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Paenibacillus polymyxa neopullulanase gene as well as method for producing neopullulanase through cloning and expression and fermentation

A technology of Bacillus polymyxa and new pullulanase, which is applied in the field of enzyme engineering, can solve the problems of less research on Bacillus polymyxa and has not yet realized industrialization, and achieve the effects of convenient cultivation, easy extraction, and improved stability

Inactive Publication Date: 2015-01-28
甘肃省商业科技研究所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, although there are more in-depth studies on the new pullulanase in Bacillus thermophiles, there are relatively few studies on Bacillus polymyxa, and the industrialization has not yet been realized.

Method used

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  • Paenibacillus polymyxa neopullulanase gene as well as method for producing neopullulanase through cloning and expression and fermentation
  • Paenibacillus polymyxa neopullulanase gene as well as method for producing neopullulanase through cloning and expression and fermentation
  • Paenibacillus polymyxa neopullulanase gene as well as method for producing neopullulanase through cloning and expression and fermentation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] (1) Extraction of Bacillus polymyxa genomic DNA:

[0037] Cultivate Bacillus polymyxa in nutrient broth medium in a constant temperature and humidity incubator at 30°C overnight, take 0.5~2mL culture liquid (no more than 2×10 9 cells), centrifuge at 10,000 rpm for 30 seconds, discard as much supernatant as possible, and collect the bacteria.

[0038] Genomic DNA of the strain was extracted according to the bacterial DNA extraction kit (SK1201-UNIQ-10 Column Bacterial Genomic DNA Extraction Kit from Shanghai Sangon Bioengineering Technology Service Co., Ltd.).

[0039] Follow the steps below:

[0040] (1) Add 200μl buffer RB to resuspend the cells, centrifuge at 1000rpm for 30 seconds, discard the supernatant, resuspend the cells in 180μl buffer RB (Gram-negative bacteria) or 480μl 50mMNa2EDTA (pH8.0) (Gram Lambert-positive bacteria);

[0041] (2) Add 120μl lysozyme (20mg / ml in 10 mM Tris-HCl, pH 8.0) and mix well. Incubate at 37°C for 30-60 minutes. Centrifuge at 1...

Embodiment 2

[0097] Example 2: Escherichia coli-induced expression of the new pullulanase gene

[0098] (1) Extraction of PGEX 4T-1 plasmid

[0099] PGEX 4T-1 was extracted according to the instructions of the plasmid mini-extraction kit.

[0100] (2) Recovery of PCR products

[0101] Separate the target DNA fragment from other DNA as much as possible by agarose gel electrophoresis, then cut the agar block containing the target DNA fragment with a clean scalpel, and put it into a 1.5 mL centrifuge tube. (When determining the position of the DNA fragment, long-wavelength ultraviolet light should be used as much as possible, and the time of irradiation under ultraviolet light should be as short as possible).

[0102] (3) Digestion of vector and PCR product

[0103] The enzyme digestion reaction system is shown in Table 2:

[0104]

[0105] Digest overnight at 37°C (12-16 h), then inactivate at 75°C for 10 min.

[0106] (4) Purification of digested products

[0107] Cut the digeste...

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Abstract

The invention discloses a paenibacillus polymyxa neopullulanase gene as well as a method for producing neopullulanase through cloning and expression and fermentation. The method comprises the following steps: by selecting Paenibacillus polymyxa CFCC10334 as an original strain and taking escherichia coli BL21 as a genetic engineering host bacterium, directionally inserting a neopullulanase gene Npu in the paenibacillus polymyxa into an expression plasmid according to the physical map of the escherichia coli expression vector plasmid, transforming escherichia coli BL21, acquiring the neopullulanase gene when an expression product ENpu5 has normal biological activity, through cloning and transformation, performing induction expression in escherichia coli, screening positive expressor, measuring the enzyme activity, thereby obtaining a genetic engineering strain with relatively high enzyme activity, and performing fermentation production, thereby obtaining a neopullulanase product. The stability and the activity of enzyme are improved, and the production cost is lowered.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to the use of genetic engineering technology to recombine microbial neopullulanase genes, construct genetically engineered bacteria, and carry out fermentative production of neopullulanase by expressing the recombinant enzyme in a carrier. Background technique [0002] Neopullulanase , nPU , EC3.2 .1.135) belongs to one of the α-amylase family 13 (α-amylase family 13) of the glucoside hydrolase (Glycosyl hydrolase family), it is a multifunctional composite starch hydrolase, Can catalyze four reactions of starch: hydrolysis of α-(1→4) glycosidic bond, hydrolysis of α-(1→6) glycosidic bond, transglycosidation of α-(1→4) bond, α-(1→6) bonded transglycosidation. Therefore, the new pullulanase has the activity of pullulanase (pullulanase, PU, ​​EC3.2.1.41), which can break the α-(1→6) glycosidic bond of pullulan to generate maltotriose; It also has the activity o...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/70C12N1/21C12N9/44C12R1/19
Inventor 胡先望杜建泉梁宁陈朋严晓娟宋勇强张鸣明
Owner 甘肃省商业科技研究所有限公司
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