Alkaline Protease Mutant Gene Engineering for Enzyme Activity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current alkaline protease-producing Bacillus strains have limited enzyme-producing capacity and high production costs, restricting their large-scale industrial application due to relatively low enzyme activity.
Innovation Solution
Employing error-prone PCR and DNA shuffling to mutate the alkaline protease gene from Bacillus clausii, followed by expression in Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis systems to enhance enzyme activity, resulting in highly active alkaline protease mutants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Bacillus strains are used to produce alkaline protease, then the enzyme can be produced through microbial fermentation, but the enzyme-producing capacity is limited and production costs are high
Solution Approach 1:
The patent applies parameter changes by modifying the gene sequence of alkaline protease through site-directed mutagenesis. Specific amino acid residues in the enzyme structure are changed to optimize catalytic efficiency and stability, thereby increasing enzyme activity and productivity while reducing production costs through more efficient fermentation
2Reliability
If traditional chemical substances are used in leather manufacturing, then the treatment process is effective, but toxic substances harm safety and cause environmental pollution
Solution Approach 1:
The patent replaces chemical treatment methods with enzymatic treatment using alkaline protease in leather manufacturing. The enzyme specifically degrades non-fibrous proteins and non-colloid components in leather, achieving effective treatment without the toxicity and environmental pollution associated with traditional chemical substances
3Productivity
If alkaline protease activity is increased through genetic modification, then enzyme productivity improves, but the complexity of the production process increases
Solution Approach 1:
The patent applies preliminary action by conducting in-vitro directed evolution and site-directed mutagenesis before industrial production. The gene is optimized in advance to encode enzymes with improved activity and stability, allowing straightforward expression in Bacillus hosts without requiring complex process modifications during production
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The approach significantly increases the activity of alkaline protease, enabling its efficient production and application in industries such as detergents, food, and leather manufacturing, while reducing production costs and environmental impact.
Implementation Method 1
Employing error-prone PCR and DNA shuffling to mutate the alkaline protease gene from Bacillus clausii
Implementation Method 2
Employing error-prone PCR and DNA shuffling to mutate the alkaline protease gene from Bacillus clausii
Implementation Method 3
Protease, a hydrolase, can degrade protein molecules and polypeptides into small peptide chains and amino acids by catalyzing splitting of protein peptide bonds
Data Source
AI summary
An alkaline protease mutant, and a gene, engineered strain, a preparation method and application thereof are provided. The method comprises the following steps of extracting genome DNA of Bacillus clausii, performing PCR amplification to obtain a wild-type alkaline protease gene sequence, mutating the wild-type alkaline protease gene obtained by the amplification through an error-prone PCR, performing high-throughput screening to obtain a plurality of highly active alkaline protease genes, performing DNA shuffling on the highly active alkaline protease genes, and performing screening to obtain eight alkaline protease mutant genes with higher activity.