阅读说明：本技术 一种适用于谷氨酸棒杆菌分泌表达木聚糖酶的重组载体、表达系统和应用 (A kind of recombinant vector, expression system and application suitable for Corynebacterium glutamicum secreting, expressing zytase ) 是由 刘秀霞 张伟 白仲虎 杨艳坤 刘春立 于 2019-02-02 设计创作，主要内容包括：本发明提供了一种适用于谷氨酸棒杆菌分泌表达木聚糖酶的重组载体,其能实现于谷氨酸棒杆菌高分泌表达木聚糖酶。一种适用于谷氨酸棒杆菌分泌表达木聚糖酶的重组载体,其特征在于：重组载体包括可操作性连接的序列原件：AH6启动子、5’UTR及其前38bp序列、SD序列、cspB信号肽和木聚糖酶基因,AH6启动子、5’UTR及其前38bp序列和SD序列连接构成的片段其序列如SEQ ID NO.1所示,cspB信号肽的序列如SEQ ID NO.2所示。AH6启动子、5’UTR及其前38bp序列、SD序列、cspB信号肽和木聚糖酶基因构成双顺反子结构,能够实现木聚糖酶于谷氨酸棒杆菌的高分泌表达,酶活达到486.2 U/ml。(The present invention provides a kind of recombinant vector suitable for Corynebacterium glutamicum secreting, expressing zytase, it is able to achieve in Corynebacterium glutamicum hypersecretion expressed xylanase.A kind of recombinant vector suitable for Corynebacterium glutamicum secreting, expressing zytase, it is characterized by: recombinant vector includes the sequence original part of operability connection: AH6 promoter, 5 ' UTR and its preceding 38bp sequence, SD sequence, cspB signal peptide and xylanase gene, its sequence of segment that AH6 promoter, 5 ' UTR and its preceding 38bp sequence and SD sequence connect and compose is as shown in SEQ ID NO.1, and the sequence of cspB signal peptide is as shown in SEQ ID NO.2.AH6 promoter, 5 ' UTR and its preceding 38bp sequence, SD sequence, cspB signal peptide and xylanase gene constitute bicistronic construct, and the hypersecretion that can be realized zytase in Corynebacterium glutamicum is expressed, and enzyme activity reaches 486.2 U/ml.)

1. a kind of recombinant vector suitable for Corynebacterium glutamicum secreting, expressing zytase, it is characterised in that: the recombination carries Body includes the sequence original part of operability connection: AH6 promoter, 5 ' UTR and its preceding 38bp sequence, SD sequence, cspB signal peptide And xylanase gene, its sequence of segment such as SEQ that AH6 promoter, 5 ' UTR and its preceding 38bp sequence and SD sequence connect and compose Shown in ID NO.1, the sequence of cspB signal peptide is as shown in SEQ ID NO.2.

2. a kind of recombinant vector suitable for Corynebacterium glutamicum secreting, expressing zytase according to claim 1, Be characterized in that: the zytase is zytase xynA, and the zytase xynA gene is as shown in SEQ ID NO.3.

3. a kind of recombinant vector suitable for Corynebacterium glutamicum secreting, expressing zytase according to claim 2, Be characterized in that: the downstream connection of the xylanase gene has histidine tag.

4. a kind of recombinant vector suitable for Corynebacterium glutamicum secreting, expressing zytase according to claim 3, Be characterized in that: the sequence of the recombinant vector is as shown in SEQ ID NO.4.

5. recombinant vector as claimed in claim 4 is preparing the application in zytase.

6. a kind of expression system of zytase, the expression system include corynebacterium glutamicum, the Corynebacterium glutamicum Conversion has recombinant vector as claimed in claim 4.

7. expression system as claimed in claim 6 is preparing the application in zytase.

8. application according to claim 7, it is characterised in that: the application is the following steps are included: culture thallus, collection bacterium Body culture solution and purifying.

9. application according to claim 8, it is characterised in that: the collection bacterial culture fluid uses centrifugation, condition For 12000 rpm of revolving speed, 4 DEG C of 5 min of centrifugation, supernatant is collected.

10. application according to claim 8, it is characterised in that: the purifying carries out affinity chromatography using nickel column, then adopts With desalting column desalination, in affinity chromatography for balancing, the buffer solution A of loading are as follows: 300mM NaCl, 20mM Tris, pH8.0's Buffer, the buffer solution B for elution are as follows: 300mM NaCl, 250mM imidazoles, the buffer of 20mM Tris, pH8.0.

Technical field

The present invention relates to it is a kind of suitable for the recombinant vector of Corynebacterium glutamicum secreting, expressing zytase, expression system and Using.

Background technique

Zytase is a kind of general name that can be catalyzed xylan and generate the class of enzymes of xylo-oligosaccharide or wood oligose, cutting Position is β-Isosorbide-5-Nitrae glycosidic bond of substrate.Zytase belongs to important industrial enzyme, has in the industries such as food and papermaking biggish Application value.

Corynebacterium glutamicum as amino acid and organic acid in addition to producing or a kind of very promising foreign protein table Up to host.Relative to Bacillus coli expression host, Corynebacterium glutamicum expression system has the advantage that endotoxin-free, more Safety；Soluble secreting, expressing, the foreign protein of expression can be secreted into extracellular, and purifying is relatively easy to.Utilizing glutamic acid rod Bacillus carries out having accumulated bulk fermentation technology and experience during amino acids production, can mention for the large-scale production of foreign protein For basis and use for reference.

For these reasons, it is proposed that on the one hand may be implemented using Corynebacterium glutamicum secreting, expressing zytase Secreting, expressing, on the other hand again can in order to avoid endotoxin caused by apply upper limitation.But existing carrier cannot achieve xylan Enzyme is in Corynebacterium glutamicum secreting, expressing.

Summary of the invention

In view of the above-mentioned problems, the present invention provides a kind of recombinations suitable for Corynebacterium glutamicum secreting, expressing zytase Carrier is able to achieve in Corynebacterium glutamicum hypersecretion expressed xylanase.

Its technical solution is a kind of such, recombinant vector suitable for Corynebacterium glutamicum secreting, expressing zytase, It is characterized by: the recombinant vector includes the sequence original part of operability connection: AH6 promoter, 5 ' UTR and its preceding 38bp sequence Column, SD sequence, cspB signal peptide and xylanase gene, AH6 promoter, 5 ' UTR and its preceding 38bp sequence are connected with SD sequence Its sequence of the segment of composition is as shown in SEQ ID NO.1, and the sequence of cspB signal peptide is as shown in SEQ ID NO.2.

Further, the zytase is zytase xynA, the zytase xynA gene such as SEQ ID NO.3 It is shown.

Further, the downstream connection of the xylanase gene has histidine tag.

Further, the sequence of the recombinant vector is as shown in SEQ ID NO.4.

The present invention also provides a kind of expression system of zytase, the expression system includes corynebacterium glutamicum, The Corynebacterium glutamicum conversion has above-mentioned recombinant vector.

The present invention also provides above-mentioned recombinant vectors to prepare the application in zytase.

The present invention also provides above-mentioned expression systems to prepare the application in zytase.

Further, the application is the following steps are included: culture thallus, collection bacterial culture fluid and purifying.

Further, the collection bacterial culture fluid uses centrifugation, and condition is 12000 rpm of revolving speed, 4 DEG C of centrifugations 5 min collect supernatant.

Further, the purifying carries out affinity chromatography using nickel column, desalting column desalination is then used, in affinity chromatography For balancing, the buffer solution A of loading are as follows: the buffer of 300mM NaCl, 20mM Tris, pH8.0, the buffer solution B for elution Are as follows: 300mM NaCl, 250mM imidazoles, the buffer of 20mM Tris, pH8.0.

In recombinant vector of the invention, AH6 promoter, 5 ' UTR and its preceding 38bp sequence, SD sequence, cspB signal peptide and Xylanase gene constitutes bicistronic construct, and the hypersecretion that can be realized zytase in Corynebacterium glutamicum is expressed, enzyme activity Reach 486.2 U/ml.

Detailed description of the invention

Fig. 1 is the structure chart of carrier pXMJ19-AH6-SD-cspB-xynA.

Fig. 2 is the structure chart of carrier pXMJ19-AH6-SD-0949-xynA.

Fig. 3 is that the SDS-PAGE of expressed xylanase schemes, and wherein swimming lane M is albumen Marker, and swimming lane 1 is wild type culture Object supernatant, swimming lane 2 are the culture supernatant with pXMJ19 empty plasmid, and swimming lane 3 is with pXMJ19-AH6-0949-xynA's Culture supernatant, swimming lane 4 are the culture supernatant with pXMJ19-AH6-cspB-xynA, and swimming lane 5 is the zytase of purifying, Arrow meaning is xylanase protein band.

Fig. 4 is that the Western-blot of expressed xylanase schemes, wherein swimming lane 1 is with pXMJ19-AH6-SD-0949- The culture supernatant of xynA, swimming lane 2 are the culture supernatant with pXMJ19-AH6-SD-cspB-xynA, and swimming lane 3 is purifying Zytase.

Specific embodiment

Corynebacterium glutamicum, deposit number CGMCC1.15647, the deposit date is in March, 2016, and it is general to be preserved in China Logical Microbiological Culture Collection administrative center, preservation mode are open deposit, and those skilled in the art can obtain.

Bacillus coli DH 5 alpha is purchased from Chinese Universities ' microbial resources data platform.

Plasmid extraction kit, plastic recovery kit are purchased from Axygen, XhoI and EcoRI enzyme, T4 ligase are purchased from Thermo；

Bacillus coli DH 5 alpha with pXMJ19-AH6-SD-cspB, the bacillus coli DH 5 alpha with pXMJ19-AH6-SD-0949 Purchased from the Suzhou biotech inc Hong Xun.