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Coding gene of heat-resistant type pullulanase as well as recombinant expression thereof and application thereof

A technology of pullulanase and coding gene, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems that there is no detailed report of pullulanase from Geobacillus stearothermophilus, and achieve good thermal stability

Inactive Publication Date: 2018-10-16
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are currently no detailed reports on pullulanase from Geobacillus stearothermophilus

Method used

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  • Coding gene of heat-resistant type pullulanase as well as recombinant expression thereof and application thereof
  • Coding gene of heat-resistant type pullulanase as well as recombinant expression thereof and application thereof
  • Coding gene of heat-resistant type pullulanase as well as recombinant expression thereof and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Amplification of the pullulanase gene

[0029] 1.1 Strains and their cultivation

[0030] The pullulanase gene of the present invention is cloned from Geobacillus stearothermophilus.

[0031] Geobacillus stearothermophilus (DSMZ456) of the present invention can be purchased directly from the German Culture Collection of Microorganisms, the strain number is DSMZ 456, and its original source is extracted from sugar beet juice in Austria. Therefore, the Geobacillus stearothermophilus described in the present invention can be obtained through commercial means, and can also be obtained through field collection or other means.

[0032] Glycerol bacteria were inoculated into No. 1 medium, cultured at 55°C for 2 days, and the bacteria were collected for genome extraction.

[0033] 1.2 Genome Extraction

[0034] Refer to the instructions of the Generay Bacterial Genomic DNA Rapid Extraction Kit.

[0035] 1.3 Amplification of the pullulanase gene from Geobacillus stearothermo...

Embodiment 2

[0045] Construction and Expression of Escherichia coli Recombinant Expression Vector Containing Pullulanase

[0046] 2.1 Recombinant expression vector pET-28a-Plu GS build

[0047] A large number of pMD19-TSimple Vectors containing the pullulanase gene of Geobacillus stearothermophilus were cloned in Escherichia coli DH5α host bacteria, and the plasmid was extracted with the AxyPrep plasmid DNA extraction kit (operating steps refer to the instructions), and EcoRI and XhoI (purchased from Themo ) for double enzyme digestion, gel cutting to recover the pullulanase gene fragment, and then ligated with the Escherichia coli expression vector pET-28a that was also digested with EcoRI and XhoI at 16°C overnight, and transformed into Escherichia coli DH5α (purchased from Tiangen Biotechnology (Beijing) Co., Ltd.), positive clones were verified by PCR and sequenced; the sequencing results showed that no frameshift and other base mutations occurred, indicating that the constructed reco...

Embodiment 3

[0052] Pullulanase activity assay

[0053] 3.1 Standard curve drawing

[0054] Take a clean test tube, label the test tube, prepare a glucose concentration gradient solution, add 0.2-1.4mL (at 0.2mL intervals) of 1% glucose solution to the test tube, and use the test tube without glucose as a blank control. Three parallel samples were made for each tube. Add ddH to the test tube respectively 2 O to a total volume of 2.0mL, then add 3mL DNS reagent to the test tube, boil for 15min, immediately add 10mL ddH 2 0 and pre-cooling, at a wavelength of 550nm, measure with a spectrophotometer colorimetrically, and write down the optical density value of the sample corresponding to each test tube to find its average value, then draw the glucose standard curve, and the results are shown in Figure 1.

[0055]3.2 Determination of enzyme activity

[0056] The enzymatic activity of recombinant pullulanase was measured by DNS termination method.

[0057] (1) Appropriately dilute the crud...

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Abstract

The invention relates to a coding gene of heat-resistant type pullulanase from geobacillus stearothermophilus as well as recombinant expression thereof and application thereof. The coding gene successfully constructs a recombinant plasmid containing PluGS, so that various highly-expressed bacterial stains which comprise escherichia coli, bacillus, saccharomycete and mycete are obtained. A recombinant host cell is suitable for expressing a gene of the heat-resistant type pullulanase. The optimal pH value of pullulanase is 6.0, and relatively high enzyme activity is kept when the pH value is 5.0-7.0. The optimal temperature is 65 DEG C, thermal stability is good at a temperature of 60-70 DEG C, the heat is preserved for 3 hours at the temperature of 60 DEG C, and the pullulanase still keep 90% or more of enzyme activity, and is suitable for industrial application needs. The pullulanase which is in recombinant expression is identified as I type pullulanase through thin-layer chromatography and efficient liquid-phase chromatographic analysis, can be used for hydrolyzing pulullan, amylopectin and alphla-1,6-glucosidic bond of soluble starch, and can not hydrolyze amylopectin and cyclodextrin.

Description

technical field [0001] The invention relates to the fields of biotechnology and food, in particular to a clone of a pullulanase derived from Geobacillus stearothermophilus with strong thermostability, and the invention also provides the construction of a recombinant plasmid encoding the pullulanase gene , expression and enzymatic properties. Background technique [0002] Pullulanase (EC 3.2.1.41) is a starch debranching enzyme that has been used to catalyze the hydrolysis of amylopectin, pullulan, α-1,4- and α- 1,6-glycosidic bond. Pullulanase is a major debranching enzyme that specifically hydrolyzes branch points, and when used together with glucoamylase, it can significantly increase the utilization of amyloglucosidase and greatly reduce glucoamylase (Bertoldo, C., Antranikian, G., Starch-hydrolyzing enzymes from thermophilic archaea and bacteria. Curr. Opin. Chem. Biol., 2002, 151-160). In the wheat fermentation industry, pullulanase is used in combination with fungal...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/44C12N15/63
CPCC12N9/2457C12Y302/01041
Inventor 李聆梦魏巍董凤英魏东芝
Owner EAST CHINA UNIV OF SCI & TECH
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