Phi29 DNA Polymerase Mutants for Thermostability

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Solution Overview

Problem

The mesophilic nature of phi29 DNA polymerase results in poor stability, leading to a short storage period and increased production, storage, and transportation costs, as well as limited validity and decreased activity over time.

Innovation Solution

Mutating the amino acid sequence at specific positions, such as 470 and 474, to enhance thermal stability, with the use of recombinant vectors and expression cassettes to produce more stable phi29 DNA polymerase mutants, including V470K and I474K, which exhibit lower activity loss and higher remaining activity ratios compared to wild-type phi29 DNA polymerase.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If phi29 DNA polymerase is stored at low temperatures (-20°C or -80°C), then stability is improved, but storage cost and operation complexity increase

Engineering Contradiction:

ImprovestabilityVSAvoidoperation complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent applies parameter changes by mutating specific amino acid residues in the phi29 DNA polymerase sequence to alter its thermal stability properties. The mutant polymerase maintains stability at higher temperatures, eliminating the need for cold storage infrastructure and simplifying operational procedures while preserving enzyme activity.

Inventive Principle:

Principle #35Parameter changes

2Reliability

If phi29 DNA polymerase is stored at low temperatures, then stability is improved, but validity period is limited and activity decreases over time

Engineering Contradiction:

ImprovestabilityVSAvoidvalidity period

Core Design Contradiction:

ReliabilityVSDuration of action of stationary object

Solution Approach 1:

The patent changes the amino acid composition parameters of the polymerase through site-directed mutagenesis, creating variants with enhanced thermal stability. This allows the enzyme to maintain its catalytic activity and structural integrity for extended periods at higher storage temperatures, effectively prolonging the validity period.

Inventive Principle:

Principle #35Parameter changes

3Manufacturing precision

If phi29 DNA polymerase is purified using refrigerator or chromatography cabinet, then purity is improved, but preparation cost increases

Engineering Contradiction:

ImprovepurityVSAvoidpreparation cost

Core Design Contradiction:

Manufacturing precisionVSEase of manufacture

Solution Approach 1:

The patent modifies the polymerase's amino acid sequence to enhance its inherent stability, allowing the enzyme to maintain purity and activity without requiring expensive refrigerated storage infrastructure during purification and handling. This reduces capital equipment costs and operational expenses.

Inventive Principle:

Principle #35Parameter changes

4Reliability

If additives like trehalose or BSA are added to stock solution, then stability is improved for low enzyme concentration, but stability does not improve for high enzyme concentration

Engineering Contradiction:

ImprovestabilityVSAvoidconcentration adaptability

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent changes the fundamental parameter of the polymerase molecule itself through amino acid substitution, rather than relying on external additives. This intrinsic modification provides concentration-independent stability enhancement, making the enzyme stable across a wide range of concentrations without requiring formulation optimization.

Inventive Principle:

Principle #35Parameter changes

Data Source

PatentEP3617308B1Phi29 DNA polymerase mutant with increased thermostability

Publication Date: 2023.10.25 MGI TECH CO LTD

Figure 1~2A
Figure 2B~3A
Figure 3B

AI summary

Provided are a phi29 DNA polymerase mutant with increased thermo stability, a method for preparing the mutant, the use of the mutant, and a method for increasing the stability of the phi29 DNA polymerase.