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Marine low temperature alpha-amylase gene engineering bacteria, recombinant enzyme and application

A technology of genetically engineered strains and amylase, which is applied in the field of bioengineering, can solve the problems of reduced efficiency of microbial decomposition of organic pollutants, etc., and achieves the effects of low acting temperature, simple preparation process and high activity

Inactive Publication Date: 2010-04-07
HUAIHAI INST OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows an alpha amyiases called beta amygdalus BAYL1 that breaks down starchy materials into smaller molecules like sugars or oligo sacchains instead of making them themselves. These small molecule fragments are then used together with other components such as proteins to create new products. Overall this makes it possible to produce biomolecular compounds at lower temperatures than traditional methods while maintaining their effectiveness.

Problems solved by technology

This patents discuss different types of processes involved during manufacturing various products like bread or vegetables due to the use of low -temperature proteins called alpha-aminobacillus acidification protein). However, these techniques may result in decreased productivity caused by reduced cellular metabolism leading to increased refrigerator load on downstream equipment. Therefore there needs further improvement in process performance without compromising quality attributes associated with beta-amyloid peptides found naturally occurring within yeast cells.

Method used

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  • Marine low temperature alpha-amylase gene engineering bacteria, recombinant enzyme and application
  • Marine low temperature alpha-amylase gene engineering bacteria, recombinant enzyme and application
  • Marine low temperature alpha-amylase gene engineering bacteria, recombinant enzyme and application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1. Construction of the cloning plasmid pMD18-T-amy.

[0040] Low-temperature α-amylase-based primers, forward primer 1 and reverse primer 2, were designed. An EcoR I restriction site was added to primer 1, and a Sal I restriction site was added to primer 2.

[0041] Primer 1 sequence: 5'-GCGC GAATTC ATGAACAGGGGTATAT-3', the underlined part is the EcoR I site.

[0042] Primer 2 sequence: 5'-TA GTC GAC The underlined part of TCAGCCCACCCCACAG-3' is the Sal I site.

[0043] The genomic DNA of Pseudoalteromonas arctica GS230 was used as a template for PCR amplification, and the PCR product was electrophoresed, recovered and purified by tapping the gel with the Gel Extraction Kit kit from TaKaRa Company. Linked with the pMD18-T vector, the cloning plasmid pMD18-T-amy was constructed (see figure 1 ), transform Escherichia coli competent cells, and initially screen positive clones through blue-white colonies, and then carry out electrophoresis detection after do...

Embodiment 2

[0044] Example 2. Expression vector pEtac-His 6 -amy's build.

[0045] Recombinant T-vector (pMD18-T-amy) and pEt-28a-His 6 Carry out EcoR I and Sal I double enzyme digestion respectively, and the enzyme digestion reaction system (20 μ L) is as follows:

[0046] 10×buffer H 2μL

[0047] EcoR I (or Sal I) (10U·μL -1 ) 1μL

[0048] pEtac-His 6 (or PCR product) 14μL

[0049] Carry out EcoR I and Sal I double digestion of pMD18-T-amy, and recover a 1.5kb band from the gel; perform EcoR I and Sal I double digestion on the pEac plasmid, and recover a 5.6kb band from the gel; The products were mixed, and connected at 16°C for 18h (such as image 3 ), the expression vector pEtac-His containing six histidine tags at the N-terminal was constructed 6 -amy. When exogenous genes are expressed in E.coli, in order to simplify the purification steps of the expression products, tags such as His and MBP are usually added so that the expression products can be purified by Ni-NTA chroma...

Embodiment 3

[0050] Example 3. A kind of genetically engineered strain Escherichia coli EPGS230 (Escherichia coli EPGS230) CGMCC No.2700 for expressing the recombinant low-temperature α-amylase recombinant expression vector described in Example 2, it is the expression described in Example 2 Vector pEtac-His 6 - The genetically engineered strain Escherichia coli EPGS230 (Escherichia coli EPGS230) CGMCC No.2700 obtained by transforming amy into Escherichia coli BL21 (DE3).

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Abstract

The invention belongs to the field of bioengineering, and relates to engineering bacteria constructed by recombining low temperature alpha-amylase gene on an expression vector and converting to an expression host, recombinant enzyme expressed by utilizing the engineering bacteria and application thereof in industry. The invention adopts molecular operating technology, a strain of engineering bacteria EPGS230 is constructed by extracting and stipulating of bacterial genome, amplification of target genes, construction of the expression vector and conversion of recombinant vector, the engineering bacteria is processed by steps of culturing, inducing, centrifuging, ultrasound crushing, ion exchange, affinity chromatography and the like to obtain purified recombinant low temperature alpha-amylase. The inventive preparation technique has the advantages of simple operation, high enzyme activity, and low operative temperature.

Description

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Claims

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Application Information

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Owner HUAIHAI INST OF TECH
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