacrificing enantioselectivity. Using the best variants, two β-amino acids were synthesized with >99% ee and 86–87% yields, providing a robust framework for future β-AADH engineering and asymmetric β-amino acid synthesis.

Crystal structures and catalytic mechanism of L-erythro-3,5-diaminohexanoate dehydrogenase for rational β-amino acid synthesis Figure 2. The substrate binding and catalytic mechanism of L-erythro-3,5-diaminohexanoate dehydrogenase, the only known member of the β-amino acid dehydrogenase family, are shown to be quite different from those of its α-counterparts. Subsequent rational engineering expanded the substrate scope without reducing the enantioselectivity, enabling efficient asymmetric synthesis of β-amino acids. (Liu et al., 2021)

Case 2: Improving a Natural Enzyme Activity Through Incorporation of Unnatural Amino Acids

Bacterial phosphotriesterases efficiently hydrolyze paraoxon and were believed to be evolutionarily optimized. However, replacing tyrosine at position 309 with unnatural amino acids—L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine—led to remarkable catalytic enhancements. Kinetic analysis revealed that the deprotonated 7-hydroxyl group of Hco facilitated faster product release via electrostatic repulsion with the negatively charged product. This single rationally designed substitution yielded an 8–11-fold improvement in catalytic turnover, surpassing what was achievable through extensive natural amino acid mutagenesis. The study highlights how designer amino acids unlock novel functional and structural possibilities in enzyme engineering.

Enzyme activity improvement of bacterial phosphotriesterases using unnatural amino acids at position Y309 Figure 3. Potential rotamers (without steric clashes to other amino acid side chains or substrate) of Hco309 relative to the substrate diethyl-methoxyphenylphosphate and Tyr309 in the wild-type protein based on the crystal structure of the enzyme-substrate complex (PDB accession code 2R1N). (Ugwumba et al., 2011)

Enzyme Engineering: Frequently Asked Questions

Q: What are the main advantages of enzyme engineering compared to natural enzyme use?

A: Engineered enzymes offer superior performance, including enhanced catalytic activity, stability, and substrate versatility. They can function under extreme industrial conditions and catalyze reactions beyond the capabilities of natural enzymes.
Q: How do I choose between directed evolution and rational design?

A: Rational design is ideal when detailed structural information is available, allowing targeted mutations. Directed evolution is preferred for exploring sequence diversity without prior structural data. Many projects combine both for optimal results.
Q: What information do clients need to provide before initiating a project?

A: We typically require the target enzyme sequence, substrate details, desired modifications or target properties, and any known structural or biochemical data. If unavailable, we can perform initial bioinformatic and homology modeling analyses.
Q: Can you handle large-scale library construction and screening?

A: Yes. Our automated directed evolution platforms support libraries of up to 10¹² variants and high-throughput screening using spectrophotometric, fluorescent, or chromatographic assays.
Q: How are project timelines determined?

A: Timelines depend on project complexity, library size, and required validation. Standard enzyme optimization projects typically take 8–12 weeks, while de novo design projects may require 16–24 weeks.
Q: Are intellectual property rights protected?

A: Absolutely. Creative Enzymes maintains strict confidentiality and IP protection for all client projects. All designs, data, and materials remain the property of the client unless otherwise specified.
Q: Do you provide expression and purification services for engineered enzymes?

A: Yes. We offer full downstream support, including expression optimization, purification, stability testing, and scale-up production for both research and industrial applications.
Q: What industries benefit most from enzyme engineering?

A: Our enzyme engineering services serve the biopharmaceutical, chemical, agricultural, food, and environmental sectors, as well as research institutions pursuing novel catalytic applications.

References:

Grigorakis K, Ferousi C, Topakas E. Protein engineering for industrial biocatalysis: principles, approaches, and lessons from engineered PETases. Catalysts. 2025;15(2):147. doi:10.3390/catal15020147
Liu N, Wu L, Feng J, et al. Crystal Structures and Catalytic Mechanism of L-erythro-3,5-Diaminohexanoate Dehydrogenase and Rational Engineering for Asymmetric Synthesis of β-Amino Acids. Angew Chem Int Ed. 2021;60(18):10203-10210. doi:10.1002/anie.202017225
Ugwumba IN, Ozawa K, Xu ZQ, et al. Improving a natural enzyme activity through incorporation of unnatural amino acids. J Am Chem Soc. 2011;133(2):326-333. doi:10.1021/ja106416g

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For research and industrial use only. Not intended for personal medicinal use. Certain food-grade products are suitable for formulation development in food and related applications.

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