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Recombinant bacillus subtilis for producing pullulanase and construction thereof

A technology of Bacillus subtilis and pullulanase, which is applied in the field of microbial fermentation to produce pullulanase, can solve problems such as product safety threats, and achieve the effect of improving the ability to produce extracellular pullulanase

Inactive Publication Date: 2016-12-07
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the current research results show that pullulanase is expressed in large quantities in E. coli, in practical industrial applications, especially in the food processing industry of starch, the cell wall of E. coli contains lipopolysaccharide, which is considered to be an endotoxin. (LPS), a threat to the safety of the product

Method used

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  • Recombinant bacillus subtilis for producing pullulanase and construction thereof
  • Recombinant bacillus subtilis for producing pullulanase and construction thereof
  • Recombinant bacillus subtilis for producing pullulanase and construction thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the acquisition of pullulanase gene pul

[0027] Bacillus naganoensis CCTCC M 2012388 Medium: CaCl 2 0.25g / L, MgSO 4 ·7H 2 O 0.5g / L, (NH 4 ) 2 SO 4 0.2g / L, yeast extract 2g / L, anhydrous glucose 5g / L, KH 2 PO 4 3g / L, 1mL / L inorganic salt solution, prepared with double distilled water, pH 5.0. Inorganic salt solution: ZnSO 4 ·7H 2 O 0.1g / L, MnCl 2 ·H 2 O0.03g / L, H 3 BO 3 0.3g / L, CoCl 2 ·6H 2 O 0.2g / L, CuCl 2 2H 2 O 0.01g / L, NiCl 2 ·6H 2 O 0.02g / L, Na 2 MoO 4 2H 2 O 0.03g / L.

[0028] The Bacillus nagano strain was inoculated into a 250 mL shake flask containing 25 mL of culture medium, and cultured with shaking at 37° C. and 200 rpm for 72 h. After the cultivation, the cells were centrifuged and washed twice with physiological saline, and the cells were collected to extract genomic DNA using a genomic DNA extraction kit Genomic DNA Extraction Miniprep System (VIOGENE Company).

[0029] Synthetic Primer 1: 5'-CG GAATTC GATGGGAACACCA...

Embodiment 2

[0035] Example 2: Recombinant Bacillus subtilis B. subtilis WB800 / pMA0911-P HpaII -build of pul

[0036] (1) Double digestion of target gene pul and plasmid pMA0911

[0037] Plasmid pMA0911 was extracted using the plasmid extraction kit Plasmid Mini Kit (OMEGA BIO-TEK).

[0038] Add water, buffer, PCR product or plasmid DNA, and restriction endonuclease into the Eppendorf tube in the order, cover the tube cap, mix gently, and centrifuge in a centrifuge for 30 seconds to concentrate the liquid at the bottom of the tube. Water bath at 37°C for 3h, add 1 / 10 Loading Buffer to the tube or place the tube at 65°C for 10min to terminate the enzyme digestion reaction. The digested product was analyzed by agarose gel electrophoresis, and the target fragment was recovered by using Gel extraction Kit (OMEGA BIO-TEK Company), and concentrated appropriately.

[0039] Enzyme digestion reaction system composition: 10×K Buffer 10 μL, genomic DNA 80 μL, restriction enzyme EcoRI 2.5 μL, restr...

Embodiment 3

[0047] Example 3: Promoter optimization

[0048] (1) Gene synthesis of P43 promoter

[0049] The P43 promoter sequence (GenBank: EF473728.1) 426bp was searched in NCBI, and single enzyme cutting sites BstxI and EcoRI were introduced on both sides of the synthetic gene, and the sequence to be synthesized was sent to Shanghai Sangon Bioengineering Co., Ltd. Gene synthesis was carried out, and the synthesized gene fragment was cloned in the strain E.coil DH5α / pUC57-P43.

[0050] Utilize the plasmid extraction kit Plasmid Mini Kit (OMEGA BIO-TEK company) to extract from bacterial strain E.coilDH5α / pUC57-P43 and E.coli JM109 / pMA0911-P respectively HpaII Extract plasmids pUC57-P43 and pMA0911-P from -pul HpaII -pul.

[0051] (2) Plasmid pUC57-P43 and plasmid pMA0911-P HpaII Double digestion of -pul

[0052] According to water, buffer, plasmid pUC57-P43 or plasmid pMA0911-P HpaII Add -pul, restriction endonuclease EcoRI and BstXI (NEB company) to the Eppendorf tube in sequence,...

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Abstract

The invention discloses recombinant bacillus subtilis for producing pullulanase and construction thereof, and belongs to the technical field of microbial fermentation production of pullulanase. According to the recombinant bacillus subtilis for producing the pullulanase and the construction thereof, a bacillus naganoensis CCTCC NO:M 2012388 pullulanase gene pul is inserted into a shuttle vector pMA0911, the obtained recombinant plasmid pMA0911-PHpaII-pul is electrically converted into B.subtilis WB800 to construct a recombinant bacterium B.subtilis WB800 / pMA0911-PHpaII-pul, and the extracellular pullulanase activity is 39.6 U / mL. An original promoter PHpaII is replaced with a strong promoter, and the extracellular enzyme activity of recombinant bacterium B.subtilis WB800 / pMA0911-P43-pul is improved to be 87.3 U / mL; the constructed recombinant plasmid pMA0911-PHpaII-pul and the pMA0911-P43-pul are converted into a host bacterium B.subtilis WB600, and the extracellular enzyme activity of the obtained recombinant bacterium B.subtilis WB600 / pMA0911-PHpaII-pul and the extracellular enzyme activity of the obtained recombinant bacterium WB600 / pMA0911-P43-pul are up to 213.5 U / mL and 245.7 U / mL respectively.

Description

technical field [0001] The invention relates to a recombinant bacillus subtilis for producing pullulanase and its construction, and belongs to the technical field of microbial fermentation for producing pullulanase. Background technique [0002] Pullulanase (pullulanase, EC 3.2.1.1.41), also known as pullulan 6-glucan hydrolase, is a kind of enzyme that can specifically hydrolyze α-1,6 in the branch point of pullulan by endocutting. A starch debranching enzyme that cleaves the entire branched structure to form amylose. The characteristic that pullulanase can decompose branched chains makes it possible to use together with glucoamylases such as α-amylase and β-amylase in the starch saccharification process to cut the smallest unit of branched chains, thereby improving the utilization rate of starch. Accelerate the saccharification process, improve product quality and reduce the total amount of amylase used. It is reported that adding pullulanase in the starch saccharificatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N9/44C12R1/125
CPCC12N9/2457C12Y302/01041
Inventor 聂尧徐岩穆晓清
Owner JIANGNAN UNIV
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