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Genetically engineered bacterium for heterologous expression of xanthan gum endonuclease and application of genetically engineered bacterium

A technology of genetically engineered bacteria and xanthan gum, applied in the field of genetic engineering, can solve problems such as side effects, poor exocrine capacity, and unsolvable disadvantages.

Pending Publication Date: 2021-07-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Escherichia coli is a pathogenic strain, which contains endotoxin, has side effects on humans and other mammals, and has poor exocrine ability. Foreign proteins are usually expressed intracellularly, and exogenous recombinant proteins are often expressed in E. coli Will form inclusion bodies and will not fold properly and efficiently
Although these problems can be solved to a certain extent through genetic engineering and optimization of fermentation conditions, when expressing enzymes used in the food industry, the disadvantage of containing endotoxins cannot be solved. Therefore, when expressing large molecular weight food industry enzymes, it is necessary Carefully select expression host strains

Method used

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  • Genetically engineered bacterium for heterologous expression of xanthan gum endonuclease and application of genetically engineered bacterium
  • Genetically engineered bacterium for heterologous expression of xanthan gum endonuclease and application of genetically engineered bacterium
  • Genetically engineered bacterium for heterologous expression of xanthan gum endonuclease and application of genetically engineered bacterium

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The construction of embodiment 1 genetically engineered bacteria

[0044] Step 1: According to the amino acid sequence of xanthan endonuclease obtained from NCBI (GenBank: ALX66163.1), the gene sequence shown in SEQ ID NO.1 is designed, and the promoter P srfA Gene sequence (SEQ ID NO.2), promoter P 43 The gene sequence (SEQ ID NO.3) was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. respectively.

[0045] Step 2: Artificially synthesized P in step 1 srfA PsrfA-F and PsrfA-R were used as primers to amplify P srfA Gene fragment; with the Bacillus subtilis WB800n genome as a template, with PhoD-F, PhoD-R as primers, PCR amplification obtains the PhoD gene fragment; with the artificially synthesized xanthan endonuclease gene EX in step 1 as a template, with EX-F and EX-R are used as primers, and the EX gene fragment is obtained by PCR amplification, and then the P srfA Gene fragment, PhoD gene fragment and EX gene fragment were ligated and amplified to obtai...

Embodiment 2

[0056] Example 2 Effects of Different Promoters and Signal Peptides on Xanthan Gum Endonuclease Enzyme Activity

[0057] (1) 2 strains of recombinant Bacillus subtilis WB800n-pHT01-P constructed in Example 1 that can successfully secrete and express xanthan endonuclease gene srfA -PhoD-EX and WB800n-pHT01-P 43 -PhoD-EX, activated culture in 50mL liquid LB medium (containing chloramphenicol resistance, 30μg / mL), cultured at 37°C, 200rpm for 12h to obtain activated seed liquid, and then The seed solution was transferred to 100 mL of liquid TB medium (containing chloramphenicol resistance, 30 μg / mL) for fermentation at an inoculum size of 1%, and cultured at 37° C. and 200 rpm for 24 hours to obtain a fermentation broth.

[0058] (2) Put the fermentation broth at 4°C, 8000r·min -1 Centrifuge for 10 minutes, discard the precipitate, and obtain the crude enzyme solution. Dialyze the crude enzyme solution in a 50kDa dialysis bag for 2-3 days, pre-freeze at -40°C, freeze-dry and co...

Embodiment 35

[0063] Embodiment 35L fermenter fermentation produces xanthan endonuclease

[0064] Two strains of recombinant Bacillus subtilis WB800n-pHT01-P constructed in Example 1 that can successfully secrete and express xanthan endonuclease gene srfA -PhoD-EX and WB800n-pHT01-P 43 -PhoD-EX, activated culture in 50mL liquid LB medium (containing chloramphenicol resistance, 30μg / mL), cultured at 37°C, 200rpm for 12h to obtain activated seed liquid, and then The seed solution was transferred to 100 mL of liquid TB medium (containing chloramphenicol resistance, 30 μg / mL) for fermentation at an inoculum size of 1%, and cultured at 37° C. and 200 rpm for 24 hours to obtain a fermentation broth.

[0065]The recombinant Bacillus subtilis WB800n-pHT01-P constructed in Example 1 43 -PhoD-EX is fermented and cultivated in a 5L fermenter equipped with TB medium. The total liquid volume of the fermenter is 3L. The fermented liquid is inoculated into the TB medium with an inoculation volume of 1% ...

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Abstract

The invention discloses a genetically engineered bacterium for heterologous expression of xanthan gum endonuclease and application of the genetically engineered bacterium, and belongs to the technical field of genetic engineering. The xanthan gum incision enzyme gene EX, the promoter P43 and the signal peptide PhoD are expressed in bacillus subtilis, so that the xanthan gum incision enzyme is efficiently expressed, and the highest enzyme activity can reach 38U / mL. According to different industrial requirements on relative molecular weights of xanthan gum degradation products, reaction conditions of enzymatic reaction of the xanthan gum incision enzyme can be adjusted, and reaction is performed for 4-16 hours under the conditions that the pH is 5-8, the temperature is 35-55 DEG C and the rotating speed is 0-200 r / min, so that low-molecular-weight xanthan gum with different relative molecular weights is obtained.

Description

technical field [0001] The invention relates to a genetic engineering bacterium expressing xanthan gum endonuclease heterologously and its application, belonging to the technical field of genetic engineering. Background technique [0002] Xanthan gum is an extracellular heteropolysaccharide secreted by Xanthomonas campestris. It consists of several basic repeating units of pentasaccharides. Glucose linked by β-1,4-glycosidic bonds forms the main chain, and mannose→glucosaldehyde Acid → mannose constitutes its side chain, and its molecular weight is generally 2×10 6 ~2×10 7 Da between. Low-molecular-weight xanthan gum has special biological activities such as anti-oxidation, antibacterial, anti-tumor, etc. It is used in various fields and has high market value. Therefore, the research on the degradation process of xanthan gum is of great significance for industrial production and life. Compared with physical and chemical methods, enzymatic degradation has the advantages o...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/42C12N15/75C12N1/21C12P19/14C12P19/06C12R1/125
CPCC12N9/2434C12N15/75C12P19/14C12P19/06C12N2800/22Y02A50/30
Inventor 高敏杰詹晓北杨国帅蒋芸李志涛
Owner JIANGNAN UNIV