CN105462994A - Thermophilic alkaline recombination manganese-catalase-containing gene, recombination carrier thereof and application thereof - Google Patents

Abstract

The invention relates to the field of gene engineering, in particular to a thermophilic alkaline recombination manganese-catalase-containing gene, a recombination carrier thereof and an application thereof. The thermophilic alkaline recombination manganese-catalase-containing gene is shown as SEQ ID No.1. According to the thermophilic alkaline recombination manganese-catalase-containing gene, the recombination carrier and the application, the sequence of the manganese-catalase-containing gene from metallosphaera hakonensis is subjected to codon optimization according to the use preference of escherichia coli codon, and when optimized manganese catalase is expressed in escherichia coli, the highest activity, capable of being detected in flask fermentation liquor, of the catalase is 300 U/mL.

Description

A kind of thermo philic alkali restructuring is containing manganese silicide gene and recombinant vectors thereof and application

Technical field

The present invention relates to genetically engineered field, particularly, the present invention relates to the restructuring of a kind of thermo philic alkali containing manganese silicide gene and recombinant vectors thereof and application.

Background technology

Catalase (Catalase is called for short CAT), catalysis H ₂o ₂be decomposed into H ₂o and O ₂.The CAT in various source is widely used in the industrial circles such as foodstuffs sterilisation, medical analysis and diagnosis, weaving, papermaking.In weave dyeing and finishing technique, CAT is mainly used in removing H remaining in fabric bleaching waste liquid ₂o ₂, eliminate its impact on follow-up dyeing process.Compared with traditional chemical technique, CAT has environmental friendliness, fabric follow-up printing quality advantages of higher; But it should be noted that, in view of the singularity of textile printing and dyeing process, the catalyzed reaction of CAT need be carried out usually in high temperature (T>70 DEG C) alkalescence (pH>9) environment, and this just needs CAT to possess the application characteristic of thermophilic basophilic.And the thermophilic alkali resistance CAT of bibliographical information is still less at present.

CAT can be divided into two classes by the constructional feature of protein three-dimensional crystallographic structure: the CAT of (1) catalytic center iron content porphyrin ring structure, claims iron catalase (FeCAT); (2) catalytic center replaces iron ion porphyrin structure by mn ion, is called manganese silicide (MnCAT).In recent years, investigator finds that the bacterium of respective source in high-temperature alkaline environment can produce the MnCAT with thermophilic basophilic characteristic, such as: the MnCAT that raw gold goal bacterium (Metallosphaerahakonensis) of case root is originated, in pH8.0-10.0 environment, process the loss alive of 60min enzyme and be less than 20%; At 70 DEG C, process 50min, enzyme lives residual rate about 60%; But the MnCAT in Metallosphaerahakonensis source only has a small amount of activity expression albumen in intestinal bacteria at present, and document ^[1]in do not state concrete enzyme and to live value.

Summary of the invention

The present invention in view of the foregoing, according to e. coli codon usage bias, carry out codon optimized to the manganese silicide gene order in Metallosphaerahakonensis source, a kind of thermo philic alkali is provided to recombinate containing manganese silicide gene and expression vector thereof and recombinant bacterial strain, for solving the problems of the prior art.

For achieving the above object and other relevant objects, the invention provides the encoding gene of a kind of thermo philic alkali restructuring containing manganese silicide, its sequence is as shown in SEQIDNo.1:

The manganese silicide gene nucleotide series that described encoding gene is originated according to Metallosphaerahakonensis, according to intestinal bacteria (Escherichiacoli) codon usage bias (http://gcua.schoedl.de/seqoverall_v2.html) optimization design.The manganese silicide gene nucleotide series in described Metallosphaerahakonensis source is selected from GenBank database.

Second aspect present invention providing package is containing the recombinant vectors of above-mentioned restructuring thermo philic alkali containing manganese silicide gene.Preferably, the expression vector that described recombinant vectors uses is pET series expression vector.

Preferred, described pET series expression vector is pET24a (+), obtains and comprises the recombinant vectors pET24a-MnCat of above-mentioned restructuring thermo philic alkali containing manganese silicide gene.Restructuring thermo philic alkali of the present invention is inserted between the suitable restriction enzyme site of expression vector containing manganese silicide gene, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, preferably restructuring thermo philic alkali of the present invention is inserted between NdeI and the HindIII restriction enzyme site on plasmid pET24a (+) containing manganese silicide gene, obtain recombinant expression vector pET24a-MnCAT, be kept in bacillus coli DH 5 alpha after sequence verification.

Third aspect present invention providing package is containing the recombinant bacterial strain of above-mentioned thermo philic alkali restructuring containing manganese silicide gene.

Described recombinant bacterial strain transforms acquisition by described restructuring containing manganese silicide expression vector pET24a-MnCAT.

Preferably, described recombinant bacterial strain is obtained containing manganese silicide expression vector transformation of E. coli by described restructuring thermo philic alkali.

Preferred, described intestinal bacteria are e. coli bl21 (DE3), and the recombinant bacterial strain of acquisition is e. coli bl21 (DE3)/pET24a-MnCAT.

Fourth aspect present invention provides described thermo philic alkali to recombinate containing manganese silicide MnCAT, is recombinated containing manganese silicide genes encoding by thermo philic alkali according to claim 1.

Fifth aspect present invention provides described thermo philic alkali to recombinate containing the preparation method of manganese silicide, comprises the steps:

1) with above-mentioned recombinant vectors transformed host cell, recombinant bacterial strain is obtained;

2) cultivate recombinant bacterial strain, the restructuring of induction restructuring thermo philic alkali is expressed containing manganese silicide;

3) the thermo philic alkali restructuring of reclaiming also expressed by purifying contains manganese silicide.

As preferably, above-mentioned restructuring thermo philic alkali, containing the preparation method of manganese silicide, comprises the following steps:

Described recombinant bacterial strain BL21 (DE3)/pET24a-MnCAT is activated in LB substratum after spending the night, be forwarded in TB substratum, when the dense OD600 of bacterium reaches 2, add MnCl ₂and IPTG, then inducing culture, obtain restructuring thermo philic alkali containing manganese silicide.

Enzyme activity in the bacterial cell disruption supernatant liquor of gained nutrient solution can reach 300U/mL.

Further, because carrier pET24a (+) is upper containing 6-His purification tag, the Ni-NTA resin affinity chromatography commonly used therefore is selected to carry out protein purification.

Preferably, transfer by 5% during switching.

Preferably, be forwarded in the TB substratum containing 50 μ g/mlKan.

Preferably, the final concentration of described IPTG is 0.08mmol/L, described MnCl ₂final concentration 10mmol/L.Preferably, the actual conditions of described inducing culture is: under 37 DEG C of conditions, induce 24h.

Present invention also offers the application of above-mentioned thermo philic alkali restructuring containing manganese silicide gene, the application containing manganese silicide in the high-efficiency activated expression Metallosphaerahakonensis source especially in intestinal bacteria.

Compared with the MnCAT that the MnCAT that Metallosphaerahakonensis originates and the Thermusthermophilus stated in patent CN103725699A originates, the aminoacid sequence of its albumen has the difference of about 5%; The difference of aminoacid sequence causes the direct change of two kinds of MnCAT three-dimensional conformations, thus cause the difference of its zymologic property, such as: the optimum PH range deriving from the MnCAT of Metallosphaerahakonensis is pH8.0-10.0, mild in the change of this scope inner enzyme vigor, good stability ^[1]; But the optimal pH deriving from the MnCAT of Thermusthermophilus is at pH10.0, in rising trend at pH8.0-10.0 scope inner enzyme vigor, without between flat zone ^[2].Notable difference on zymologic property, shows that two kinds of source MnCAT have its respective uniqueness.

The present invention is according to e. coli codon usage bias, carry out codon optimized to the manganese silicide gene order in Metallosphaerahakonensis source, when the MnCAT after optimizing is at expression in escherichia coli, the highlyest in shake flask fermentation liquid detect that CAT enzyme is lived as 300U/mL, the present invention is optimized by gene codon and fermentation optimization achieve Metallosphaerahakonensis source the high-efficiency activated expression of MnCAT in intestinal bacteria, and its fermenting enzyme is lived, value reaches about 300U/mL, for the further application and development of this enzyme is laid a good foundation.

Reference

[1]ShinjiEbara,YasushiShigemori.Alkali-toleranthigh-activitycatalasefromathermophilicbacteriumanditsoverexpressioninEscherichiacoli,Proteinexpressionandpurification,2008.57(2):255-260.

[2] He Xiaojuan, Xue Zhenglian, Zhao Zhijun, waits the expression of .ThermusthermophilusHB27 manganese silicide in intestinal bacteria and characterization analysis. Chinese biological engineering magazine, 2014,34 (2): 52-58.

Accompanying drawing explanation

The physical map of Fig. 1 expression vector pET24a-MnCAT.

The enzyme of Fig. 2 expression vector pET24a-MnCAT cuts qualification; Wherein,

M1:DNAMarkDL2000; M2:DNAMark λ-EcoT14Idigest; DNA fragmentation after 1-2:NdeI and HindIII double digestion.

The SDS-PAGE figure of Fig. 3 recombination bacillus coli E.coliBL21 (DE3)/pET24a-MnCAT; Wherein,

M:ProteinMarkerBM524; 1: cytoclasis supernatant liquor; 2: the centrifuged supernatant after cytoclasis supernatant liquor places 2h in 75 DEG C of environment.

Embodiment

Test materials and reagent

1, bacterial strain and carrier

Coli expression carrier pET24a (+) and bacterial strain BL21 (DE3) is purchased from Invitrogen company.

2, enzyme and other biochemical reagents

Restriction enzyme and DNA ligase and other reagent are purchase.

3, substratum: be and buy or prepare according to existing culture medium prescription.

Illustrate: in following examples, do not make the experimental methods of molecular biology illustrated, concrete grammar listed in equal reference " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book carries out, or carries out according to test kit and product description.

The structure of embodiment 1 manganese silicide MnCAT prokaryotic expression genetic engineering bacterium

1.1 based on the MnCAT gene of e. coli codon usage bias optimization design

With the manganese silicide gene nucleotide series in existing Metallosphaerahakonensis source in GenBank database for template, the preferences that uses according to intestinal bacteria (Escherichiacoli) codon ( http:// gcua.schoedl.de/seqoverall_v2.html) optimization design, the MnCAT gene after gained is codon optimized has the nucleotide sequence as shown in sequence SEQIDNo.1.

The structure of 1.2MnCAT prokaryotic expression carrier pET24a-MnCAT

At the 5 '-end and 3 '-hold and design respectively in this gene and do not possess but the restriction enzyme NdeI that carrier pET24a (+) multiple clone site has and HindIII restriction enzyme site of MnCAT gene, directly synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, MnCAT gene fragment is on NdeI and HindIII double digestion rear clone to expression vector pET24a (+) after same enzyme is cut, build recombinant expression vector pET24a-MnCAT (structure as shown in Figure 1), be kept in bacillus coli DH 5 alpha after sequence verification.

The structure of 1.3 recombinant strains BL21 (DE3)/pET24a-MnCAT

Recombinant expression plasmid pET24a-MnCAT transformation of E. coli BL21 (DE3) competent cell, coat LB flat board (Kan, 50 μ g/mL), be inverted, be cultured to the single bacterium colony of appearance restructuring in 37 DEG C, picking is recombinated single bacterium colony, LB liquid nutrient medium (Kan, 50 μ g/mL) in cultivate after 10-12h, extract plasmid and carry out NdeI and HindIII double digestion checking correct rear (as described in Figure 2), obtain recombination bacillus coli BL21 (DE3)/pET24a-MnCAT.

The abduction delivering of embodiment 3 manganese silicide MnCAT

2.1 seed culture

By genetic engineering bacterium BL21 (DE3)/pET24a-MnCAT, access is containing in the 50mLLB substratum of 50 μ g/mL kantlex, and 37 DEG C, rotating speed is 200rpm, and incubation time is 8-10 hour, and after cultivating, OD600 light absorption value is between 4 ~ 5.

Note: LB substratum (g/L) component is: peptone 10, yeast powder 5, sodium-chlor 10.

2.2 shake flask fermentations are cultivated:

Seed culture fluid is forwarded in the TB substratum containing 50mg/L kantlex (Kan) by 5%, when recombinant bacterium E.coliBL21 (DE3)/pET24a-MnCAT grows to OD ₆₀₀be worth to 2 time, add final concentration 0.08mmol/LIPTG, add final concentration 10mmol/LMnCl simultaneously ₂inducing culture 24h at 37 DEG C, after collecting thalline, smudge cells, SDS-PAGE analysis is carried out to supernatant liquor, find near 33Kda, there is obvious protein band, consistent with the MnCAT molecular weight of albumen of bibliographical information (as shown in Figure 3), carry out the order-checking of N end to obtained albumen, result meets expection.Enzyme activity determination result shows, and in broken supernatant liquor, CAT enzyme activity is up to 300U/mL.

Note: TB substratum (g/L) component is: glycerine 5, peptone 12, yeast powder 24, K ₂hPO ₄12.54, KH ₂pO ₄2.31.

In sum, the CAT enzyme activity expressed by sequence being contained manganese silicide by restructuring provided by the present invention reaches about 300U/mL, possesses good application and development potentiality.

Claims (8)

1. thermo philic alkali restructuring is containing a manganese silicide gene, and it is characterized in that, its base sequence is as shown in SEQIDNo.1.

2. comprise thermo philic alkali restructuring described in claim 1 containing the recombinant vectors of manganese silicide gene.

3. recombinant vectors according to claim 1, is characterized in that, described recombinant vectors is pET24a (+).

4. comprise thermo philic alkali restructuring described in Claims 2 or 3 containing the recombinant bacterial strain of manganese silicide gene.

5. recombinant bacterial strain according to claim 4, is characterized in that, described recombinant bacterial strain is e. coli bl21 (DE3).

6. thermo philic alkali restructuring is containing a manganese silicide MnCAT, it is characterized in that, is recombinated containing manganese silicide genes encoding by thermo philic alkali according to claim 1.

7. prepare thermo philic alkali restructuring containing a method of manganese silicide MnCAT, comprise the following steps:

1) with the recombinant vectors transformed host cell described in claim 1 or 3, recombinant bacterial strain is obtained;

2) cultivate recombinant bacterial strain, the restructuring of induction restructuring thermo philic alkali is expressed containing manganese silicide;

3) the thermo philic alkali restructuring of reclaiming also expressed by purifying contains manganese silicide.

8. thermo philic alkali restructuring described in claim 1 is containing the application of manganese silicide gene.