An Alkaline Pectinase Mutant with Improved Secretion Performance
A technology of secretion performance and pectinase, which is applied in the field of bioengineering, can solve the problems of high-potency commercial alkaline pectinase, which cannot be ignored in research, and achieve enhanced heat resistance, increased expression of extracellular secretion, and enzymatic properties Improved effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0027] The construction of embodiment 1 mutant expression plasmid and the acquisition of recombinant Bacillus subtilis
[0028] 1. Construct a mutant expression vector using the pET-20b(+)-pgl plasmid as a template
[0029] The nucleotide sequence of the gene encoding the wild-type alkaline pectinase and the signal peptide consisting of 21 amino acids is shown in SEQ ID NO.1, and the amino acid sequence of the wild-type mature alkaline pectinase is shown in SEQ ID NO.2. By analyzing the three-dimensional structure of alkaline pectinase, it is speculated that the isoleucine I at position 58 has a greater impact on the secretion and expression of alkaline pectinase, and a mutation experiment was designed to mutate the isoleucine I at position 58 into valine Acid V.
[0030] Using the pET-20b(+)-pgl plasmid as a template, the plasmid containing the mutant gene was amplified in vitro by PCR (polymerase chain reaction), and the isoleucine at position 58 was mutated into valine.
...
Embodiment 2
[0046] Expression of embodiment 2 mutant PGL
[0047] Seed medium composition (g / L): yeast powder 5, tryptone 10, NaCl 10, glucose 20, pH 7.0.
[0048] Composition of fermentation medium: yeast powder 24g / L, tryptone 12g / L, glycerol 5g / L, K 2 HPO 4 72mmolL -1 , KH 2 PO 4 17mmolL -1 .
[0049] Inoculate the recombinant bacteria E.coliBL21(DE3) (pET-20b(+)-pglI58V) containing the mutant expression vector pET-20b(+)-pglI58V into 100μgmL -1 In the seed medium of ampicillin, the filling volume is 20mL / 250mL. Culture temperature 37℃, 200rpmmin -1 Incubate on a shaker for 10 h.
[0050] The seed solution cultivated for 10h was inoculated with 100μgmL with a 3% (V / V) inoculum -1 In the fermentation medium of ampicillin, the filling volume is 50mL / 500mL, at 37°C, 200rmin -1 to cultivate. Bacteria grow to a certain stage (OD 600 =0.6), adding a final concentration of 0.4mMIPTG for induction, while adjusting the temperature to 30°C, and inducing fermentation for 48h.
Embodiment 3
[0051] Example 3 PGL extracellular enzyme activity before and after mutation
[0052] According to the method described in Example 2, E.coliBL21(DE3) containing the unmutated expression vector pET-20b(+)-pgl and the mutant strain E.coliBL21(DE3)(pET-20b(+)-pglI58V ) for fermentation.
[0053] figure 1 SDS-PAGE protein electrophoresis showed that the extracellular secretion expression of alkaline pectinase I58V was significantly increased after mutation, the extracellular enzyme activity of alkaline pectinase was 129.65U / mL before mutation, and the extracellular enzyme activity of alkaline pectinase increased after mutation It was 337.58U / mL, which was 2.60 times that before the mutation. Combined with Table 1, the specific enzyme activity of alkaline pectinase after mutation increased from 179.14U / mg to 295.63U / mg, which was 1.65 times that before mutation. It shows that after the mutation, the secretion ability of alkaline pectinase I58V is enhanced, and the enzyme activit...
PUM
![No PUM](https://static-eureka-patsnap-com.libproxy1.nus.edu.sg/ssr/23.2.0/_nuxt/noPUMSmall.5c5f49c7.png)
Abstract
Description
Claims
Application Information
![application no application](https://static-eureka-patsnap-com.libproxy1.nus.edu.sg/ssr/23.2.0/_nuxt/application.06fe782c.png)
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap