Phytase mutant yeappa-l162v and its coding gene and application
A technology encoding a gene and phytase, applied to the phytase mutant YeAPPA-L162V and its encoding gene and its application field, can solve the problems of low catalytic efficiency, poor thermal stability, and narrow pH range
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0027] Embodiment 1: Obtaining of mutant genes
[0028] A mutation was introduced into the gene sequence (SEQ ID NO. 2) of the phytase YeAPPA derived from Yersinia enterocolitica through two rounds of PCR reactions using the Overlap PCR method to obtain the mutant gene YeAPPA-L162V. The PCR template is the pEASY-T3-YeAPPA recombinant plasmid. The upstream and downstream primers for amplifying the complete coding sequence of the mutated gene carried EcoR I and Not I recognition sequences, respectively YeAPPA Forward:5'-CGCGAATTCGCACCGCTTGCAGCACAATCTAC-3' and YeAPPAReverse:5'-GATGCGGCCGCTTAAATATGGCAGGCTGGCTCG-3'. The upstream and downstream primers for introducing mutations at specific positions were YeAPPA L162V Forward: 5’-GTCTGTAAAGTGGACTCAGCGAAAACTC-3’ and YeAPPA L162VReverse: 5’-GAGTTTTCGCTGAGTCCACTTTACAGAC-3’, respectively. The desired mutant gene was connected to the pEASY-T3 vector and verified by the company's sequencing.
Embodiment 2
[0029] Example 2: Expression and purification of mutant enzyme YeAPPA-L162V and wild enzyme YeAPPA in bacteria
[0030] The wild-type and mutant phytases were removed from the signal peptide sequence, cloned between the EcoRI and NotI sites of the expression vector pET-22b (+), and subjected to 2 mM IPTG (isopropyl -β-D-galactoside) induced expression. The crude enzyme solution was purified by nickel-nitrilotriacetic acid (Ni-NTA) column and diethylaminoethyl (DEAE) column, and the purified enzyme was separated by 10% SDS-PAGE electrophoresis to analyze its apparent molecular weight. The wild enzyme and the mutant enzyme have a total length of 441 amino acids, the N-terminal 23 amino acids are signal peptide sequences, and the theoretical molecular weight of the mature protein is 48.6 kDa. The coding region sequence except the signal peptide was prokaryotically expressed and purified, and a specific band of about 46 kDa was detected on SDS-PAGE electrophoresis (data not shown...
Embodiment 3
[0031] Embodiment 3: Enzymatic property comparison of mutant enzyme and wild enzyme
[0032] Purified wild-type and mutant phytase were reacted for 30 minutes at different pH (1-12) and different temperature (30-80° C.), and their optimum pH and optimum temperature were detected according to enzyme activity. The buffer used includes 0.1mol / L glycine-hydrochloric acid buffer (pH1-3), 0.1mol / L sodium acetate-acetic acid buffer (pH3-6), 0.1mol / L Tris-hydrochloric acid buffer (pH6-8) And 0.1mol / L glycine-sodium hydroxide buffer solution (pH8-12). The purified enzyme solution was treated at 37°C for 1 hour at pH 1-9 to study the pH stability of the enzyme. At a certain temperature, the purified enzyme solution was treated for 0, 2, 5, 10, 20, 30, and 60 minutes, and then its thermal stability was measured. Phytase activity was determined using the ferrous sulfate molybdenum blue method. With 1.5mmol / L sodium phytate as substrate, react at 37°C for 30min, terminate the reaction w...
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More