阅读说明：本技术 一种定点突变改造的β-半乳糖苷酶及其构造方法 (Site-directed mutagenesis modified beta-galactosidase and construction method thereof ) 是由 盖宏伟 金潇婷 张业旺 于 2020-06-17 设计创作，主要内容包括：本发明提供了一种定点突变改造的β-半乳糖苷酶,属于基因工程领域。本发明的β-gal突变体是由野生型β-gal的氨基酸序列基础上,发生第229位Ala突变为Cys。并将该突变酶转化到大肠杆菌,进行异源表达。相较于野生酶,突变酶的酶活力提高了1倍左右。β-gal进行定点突变改造后,暴露在蛋白质表面的巯基,与修饰巯基的磁纳米颗粒之间,通过巯基-二巯基交换反应,完成酶的共价固定化。(The invention provides a beta-galactosidase modified by site-directed mutagenesis, belonging to the field of genetic engineering. The beta-gal mutant of the invention is characterized in that the 229 th Ala is mutated into Cys based on the amino acid sequence of wild beta-gal. And transforming the mutant enzyme into Escherichia coli for heterologous expression. Compared with wild enzyme, the enzyme activity of the mutant enzyme is improved by about 1 time. After the beta-gal is subjected to site-directed mutagenesis modification, the thiol exposed on the surface of the protein and the magnetic nanoparticle for modifying the thiol are subjected to thiol-dimercapto exchange reaction to complete covalent immobilization of the enzyme.)

1. A β -gal mutant, characterized in that: the amino acid sequence is shown in SEQ ID No. 1.

2. A gene encoding the mutant of claim 1.

3. A recombinant plasmid vector characterized by: the recombinant plasmid vector contains the gene according to claim 2.

4. A host cell, characterized in that: the host cell contains the gene of claim 2 or the recombinant plasmid vector of claim 3.

5. The host cell of claim 4, wherein: the host cell is e.

6. The method of constructing the β -gal mutant of claim 1, comprising the steps of:

(1) constructing a recombinant plasmid vector containing the coding gene of the beta-gal, wherein the recombinant plasmid vector takes escherichia coli as a host;

(2) using the recombinant vector in the step (1) as a template, and performing reverse PCR amplification by using primer pairs shown as SEQ ID No.5 and SEQ ID No.6 to obtain a PCR product, namely a circular plasmid, containing a base sequence shown as SEQ ID No. 2;

(3) and transforming the circular plasmid into a host cell, namely E.coliBL21, so as to obtain the gene engineering bacteria containing the beta-gal mutant.

Technical Field

The invention relates to genetic engineering, in particular to a beta-galactosidase modified by site-directed mutagenesis and a construction method and application thereof.

Background

Beta-galactosidase (beta-galactosidase, beta-Gal, EC 3.2.1.23), often referred to as lactase, is widely present in various animals, plants and microorganisms, and is an important hydrolase with physiological and pathological effects. Its main physiological function is to catalyze hydrolysis of glycosidic bond and convert lactose into galactose, which plays a very important role in maintaining normal life activities, and the abnormality of β -gal activity and content is usually closely related to cancer.

In biochemical analysis, the increase in absorbance at 405nm after the enzymatic reaction (minus the absorbance value of the control) is used to detect the level of beta-gal activity, usually based on the principle that beta-gal can catalyze the hydrolysis of ONPG to produce ONP, which has a maximum absorbance peak at 405 nm. Furthermore, FDG is considered to be one of the most sensitive fluorogenic substrates that can be used for the detection of β -gal. Colorless and non-fluorescent FDG is hydrolyzed to highly fluorescent fluorescein with excellent spectral properties (Ex/Em 492/520nm) matching the optimal detection window for most fluorescent instruments. The β -gal catalyzed hydrolysis of FDG may be followed by an increase in fluorescence around 520 nm. The activity of beta-gal can be determined by fluorescence intensity.

At present, research shows that the fixed-point immobilization of enzyme can be successfully realized through a sulfhydryl-dimercapto exchange reaction. The thiol-dimercapto exchange reaction is one of the enzyme coupling reactions, and refers to a carrier with-SH or-S-through the thiol-dimercapto exchange reaction and the coupling of non-essential thiol on the enzyme molecule. The 613 th Cys of beta-gal is exposed on the protein surface, is closely indistinguishable from the related properties of beta-gal, and is reductive and easily oxidized, so the thiol group in Cys is not an unnecessary thiol group on the enzyme molecule. Thus, we attempted to complete the thiol-dimercapto exchange reaction by site-directed mutagenesis.

Site-directed mutagenesis techniques involve the precise alteration of one or more bases in a known nucleotide sequence, thereby altering one or more amino acid residues that make up a protein, in order to study the structural and functional relationships of the protein. The site-directed mutagenesis technique plays a great role in genetic engineering modification, and has very beneficial effects on the aspects of improving enzyme activity, improving the catalytic characteristics of the enzyme and the like. It has been used to mutate one or more amino acid residues on the surface of a protein to Cys by site-directed mutagenesis to achieve site-directed immobilization of the enzyme by thiol-dimercapto exchange reaction (J.R.Simons)^a,M.Mosisch^b,A.E.Torda^b,L.Hilterhaus^a,*Journal of Biotechnology 167(2013) 1-7) but the enzyme immobilization was accomplished by thiol-dimercapto exchange reaction with mutation of the β -gal surface amino acid to Cys and no application to date.

Based on the background, the invention intends to disclose a site-directed mutagenesis modified beta-gal and a construction method and application thereof.

Disclosure of Invention

The invention aims to mutate one or more amino acid residues of beta-gal into Cys by a site-directed mutagenesis technology, so that the Cys is immobilized on a magnetic nanoparticle modified with sulfydryl through a sulfydryl-dimercapto exchange reaction, and the site-directed immobilization of enzyme is realized.

As a first aspect of the present invention, there is provided a β -gal mutant, whose amino acid sequence is shown in SEQ ID No. 1.

As a second aspect of the present invention, there is also provided a gene encoding the above mutant.

As a third aspect of the present invention, there is also provided a recombinant plasmid vector containing the above gene.

The present invention also provides a host cell containing the above gene or the above recombinant plasmid vector, which constitutes the fourth aspect of the present invention.

Preferably, the host cell is e.

As a fifth aspect of the present invention, there is also provided a method for constructing the above β -gal mutant, comprising the steps of:

(1) constructing a recombinant plasmid vector containing the coding gene of the beta-gal, wherein the recombinant plasmid vector takes escherichia coli as a host;

(2) using the recombinant vector in the step (1) as a template, and performing reverse PCR amplification by using primer pairs shown as SEQ ID No.5 and SEQ ID No.6 to obtain a PCR product, namely a circular plasmid, containing a base sequence shown as SEQ ID No. 2;

(3) and transforming the circular plasmid into a host cell, namely E.coliBL21, so as to obtain the gene engineering bacteria containing the beta-gal mutant.

Compared with the prior art, the invention has the beneficial effects that:

compared with wild enzyme, the enzyme activity of the mutant enzyme is improved by about 1 time, and the immobilization of the enzyme is successfully completed through the mercapto-dimercapto exchange reaction between the mercapto group exposed on the surface of the protein and the magnetic nano-particles modified with the mercapto group after the beta-gal is subjected to site-specific mutagenesis modification.

Description of the drawings:

FIG. 1 is an SDS-PAGE analysis of β -gal before and after engineering, where M: a protein Marker; 1: affinity purified WT β -gal; 2: affinity purified β -gal-A229C;

FIG. 2 is a graph showing fluorescence spectra of WT β -gal and β -gal-A229C after reaction with FDG as a substrate

FIG. 3 is a graph showing the color change of solutions after binding of WT β -gal (right) and β -gal-A229C (left) to a substrate FDG after binding to a carrier Purimag Si-SH, respectively.

Detailed Description