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Enzyme Engineering by Random Mutagenesis and DNA Shuffling

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Creative Enzymes provides comprehensive enzyme engineering services designed to enhance or modify enzyme performance through random mutagenesis and DNA shuffling. By integrating advanced molecular biology techniques with efficient high-throughput screening, we enable the generation of diverse mutant libraries and the rapid identification of superior enzyme variants. Our services are ideal for improving enzymatic properties such as activity, selectivity, substrate specificity, stability, and solvent tolerance.

With a deep understanding of the relationship between sequence, structure, and function, Creative Enzymes ensures reliable, cost-effective, and high-quality outcomes for your enzyme engineering needs.

Background: Understanding Random Mutagenesis and DNA Shuffling

Enzyme engineering is a powerful approach for tailoring biocatalysts to meet industrial and research demands. Among the available strategies, random mutagenesis and DNA shuffling are proven and highly effective methods for generating functional diversity without prior structural information.

Random mutagenesis introduces random point mutations throughout a target gene, creating large genetic libraries that explore a wide range of sequence space. Beneficial mutations can enhance catalytic activity, alter substrate affinity, or improve enzyme stability under extreme conditions.

DNA shuffling, on the other hand, mimics natural recombination by fragmenting and reassembling homologous genes from related sequences. This process combines beneficial mutations from multiple parent sequences, accelerating the evolution of improved enzymes. When applied iteratively, DNA shuffling enables the discovery of high-performance biocatalysts that might be unattainable by rational or single-gene mutagenesis approaches alone.

Together, these two techniques represent the foundation of directed evolution, offering an efficient and unbiased means to evolve enzymes toward desired traits. Creative Enzymes has extensive experience in applying these strategies to diverse enzyme families for applications in pharmaceuticals, agriculture, biofuels, and fine chemicals.

Principle and workflow of enzyme random mutagenesis using DNA shuffling for variant generationFigure 1. Random mutagenesis by DNA shuffling. (Mau Goh et al., 2012)

What We Offer: Random Mutagenesis and DNA Shuffling

Creative Enzymes provides end-to-end services covering every stage of enzyme engineering by random mutagenesis and DNA shuffling—from gene preparation and library construction to screening and final characterization. Our expertise ensures both high-quality data and efficient project timelines.

Category Services Timeline Price
Upstream Services for Random Mutagenesis and DNA Shuffling Template DNA Sequencing
Accurate verification of the initial gene template prior to mutagenesis. 		1-2 weeks Get a quote
Gene Synthesis
De novo synthesis of genes optimized for expression in your preferred host.
Expression Cloning
Cloning of target genes into suitable expression vectors for efficient production.
Core Random Mutagenesis and DNA Shuffling Services Mutagenesis Library Construction
Generation of large, diverse libraries using proven error-prone PCR, chemical mutagenesis, or recombination-based methods. 		Inquiry Get a quote
Design of Activity Assay Methods
Custom design of functional assays suited for activity, selectivity, or stability screening.
Library Screening
High-throughput screening to identify mutants with improved enzymatic properties.
Downstream Services for Random Mutagenesis and DNA Shuffling Expression and Purification
Production and isolation of promising enzyme variants for further evaluation. 		Inquiry Get a quote
Optimization of Reaction and Expression Conditions
Fine-tuning conditions to maximize enzyme performance and yield.
Structural and Mechanistic Analysis
Characterization of molecular changes driving improved function through kinetic and structural studies.

All services can be tailored to specific project requirements. We also provide standalone modules or full-package workflows, depending on client preferences.

Service Workflow

Service workflow for enzyme engineering through random mutagenesis and DNA shuffling

Contact Our Team

Why Partner with Creative Enzymes

Comprehensive Technical Expertise

Our scientists specialize in enzyme evolution, protein design, and high-throughput mutagenesis, ensuring optimal strategies for every project.

High-Diversity, High-Accuracy Libraries

We generate libraries with exceptional diversity and fidelity, maximizing the likelihood of discovering improved variants.

Customized Screening Solutions

Each project benefits from tailored assay systems and screening strategies aligned with the enzyme's functional requirements.

Integrated Upstream & Downstream Services

From template preparation to kinetic analysis, we provide complete support under one roof for seamless project execution.

Data Transparency and Fast Turnaround

Clients receive detailed progress updates, clear data analysis, and timely delivery of results.

Cost-effective and Scalable

Competitive pricing, scalable workflows, and flexible project scopes make our services suitable for both research and industrial applications.

Case Studies and Real-World Applications

Case 1: Directed Evolution and Activity Characterization of Synthetic Plant GSTs

Glutathione transferases (GSTs) are versatile enzymes crucial for detoxification and have broad biotechnological potential. In this study, a synthetic GST library was generated from Phaseolus vulgaris and Glycine max cDNAs after abiotic stress induction. Using degenerate primers and reverse transcription-PCR, the library was further diversified through DNA shuffling and directed evolution. Activity screening identified a novel tau class GST, PvGmGSTUG, which was purified, kinetically characterized, and structurally analyzed via X-ray crystallography. PvGmGSTUG exhibited enhanced glutathione hydroperoxidase activity and unusual cooperative kinetics toward CDNB. This approach enables simultaneous gene shuffling from multiple plants, providing a versatile platform for creating synthetic GSTs with tailored biochemical properties for industrial applications.

DNA shuffling-based directed evolution expanding plant GST enzymes with improved catalytic and binding propertiesFigure 2. Activity screening (A). Activity screening of different colonies obtained following DNA shuffling. The graph depicts only the colonies with detectable activity toward the substrate system CDNB/GSH. (B) Distribution of the specific activity of different colonies. (Chronopoulou et al., 2018)

Case 2: Enhanced catalytic efficiency of CotA-laccase by DNA shuffling

Bacterial CotA-laccases are promising green catalysts for industrial dye decolorization due to their high activity under alkaline and high-salt conditions, but their low yield and catalytic efficiency limit applications. Using DNA shuffling, a random mutation library was generated, leading to the identification of mutant 5E29 (T232P/Q367R) with improved activity. This variant showed a 1.21-fold increase in catalytic efficiency compared with wild type, with KM and kcat values of 20.3 µM and 7.6 s-1 for SGZ, and slightly enhanced thermal stability. Mutant 5E29 efficiently decolorized Indigo Carmine and Congo Red at pH 9, making it a strong candidate for biotechnological applications under alkaline conditions.

Improved catalytic efficiency of CotA laccase achieved through DNA shuffling-driven evolutionFigure 3. Local structure of the wild-type laccase and its variants. (a) The position of Gln367 and neighboring residues of the wild-type laccase (b) Gln-367-Arg. (Ouyang and Zhao, 2019)

FAQs: Random Mutagenesis and DNA Shuffling

  • Q: What is the difference between random mutagenesis and DNA shuffling?

    A: Random mutagenesis introduces point mutations throughout a gene without requiring prior structural knowledge, while DNA shuffling recombines fragments from related sequences to merge beneficial mutations into a single gene. Together, these techniques enable rapid evolution of enzymes with improved or novel properties.

  • Q: What types of properties can be improved using these methods?

    A: They can enhance catalytic activity, enantioselectivity, thermostability, solvent resistance, substrate range, and pH tolerance, among others.

  • Q: How large are the libraries generated by Creative Enzymes?

    A: Depending on the chosen method, we can generate libraries ranging from 104 to 108 variants, ensuring sufficient diversity to capture beneficial mutations.

  • Q: Can you combine random mutagenesis with rational design?

    A: Yes. We frequently apply semi-rational approaches, using computational insights to focus mutagenesis on hotspots, increasing efficiency while preserving the exploratory power of randomness.

  • Q: Do you provide expression and purification of selected variants?

    A: Absolutely. Our downstream services include expression, purification, and biochemical characterization to fully validate performance improvements.

  • Q: What information do clients need to provide to initiate a project?

    A: We typically require the target gene sequence, the expression host (if applicable), desired enzyme properties for improvement, and any existing activity or stability data.

References:

  1. Chronopoulou EG, Papageorgiou AC, Ataya F, Nianiou-Obeidat I, Madesis P, Labrou NE. Expanding the plant GSTome through directed evolution: DNA shuffling for the generation of new synthetic enzymes with engineered catalytic and binding properties. Front Plant Sci. 2018;9:1737. doi:10.3389/fpls.2018.01737
  2. Mau Goh K, Poh Hong G, Pearly Ng NHC, Kian Piaw C, Raja Abdul Rahman RNZ. Trends and tips in protein engineering, a review. Jurnal Teknologi. 2012;59(1). doi:10.11113/jt.v59.1574
  3. Ouyang F, Zhao M. Enhanced catalytic efficiency of CotA-laccase by DNA shuffling. Bioengineered. 2019;10(1):182-189. doi:10.1080/21655979.2019.1621134
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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.

Services
Online Inquiry

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.