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Novel pullulanase and gene and preparation methods thereof and engineering bacteria expressing same

A technology of pullulanase and engineering bacteria, which is applied in the field of enzyme genetic engineering and can solve the problem of low expression level of pullulanase

Active Publication Date: 2019-02-12
SHANDONG LONGKETE ENZYME PREPARATION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although genetic engineering technology has made some achievements in the research of pullulanase, in general, the expression level of pullulanase is not high

Method used

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  • Novel pullulanase and gene and preparation methods thereof and engineering bacteria expressing same
  • Novel pullulanase and gene and preparation methods thereof and engineering bacteria expressing same
  • Novel pullulanase and gene and preparation methods thereof and engineering bacteria expressing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Acquisition of a Novel Pullulanase Mature Peptide Gene of Pullulan Bacillus Nagano

[0051] 1. The wild-type pullulanase mature peptide gene is derived from Pullulanbacterium naganolyticum screened by the applicant's laboratory, and the gene can be amplified by gene synthesis or PCR (shown in SEQ ID NO.3). Wherein, the extraction steps of the genomic DNA of Pullulanella nagano are as follows:

[0052] (1) Inoculate and streak on LB solid plates from glycerol tubes, and culture at 37°C for 12 hours;

[0053] (2) Pick a single colony from the culture plate and inoculate it in 5mL liquid LB medium, and culture it at 220r / min, 37°C for 12h;

[0054] (3) Dispense the bacterial liquid into sterilized 1.5mL microcentrifuge tubes, centrifuge at 12000r / min for 1min to collect the bacterial cells, and discard the supernatant;

[0055] (4) Resuspend the pellet in 200 μL of pre-cooled Solution I (OMEGA Genomic DNA Extraction Kit) and repeatedly pipette and mix with a p...

Embodiment 2

[0074] Example 2: Mutation of the pullulanase gene

[0075] 1. The wild-type pullulanase gene is connected to the pET-22b(+) vector.

[0076] The purified pul was ligated with the pET-22b(+) vector, and then the recombinant plasmid was transformed into Escherichia coli DH 5α, and it was successfully verified that the wild-type pullulanase gene had been cloned into pET- The recombinant plasmid pET-pul was constructed on the 22b(+) vector.

[0077] 2. Error-prone PCR: Using the recombinant plasmid pET-pul constructed above as a template, the reaction system is as follows:

[0078] ddH 2 O

21μL

Recombinant plasmid pET-pul (5ng / μL)

1μL

Upstream primer P1 (10μmol / L)

2μL

Downstream primer P2 (10μmol / L)

2μL

Taq DNA polymerase

0.5μL

10×Taq buffer

5μL

dATP (10mmol / L)

1μL

dGTP (10mmol / L)

1μL

dTTP (10mmol / L)

5μL

dCTP(10mmol / L)

5μL

MgCl 2 (25mmol / L)

10 μL

...

Embodiment 3

[0108] Example 3: Construction of a novel pullulanase recombinant bacterium with high stability in Bacillus subtilis

[0109] 1. Construction of expression vector pBSA43

[0110] pBSA43 is obtained by using the E. coli-Bacillus subtilis shuttle cloning vector pBE2 as the backbone, cloning into a strong Bacillus constitutive promoter P43, and the fructan sucrase signal sequence sacB that can directly secrete the recombinant protein into the medium . it comes with amp r Gene that can use ampicillin resistance as a selectable marker in E. coli; also has Km r Gene, kanamycin resistance can be used as a selection marker in Bacillus subtilis and Bacillus licheniformis.

[0111] 2. Construction of a novel pullulanase expression vector pBSA43-pulm

[0112] Ligate the novel pullulanase gene (pulm) amplified by PCR and recovered after EcoR I and Not I double-digestion with the same double-digestion Bacillus subtilis expression vector pBSA43, and transform the ligated product into th...

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Abstract

The invention belongs to the technical field of genetic engineering of enzymes, and particularly relates to novel pullulanase and a gene, an amino acid sequence and preparation methods thereof. According to the technical scheme, by screening bacteria, a strain of bacillus naganoensis which can generate pullulanase is obtained, and through an error-prone PCR technology, the pullulanase gene which is obtained through screening is modified, so that a piece of modified gene of the pullulanase is obtained, and then the novel pullulanase gene is expressed in a bacillus subtillis expression system, abacillus amyloliquefaciens expression system and a bacillus licheniformis expression system for expression separately to achieve the different preparation methods of the novel pullulanase. Meanwhile,the novel pullulanase has good application in the aspects of the food industry and the medical industry.

Description

Technical field: [0001] The present invention relates to a novel pullulanase and its gene, engineering bacteria and preparation method, in particular to a recombinant expression strain expressing the novel pullulanase obtained by means of genetic engineering technology and molecular biology, and the bacterial pullulan The industrial application of enzyme protein belongs to the technical field of enzyme genetic engineering. Background technique: [0002] Pullulanase, also known as α-dextrin endo-1,6-α-glucosidase, or pullulan 6-glucohydrolase. It was first discovered that it was named pullulanase because it can hydrolyze the α-1,6-glucosidic bond in pullulan polysaccharide. It can specifically cut the α-1,6-glucosidic bond of the branch point in amylopectin, cut off the branch chain structure and make amylopectin to form amylose. [0003] In the enzyme classification database, pullulanase belongs to the 13th family of glycosyl hydrolases. It widely exists in animals, plant...

Claims

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Application Information

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IPC IPC(8): C12N9/44C12N15/75C12N1/21C12R1/125C12R1/07C12R1/10
CPCC12N9/2457C12N15/75C12Y302/01041
Inventor 王兴吉刘逸寒王克芬路福平刘文龙
Owner SHANDONG LONGKETE ENZYME PREPARATION
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