Esterase mutant Est8-XL with improved activity and application thereof

A technology of est8-xl and mutants, applied to esterase mutant Est8-XL and its application field

Pending Publication Date: 2021-11-12
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims to provide a kind of esterase mutant Est8-XL with improved deacetylation activity of GDSL

Method used

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  • Esterase mutant Est8-XL with improved activity and application thereof
  • Esterase mutant Est8-XL with improved activity and application thereof
  • Esterase mutant Est8-XL with improved activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Construction of embodiment 1 mutant library

[0034] The esterase gene est8 in the thermophilic Bacillus sp. K91 strain was amplified by error-prone PCR to obtain the mutant sequence. Error-prone PCR was performed according to the instructions of the GeneMorph II EZClone Domain Mutagenesis Kit Random Mutagenesis Kit (purchased from Agilent Technologies Co., Ltd.).

[0035] 1) Est8 mutation large primer PCR

[0036] Using the recombinant plasmid containing the gene est8 extracted from the E.coliBL21(DE3) expression recombinant strain as the template for PCR, design primers:

[0037] Upstream primer est8F: 5'-GCAAATCATATTTATCTTGC-3';

[0038] Downstream primer est8R: 5'-CCTTTCTTTGATGATCGATTC-3'.

[0039] The PCR reaction system is as follows:

[0040]

[0041]

[0042] PCR reaction parameters: pre-denaturation at 95°C for 5 minutes; then denaturation at 95°C for 30 sec, annealing at 42°C for 30 sec, extension at 72°C for 1 min, and incubation at 72°C for 10 min ...

Embodiment 2

[0058] Example 2 Screening of mutants with improved deacetylation activity

[0059] 1) Primary screening: 480 mutants were screened.

[0060] Add 100 μL of substrate / well (the substrate is 50 mM 7-ACA, 0.02% BTB, and 50 mM phosphate buffer at pH 7.3) to 96-well ELISA plate; add 50 μL of cell lysate / well to 96 After mixing in the microplate plate, react at 25°C for 10 minutes, and then detect the OD value under a microplate reader with an absorbance of 616nm. Here, the colorimetric method is used to screen mutants: since esterase can catalyze 7-ACA to generate D-7-ACA and acetic acid, the pH of the solution drops at this time, and when there is an indicator of 0.02% BTB, the solution will turn from blue to blue. Green and then yellow, the more yellow the solution, the higher the activity of the enzyme.

[0061] 2) Twice re-screening: screen the 40 mutants screened out initially

[0062] The screened 40 mutants with high 7-ACA deacetylation activity were picked on a 96-well p...

Embodiment 3

[0064] Embodiment 3 preparation of wild enzyme Est8 and mutant Est8-XL

[0065] The recombinant strains containing the wild enzyme Est8 and the mutant Est8-XL were added to the test tubes containing 5 mL of Amp-LB liquid medium at an inoculation amount of 1‰ respectively (37 ° C, 180 rpm); after 15 hours, 4 mL was added to 400 mL Cultivate in Amp-LB liquid medium for 2.5h; add Isopropylβ-D-Thiogalactoside (IPTG) at a final concentration of 1.4mM to induce for 21h at 20°C and 150rpm; use a refrigerated centrifuge Collect the bacteria (4°C, 5000rpm, 10min); resuspend the bacteria with Tris-HCl 8.0 buffer and break the cells with an ultrasonic cell crusher; centrifuge at 12000rpm, 4°C for 20min to collect the supernatant; The supernatant protein solution was added to a Nickel-NTAAgarose purification column and eluted with a gradient elution solution containing 0-500mM imidazole to purify the target protein.

[0066] The SDS-PAGE results showed that both the mutant enzyme Est8-XL...

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Abstract

The invention discloses an esterase mutant Est8-XL with improved activity and application thereof. The amino acid sequence of the esterase mutant Est8-XL is as shown in SEQ ID NO. 1. The mutant esterase Est8-XL with improved activity is obtained through three rounds of screening by adopting error-prone PCR (Polymerase Chain Reaction) and a high-throughput screening method based on color change of a bromothymol blue (BTB) indicator. Compared with the wild esterase Est8, the mutant esterase Est8-XL has the advantages that the deacetylation activity on 7-aminocephalosporanic acid is improved, and the activity of the mutant esterase Est8-XL in a buffer solution with the pH value of 9.5 to 10.0 and the activity of the mutant esterase Est8-XL in a 10 mM NaCl solution, a 10 mM KCl solution, a 10 mM LiCl solution, a 10 mM CuSO4 solution, a 10 mM NiSO4 solution and a 10 mM ZnSO4 solution are improved. The mutant esterase Est8-XL disclosed by the invention can be applied to the technical field of biological medicines.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and enzyme engineering, and in particular relates to an esterase mutant Est8-XL with improved activity and its application. Background technique [0002] my country is a big country in the use of antibiotics and a big country in the production of antibiotics. Cephalosporin antibiotics are the largest family of antibiotics at home and abroad. Because of their advantages of high efficiency, low toxicity, broad spectrum and resistance to β-lactamase, they play an important role in the medical field. However, due to the overuse of antibiotics, there is a serious problem of the spread of drug-resistant bacteria, so the development of new antibiotics has become the general trend. [0003] Deacetyl-7-amino-cephalosporic acid (Deacetyl-7-amino-cephalosporic acid, D-7-ACA), an intermediate in the synthesis of new antibiotics, can remove the acetyl group at the 3-position under the action of d...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/55C12N15/70C12N1/21C12P35/00C12R1/19
CPCC12N9/16C12N15/70C12P35/00
Inventor 丁俊美王晓亮主虎杰黄遵锡刘艳周峻沛韩楠玉
Owner YUNNAN NORMAL UNIV
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