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Preparing purification method for thermophilic alpha-amylase

A technology of amylase and thermophilic archaea, applied in the field of bioengineering

Inactive Publication Date: 2007-10-31
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition to the factors of yield, the glycerol extraction method also has problems in process amplification and other aspects

Method used

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  • Preparing purification method for thermophilic alpha-amylase
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  • Preparing purification method for thermophilic alpha-amylase

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Experimental program
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Embodiment 1

[0062] Preparation of thermophilic alpha-amylase PFA

[0063] Materials and Methods

[0064] 1. Materials

[0065] Strains and vectors: Genomic DNA of Pyrococcus furiosus DSM3638 ATCC No. 43587 TM ; Escherichia coli E.coli DH5α, E.coli BL21(DE3) and E.coli BL21-CodonPlus(DE3)-RIL were purchased from Stratagen; pET21a expression vector was purchased from Novagen;

[0066] Molecular biology reagents: Restriction enzymes Nde I, HindIII, Pyrobest DNA polymerase, T4 DNA polymerase, T4 DNA ligase were purchased from TaKaRa Company; protein molecular quality standards were purchased from Shanghai Institute of Biochemistry; Phenyl Sepharose 6 Fast Flow chromatography The column material was purchased from Novagen; the gel recovery kit was purchased from Huashun Bioengineering Co., Ltd.; the primer synthesis was completed by Shanghai Bocai Biotechnology Co., Ltd.; the DNA sequencing was completed by Yingjun Biotechnology Co., Ltd.; other reagents were of analytical grade.

[0067] 2...

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Abstract

The invention discloses a preparing method of thermophilic alpha-amylase (PFA), which comprises the following steps: heating and dissolving recombinant protein inclusion body under the condition of alkalinity; diluting; compositing; separating and purifying through column layer. This method possesses high reclaiming ratio and high alpha-amylase activity.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to the extraction and purification of recombinant proteins and thermophilic enzymes. Background technique [0002] High temperature resistant α-amylase is one of the most commonly used enzyme preparations in industry, and is widely used in starch processing in food industry and chemical industry. The search and development of high heat-tolerant amylases has long been a key and hot topic in the field of industrial enzyme research. The global starch processing enzyme market is about 150 million US dollars, of which about 24% are used in the starch liquefaction process. Therefore, any improvement in amylase production yield, heat resistance and enzyme activity has very important theoretical significance and direct commercial value. [0003] At present, the heat-resistant amylase commonly used in industry comes from Bacillus licheniformis. The optimum catalytic temperature of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12P19/14C12P19/02
Inventor 张毅王丽萨杨胜利
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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