Enzyme Engineering by Site-directed Mutagenesis

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Creative Enzymes is a pioneer in enzyme engineering, providing advanced site-directed mutagenesis (SDM) services for precise modification of enzymes. Leveraging state-of-the-art computational analysis, rational design strategies, and efficient experimental techniques, we enable the introduction of virtually any desired mutation in cloned DNA. Our comprehensive services cover upstream preparation, mutagenesis execution, and downstream characterization, ensuring fast, accurate, and reliable results. With extensive experience in designing and generating mutants for academic and industrial research, Creative Enzymes offers a cost-effective and high-quality solution for enhancing enzyme properties, studying structure-function relationships, or developing novel biocatalysts.

Understanding Site-directed Mutagenesis

Site-directed mutagenesis (SDM) is a cornerstone technique in modern enzyme engineering, enabling precise, targeted modifications of nucleotides in a gene to alter specific amino acid residues in the corresponding protein. Since its introduction in the late 1970s, SDM has revolutionized protein science by allowing researchers to explore structure-function relationships, improve enzymatic properties, and create novel biocatalysts tailored for specific applications.

SDM offers unparalleled control over protein sequence, including point mutations, insertions, deletions, and multiple nearby substitutions, making it an essential tool for fine-tuning enzymes. By altering key residues, scientists can enhance catalytic activity, substrate specificity, thermostability, solubility, or resistance to inhibitors, while retaining overall protein integrity. Beyond industrial applications, SDM has become instrumental in therapeutic enzyme development, drug discovery, and mechanistic studies of enzyme function.

Several well-established PCR-based strategies are commonly applied in enzyme engineering, including overlap extension PCR, complementary primer-based mutagenesis, partially complementary primer or fragment-based recombinational ligation, and inverse PCR. Each method enables precise sequence alteration, efficient mutation incorporation, and compatibility with downstream cloning and expression workflows. Together, these techniques form the methodological backbone of modern enzyme engineering, providing a flexible and reliable route for constructing targeted variants and testing functional hypotheses.

Diagram of PCR-based strategies used for enzyme site-directed mutagenesis Figure 1. A summary showing the methods established for PCR-based site-directed mutagenesis. (A) Overlap-extension PCR followed by restriction digestion and cloning using mutation-specific and cloning primers. (B) Mutagenesis using fully complementary primers, PCR amplification, DpnI digestion, and transformation. (C) Approaches using partially complementary primers or fragments, followed by DpnI digestion or recombinational ligation. (D) Mutagenesis with inverse primers, phosphorylation, ligation, and transformation. Mutated bases are indicated by red stars. (Zhang et al., 2021)

By leveraging advanced SDM techniques, Creative Enzymes empowers researchers to accelerate enzyme optimization and rational design, opening avenues for next-generation biocatalysts and customized enzymatic solutions for both academic and industrial projects.

Comprehensive Site-directed Mutagenesis Offerings

Creative Enzymes provides a full-service SDM platform, including:

Category	Services	Timeline	Price
Upstream Services for Site-directed Mutagenesis	Template DNA Sequencing Verify your gene constructs to ensure accurate starting material.	1-2 weeks	Get a quote
	Gene Synthesis Custom DNA sequences designed to match your project requirements.
	Cloning into Expression Vectors Flexible insertion into suitable vectors for downstream expression.
Site-Directed Mutagenesis Services	Mutagenesis from Creative Enzymes-Synthesized Templates Reliable and optimized templates designed in-house.	2-3 weeks	From $99/mutation Get a quote
	Mutagenesis from Customer-Provided Templates Flexible support for your existing constructs.
Downstream Services for Site-directed Mutagenesis	Expression and Purification High-quality recombinant protein ready for testing or application.	Inquiry	Get a quote
	Activity Assays Measure catalytic efficiency, substrate specificity, or enantioselectivity.
	Structural and Mechanistic Analysis Understand how mutations affect enzyme stability, folding, and function.

We can design and execute single-site or multi-site mutagenesis, tailored to your research goals. Multi-site strategies allow mutations spread across hundreds of base pairs to be introduced efficiently in a single workflow, avoiding cumbersome subcloning steps.

Site-directed Mutagenesis Workflow

Workflow illustration for site-directed mutagenesis

Technical Highlights

Creative Enzymes' site-directed mutagenesis platform combines precision, flexibility, and efficiency to deliver reliable results for enzyme engineering projects:

Targeted Mutations Anywhere: Introduce mutations at any nucleotide or amino acid position with high accuracy.
Efficient Multi-Site Mutagenesis: Simultaneously modify distant residues across a gene, saving time and experimental effort.
Flexible Template Compatibility: Work with DNA templates synthesized by Creative Enzymes or provided by customers.
Integrated Computational & Experimental Design: Leverage in silico analysis alongside proven laboratory techniques to maximize success and minimize errors.

Contact Our Team

Why Partner with Creative Enzymes

Comprehensive SDM Platform

Covers upstream preparation, mutagenesis, and downstream characterization.

Precision and Accuracy

Mutations introduced with minimal off-target effects.

Flexible Mutagenesis Options

Single or multiple site mutations, including distant residues.

Rapid Turnaround

Optimized workflows ensure fast delivery without sacrificing quality.

Expert Team

Skilled scientists in computational design, molecular biology, and enzyme characterization.

Cost-Effective Solutions

Competitive pricing with reliable and reproducible outcomes.

Practical Insights: Site-Directed Mutagenesis

Case 1: Enhancing SML Lipase via Site-Directed Mutagenesis

Site-directed mutagenesis was applied to improve the activity and thermostability of Serratia marcescens lipase (SML), a key biocatalyst in pharmaceutical and industrial applications. Four mutant lipases (MutG2P, MutG59P, MutH279K, and MutL613WA614P) were designed using computational protein modeling and expressed in E. coli. Purified mutants were analyzed using circular dichroism, differential scanning calorimetry, and kinetic assays. MutG2P and MutG59P exhibited enhanced thermal stability, with T1/2 increases of 2.3- and 2.9-fold, respectively, while MutH279K showed a twofold improvement in catalytic efficiency (k_cat/K_M). These results demonstrate that rational site-directed mutagenesis guided by modeling can effectively enhance enzyme performance for industrial use.

Activity and thermostability enhancement of Serratia marcescens lipase A through site-directed mutagenesis Figure 2. Glycine 2 location in SML (A) and potential mutation to proline 2 (B). Glycine 59 location in SML (C) and potential mutation to proline 59 (D). (Mohammadi et al., 2016)

Case 2: Overcoming Trade-Offs in Esterase Engineering

Directed evolution can enhance specific enzyme properties, but often at the cost of other essential functions. A previously evolved esterase mutant, CVH, exhibited high enantioselectivity but reduced catalytic activity compared to another mutant, YH. To address this trade-off, site-directed saturation mutagenesis was performed on four key residues—three hot spots (Asn62, Met121, Leu145) identified by directed evolution and one additional residue (Tyr27) to bridge spatial gaps. The resulting mutant, HMVY, combined high enantioselectivity with activity comparable to YH. Kinetic analysis and molecular dynamics revealed that the improvements arose from enhanced catalysis of the preferred substrate, providing a strategy to balance activity and selectivity in enzyme evolution.

Compensating the enantioselectivity–activity trade-off in a Rhodobacter sphaeroides esterase using site-directed saturation mutagenesis Figure 4. The conformations of the preferred substrate binding to the active pocket of the enzyme. (S)-methyl mandelate was shown in purple. The amino acid residues were shown in gray and the surface was shown in white with the 50 % transparency. a | (S)-methyl mandelate binding to the wild type. b | (S)-methyl mandelate binding to YH. c | (S)-methyl mandelate binding to CVH. d | (S)-methyl mandelate binding to HMVY. e and f | Sketch maps of the substrate-binding pocket of WT and HMVY indicating the steric hindrance of Met121. (Guo et al., 2013)

Site-directed Mutagenesis FAQs

Q: Which method should I choose for site-directed mutagenesis?
A: We offer two major approaches:
- Single-site mutagenesis: Ideal for point mutations, insertions, deletions, and nearby substitutions. Optimized protocols reduce time and cost.
- Multiple-site mutagenesis: Suitable for distant mutations across hundreds of base pairs. Efficient assembly avoids restriction enzyme use and introduces all desired mutations in one step.
Q: Can I provide my own DNA template?

A: Yes, SDM can be performed using customer-provided templates or DNA synthesized by Creative Enzymes.
Q: How many mutations can be introduced simultaneously?

A: Multiple-site SDM allows several distant mutations to be introduced efficiently in a single workflow.
Q: Do you provide downstream characterization?

A: Yes, we offer expression, purification, activity assays, and structural/mechanistic studies to ensure the mutant enzyme meets your research goals.
Q: How fast can I get the results?

A: Typical timelines: 1–2 weeks for upstream services, 2–3 weeks for mutagenesis, and inquiry-based timelines for downstream analyses.
Q: Are your SDM services cost-effective?

A: Yes, our competitive pricing starts from $99 per mutation, with high accuracy and reliability that saves time and reduces experimental repetition.

References:

Guo F, Xu H, Xu H, Yu H. Compensation of the enantioselectivity-activity trade-off in the directed evolution of an esterase from Rhodobacter sphaeroides by site-directed saturation mutagenesis. Appl Microbiol Biotechnol. 2013;97(8):3355-3362. doi:10.1007/s00253-012-4516-z
Mohammadi M, Sepehrizadeh Z, Ebrahim-Habibi A, Shahverdi AR, Faramarzi MA, Setayesh N. Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis. Enzyme and Microbial Technology. 2016;93-94:18-28. doi:10.1016/j.enzmictec.2016.07.006
Zhang K, Yin X, Shi K, et al. A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation. Sci Rep. 2021;11(1):10454. doi:10.1038/s41598-021-89884-z

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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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