Pullulanase enzyme production gene, carrier containing same and application of carrier

A technology of pullulanase and enzyme gene, applied in the preparation of pullulanase, in the field of pullulanase enzyme production gene, can solve the restriction of pullulanase industrial application, pullulanase enzyme activity is not high , can not adapt to the acidic high temperature environment and other problems

Pending Publication Date: 2015-12-30
HENAN YANGSHAO BIOCHEM ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Pullulanase is widely used in industry, but the enzyme activity of most pullulanase is not high, or cannot adapt to the acidic and high temperature environment, these factors seriously restrict the industrial application of pullulanase

Method used

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  • Pullulanase enzyme production gene, carrier containing same and application of carrier
  • Pullulanase enzyme production gene, carrier containing same and application of carrier
  • Pullulanase enzyme production gene, carrier containing same and application of carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] The pullulanase enzyme-producing gene provided by the present invention is compatible with Klebsiella mutabilis HN7 ( Klebsiella variicola Compared with the wild-type pullulanase gene of HN7), the N-terminus of the de-signal peptide lacks the gene sequence corresponding to 31 amino acids, and the specific sequence is shown in the sequence table.

[0072] Because the pullulanase enzyme production gene of the present invention and Klebsiella mutabilis HN7 ( Klebsiella variicola HN7) is relatively close to the wild-type pullulanase gene, so the applicant uses Klebsiella mutabilis HN7 ( Klebsiella variicola HN7) wild type as the basis, and the vector containing the pullulanase enzyme production gene was constructed at the same time, and the specific construction process is introduced as follows.

[0073] (1) Genomic DNA extraction Specifically, the Klebsiella mutabilis HN7 ( Klebsiella variicola HN7) was inoculated into the seed medium, the bacteria were collected, and ...

Embodiment 2

[0135] The carrier containing the pullulanase enzyme-producing gene prepared in Example 1 is used in the preparation of pullulanase, and the specific steps are as follows:

[0136] (1) Heat shock transformation to construct pullulanase truncated mutant engineered bacteria ,Specifically,

[0137] The pET21a-pulA-N31 expression vector constructed in Example 1 was transformed into Escherichia coli BL21 (DE3) competent cells by heat shock method, and after recovery, the competent cells were spread on LB plates containing ampicillin (Amp), Cultivate; in detail:

[0138] This example is a period of simple operation, directly add 10 μL of the ligation product to 200 μL of Escherichia coli BL21 (DE3) competent cells, gently pipette evenly, place on ice for 30 minutes, then heat shock at 42°C for 90 seconds, and then place on ice for 90 seconds. Spread the competent cells on LB plates with a final concentration of Amp of 0.1 mg / mL, and incubate at 37°C for 12-16 hours;

[0139] Aft...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a pullulanase enzyme production gene capable of improving the specific activity and heat stability of pullulanase, a carrier containing the same and the application of the carrier in preparation of pullulanase. Compared with a klebsiella variicola HN7 wild pullulanase gene the preservation number of which is CGMCC NO.10357, base sequences corresponding to 31 amino acids are omitted at the N end after signal peptide is removed in the pullulanase enzyme production gene, and the specific sequence is shown in the sequence table. The invention further provides the carrier containing the gene by means of the gene engineering method. Compared with the original strain klebsiella variicola HN7, the pullulanase prepared by means of the carrier has the advantages that the specific activity, heat stability and affinity for pullulan substrates of the pullulanase are improved greatly, and the pullulanase has broad application prospects.

Description

Technical field [0001] The present invention is a genetic engineering technology field, which involves a Puluran enzyme gene that can improve the Perunase enzyme activity and thermal stability, the carrier containing the gene, and the carrier in the preparation of Prulandinase preparationApplications. Background technique [0002] Starch is a relatively abundant biomass resources on the planet. Its main component unit is glucose.In addition to being directly used as food raw materials, starch can also generate other valuable substances through chemical or biological methods, including: amino acids, glucose, organic acid, alcohol, etc.Starch is a mixture of direct chain starch and branches starch. The straight chain starch is formed by the glucose monomer through the α-1,4 glycoside bond.It is formed by the α-1,6 glycoside bond. [0003] The starch content in industrial application starch is as high as 70%to 95%, of which the alpha-1,6 keys are 4%to 5%.α-amylase and glycated enzym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/44C12N15/70
Inventor 王明道郭双聂慧慧张雨杭焦国宝陈晓慧孙利鹏邱立友时延光王红阳郜峰原增艳付香斌
Owner HENAN YANGSHAO BIOCHEM ENG
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