Bacillus subtilis expression vector of efficient secreting expression recombination lipoxygenase and application thereof

A technology of lipoxygenase and Bacillus subtilis, which is applied in the direction of oxidoreductase, recombinant DNA technology, and the use of vectors to introduce foreign genetic materials, etc., can solve the problem that the complete removal of E. coli pyrogens cannot be guaranteed.

Inactive Publication Date: 2012-07-04
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzymes for food are bulk industrial products, and the downstream generally adopts low-cost separation and extraction methods, which cannot guarantee the complete removal of pyrogens such as E. coli; if complex purification methods are used, the cost becomes the limiting factor for application

Method used

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  • Bacillus subtilis expression vector of efficient secreting expression recombination lipoxygenase and application thereof
  • Bacillus subtilis expression vector of efficient secreting expression recombination lipoxygenase and application thereof
  • Bacillus subtilis expression vector of efficient secreting expression recombination lipoxygenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Cloning of Anabaena sp.PCC 7120 lipoxygenase gene

[0031] Anabaena sp. PCC 7120 cells were collected by centrifugation, and the genomic DNA of Anabaena sp. was extracted with the Shanghai Sangon Genomic DNA Extraction Kit. According to the genome sequence published by NCBI, two primers were designed: ana-LOX-F (SEQ ID NO.12) and ana-LOX-R (SEQ ID NO.13). Using the genomic DNA of Anabaena as a template, PCR amplified To increase the ana-LOX gene, the reaction system is as follows:

[0032]

[0033] The PCR program is: 94°C 2min; 30×(94°C 45s; 58°C 50s; 72°C 4min); 72°C 10min.

[0034]The 1368bp gene fragment obtained by PCR amplification was recovered by tapping and ligated into the pMD19-T cloning vector. After the sequence was correct, it was named pMD 19-ana-LOX and stored at -20°C for future use.

[0035] Using the computer software DNAMAN to analyze the sequencing results, the Anabaena lipoxygenase gene (ana-LOX), whose sequence is SEQ ID NO.1, encod...

Embodiment 2

[0036] The construction of embodiment 2 secretion expression vector pHBSR

[0037] 2.1 The acquisition of carrier pHB-hc ( figure 1 )

[0038] In order to construct a Bacillus subtilis expression vector that can secrete foreign protein products, the Escherichia coli / Bacillus subtilis shuttle vector pHB201 was selected as the backbone, which is a stable cloning vector, and its backbone was derived from the Bacillus subtilis vector pTA1060. The multiple cloning site is located in the fusion gene cat86::lacZα, and because it is not suitable for expression, its promoter P59, cat86::lacZα region can be excised ( figure 1 ).

[0039] A pair of primers P1 (SEQ ID NO.2) and P2 (SEQ ID NO.3) are designed so that they can be paired to form double strands, and the two ends form sticky ends of ClaI and XhoI. The reaction system is as follows:

[0040] In a 200 µL PCR thin-walled tube add:

[0041]

[0042] After mixing, denature in a water bath at 94°C for 2 minutes, cool naturall...

Embodiment 3

[0060] Example 3 Construction of Anabaena sp.PCC 7120 lipoxygenase gene prokaryotic expression vector ( Image 6 )

[0061] The plasmid pMD19-ana-LOX and the expression vector pHBSR were digested with XhoI respectively. The 1368bp gene fragment (ana-LOX) and the linearized pHBSR plasmid were purified with the Shanghai Sangon PCR Product Purification Kit. After the two gene fragments were purified, they were ligated with T4 ligase and transformed into Escherichia coli DH5α. A few colonies were randomly picked from the transformation plate, inserted into LB liquid medium, cultured by shaking, a small amount of plasmid was extracted, electrophoresis, PCR verification was carried out using the plasmid lagging behind in electrophoresis as a template, and it was sent to Shanghai Sangon for sequencing after confirming that the connection was successful.

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Abstract

The invention belongs to the technical field of food industry biology and relates to a bacillus subtilis expression vector of efficient secreting expression recombination lipoxygenase and application thereof. The vector utilizes a bacillus subtilis vector pHB201 as a framework, the pHB201 enzyme cutting site is modified first through an enzyme cutting method to obtain a pHB-hc vector, composition type strong promoter P43, composition type strong promoter PamyE, molecular chaperone PrsA of an Sec path and neutral protease signal peptide SnprB are obtained by cloning in bacillus subtilis 168 genome through a polymerase chain reaction (PCR), and a bacillus subtilis secretion type expression vector pHBSR is obtained in construction. Lipoxygenase gene obtained from anabaenagenome DNA in amplification is inserted into autonomously constructed expression vector pHBSR and then electrically converted into a bacillus subtilis host WB800 to achieve secreting expression of the recombination lipoxygenase, and the largest enzyme activity in fermentation liquid can reach 76U/ml.

Description

technical field [0001] The invention belongs to the field of food industry biotechnology, and relates to a bacillus subtilis expression vector for efficiently secreting and expressing recombinant lipoxygenase and its application. Background technique [0002] Potassium bromate, as a quality improver for bread and cake flour in the baking industry, has been successfully used abroad, especially in Europe and America, for decades. As a slow oxidant, it can improve dough structure and rheology, enhance gluten and elasticity, and make baked products obtain satisfactory results. Potassium bromate was also used as a flour quality improver earlier in my country, and it was included in the GB2760 hygienic standard for use, with a limit of 0.03g / kg. However, it is stipulated that there should be no residue after baking. In the early days, it was believed that potassium bromate would decompose after roasting, but in the 1980s, Japan and the United Kingdom, after long-term research, f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/64C12N15/66C12N9/02C12R1/125
Inventor 陆兆新张充吕凤霞别小妹王昱沣赵海珍应琦
Owner NANJING AGRICULTURAL UNIVERSITY
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