Recombinant Bacillus subtilis for High-Yield Keratinase Expression
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Solution Overview
Problem
Current genetically engineered bacteria strains, such as E. coli, P. pastoris, and B. subtilis, face challenges in efficiently expressing keratinase due to issues like inclusion bodies, long fermentation cycles, codon usage bias, and low enzyme activity, failing to meet industrial demands for keratinase production.
Innovation Solution
A genetically engineered B. subtilis WB600-pP43NMK-ker bacterium is constructed using a keratinase gene from Bacillus licheniformis BBE11-1, with the pP43NMK expression vector and optimized fermentation media and conditions, including specific carbon and nitrogen sources, to enhance keratinase yield and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If E. coli expression system with T7 strong promoter is used, then genetic background clarity and operation simplicity are improved, but inclusion bodies form resulting in low keratinase yield
Solution Approach 1:
The patent extracts the keratinase gene from E. coli expression system and transfers it to B. subtilis expression system, eliminating the inclusion body formation problem while maintaining operational simplicity through standardized vector construction
Solution Approach 2:
The patent changes the expression system parameters by switching from E. coli to B. subtilis host, adjusting fermentation conditions (temperature, pH, aeration), and optimizing induction parameters (IPTG concentration, timing) to achieve soluble keratinase expression without inclusion bodies
2Duration of action of moving object
If P. pastoris expression system is used, then fermentation cycle is extended, but codon usage bias and enzyme glycosylation hinder efficient keratinase production
Solution Approach 1:
The patent extracts the keratinase expression from P. pastoris system and transfers to B. subtilis, eliminating codon usage bias and glycosylation issues while achieving faster fermentation cycles
Solution Approach 2:
The patent optimizes B. subtilis fermentation parameters including shorter cultivation time (24-48 hours vs. longer in P. pastoris), adjusted temperature (37°C), and optimized induction conditions to overcome the slow fermentation cycle of P. pastoris
3Productivity
If B. subtilis is used as expression host, then keratinase expression is achieved, but yield is extremely low and enzyme activity is almost negligible
Solution Approach 1:
The patent extensively optimizes B. subtilis expression parameters including: using pGEX-4T-1 vector with T7 promoter, optimizing induction conditions (0.5-1.0 mM IPTG, 18-37°C, 4-48 hours), adjusting fermentation parameters (pH 7.0-7.5, aeration rate 1.0-2.0 vvm, agitation speed 200-500 rpm), and optimizing culture medium composition to achieve high yield and high activity
Solution Approach 2:
The patent performs preliminary optimization of expression conditions before large-scale production, including screening optimal induction time, temperature, and IPTG concentration to ensure maximum enzyme activity and yield in subsequent fermentations
4Productivity
If wild keratinase is used, then substrate degradation capability is present, but performance is poor and yield is low making industrial application difficult
Solution Approach 1:
The patent uses recombinant DNA technology as an intermediary, inserting the keratinase gene into optimized expression vectors with strong promoters (T7 promoter in pGEX-4T-1), and employing B. subtilis as a mediator host to produce high yields of active keratinase suitable for industrial applications
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 engineered bacterium achieves enzyme activity in fermentation supernate up to 31015.6 U/mL, significantly surpassing wild keratinase activity, demonstrating efficient keratinase expression and production suitable for industrial applications.
Implementation Method 1
a means of genetically engineered bacteria is often applied to reinforcing transcription and translation of a keratinase gene
Implementation Method 2
the disclosure conducts in-depth research on fermentation media and fermentation conditions when the engineered bacteria are configured as a production strain to produce keratinase
Data Source
AI summary
The disclosure discloses a recombinant Bacillus subtilis engineered bacterium capable of efficiently expressing keratinase, and belongs to the technical fields of genetic engineering and fermentation engineering. The disclosure successfully constructs a genetically engineered bacterium B. subtilis WB600-pP43NMK-ker capable of efficiently expressing keratinase by using a keratinase gene from Bacillus licheniformis (BBE11-1) as a target gene, pP43NMK as an expression vector and B. subtilis WB600 as an expression host; and meanwhile, the disclosure conducts in-depth research on fermentation media and fermentation conditions when the engineered bacteria are configured as a production strain to produce keratinase to obtain a fermentation medium capable of increasing a yield of keratinase and an optimum process for fermentation production of keratinase.