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Fused acid thermophilic alpha-amylase gene and fused acid thermophilic alpha-amylase, preparation and application thereof

A technology that integrates genes and amylase, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve the problems of increasing production costs and processing equipment

Active Publication Date: 2012-12-26
欣雅利华生物技术(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This not only increases the production cost, but also causes problems such as the increase of subsequent processing equipment.

Method used

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  • Fused acid thermophilic alpha-amylase gene and fused acid thermophilic alpha-amylase, preparation and application thereof
  • Fused acid thermophilic alpha-amylase gene and fused acid thermophilic alpha-amylase, preparation and application thereof
  • Fused acid thermophilic alpha-amylase gene and fused acid thermophilic alpha-amylase, preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Construction of ST0926 and ST0927 engineered bacteria and protein expression, including the following steps:

[0063] (1) Thermophilic acid archaea (Sulfolobus Tokodaii strain 7 T ) Cultivation and genomic DNA extraction

[0064] The medium composition of 1L thermophilic acid archaea is: 4.5mg Na 2 B 4 O 7 ·10H 2 O, 1.8mg MnCl 2 ·4H 2 O, 1.3g(NH 4 ) 2 SO 4 , 0.28g KH 2 PO 4 , 0.25g MgSO 4 ·7H 2 O, 0.07g CaCl 2 ·2H 2 O, 0.02g FeCl 3 ·6H 2 O, 0.22mg ZnSO 4 ·7H 2 O, 0.05mg CuCl 2 ·2H 2 O, 0.03mg VOSO 4 ·2H 2 O, 0.03mg Na 2 MoO 4 ·2H 2 O, 0.01mg CoSO 4 ·7H 2 O, 1.0g yeast extract, add 1.0L double distilled water to dissolve. Use 10N H 2 SO 4 Adjust pH to 2.0, 1.034×10 5 Pa autoclaved steam sterilization for 20min.

[0065] To the thermophilic acid archaea sulfolobus Tokodaii strain 7 purchased from the Japan Collection of Microorganisms T Add 0.5 mL of the above-mentioned thermophilic acid archaea culture medium that has been sterilized to the dry cell, and transfer all...

Embodiment 2

[0080] Example 2: PCR amplification of fusion acid thermophilic α-amylase gene, construction of engineering bacteria and expression of enzyme

[0081] (1) Amplify the fusion acid thermophilic α-amylase gene ST0926-27 gene by overlap extension PCR method: Through homologous sequence analysis, find the appropriate splicing sites of ST0926 and ST0927 (ST0926 amino acid sequence from 1 to 198 is consistent with ST0927 Polypeptide chain fusion constitutes ST0926-27 fusion protein, with a total of 557 amino acid residues and a molecular weight of about 61kDa). The designed primer is (ST0926 upstream primer sequence 3: 5'CATCGGGC CATATG TTCGGACCTAG 3', the horizontal line is the Nde I restriction site; downstream primer 3: 5'AC ATCATGTAATTACCCTCTG 3', the base on the horizontal line is complementary to ST0927 upstream primer 4; ST0927 upstream primer 4: 5'TC CAGAGGGTAATTACATGAT 3', the horizontal line is complementary to the downstream primer 3 of ST0926; downstream sequence 4: 5'TTAC...

Embodiment 3

[0111] Example 3: Characteristics of Fusion Acid Thermophilic α-Amylase

[0112] (1) Optimal reaction temperature and temperature stability

[0113] Temperature is an important factor affecting enzyme catalytic activity. Under normal circumstances, the reaction activity of enzymes at high temperatures is much higher than that at low temperatures. When the temperature reaches or exceeds the optimal reaction temperature, the stability of the enzyme decreases. The temperature range for detecting the catalytic activity of the fusion acid thermophilic alpha-amylase ST0926-27 of the present invention is 20-100°C.

[0114] Using soluble starch as a substrate, the activity of the fusion acid thermophilic α-amylase ST0926-27 of the present invention was measured in the temperature range of 20-100°C, and the relative activity of the enzyme was plotted against temperature, as Figure 7 As shown, it shows that the activity of fusion acid thermophilic α-amylase ST0926-27 increases with increasin...

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Abstract

The invention belongs to the technical field of biological engineering, and relates to a fused acid thermophilic alpha-amylase gene, an engineering bacterium (Escherichia coli) JDA001 for producing fused acid thermophilic alpha-amylase, the fused acid thermophilic alpha-amylase produced by fermenting by using the genetic engineering bacterium and application of the fused acid thermophilic alpha-amylase to the deep processing of starch. The purity of a final protein product which is obtained through separation and purification by the method is over 98 percent, and the specific activity is 274.97U / mg. The fused acid thermophilic alpha-amylase gene ST0926-27 is obtained through overlap extension polymerase chain reaction (PCR) amplification, is connected to an expression vector, and is successfully expressed in the Escherichia coli; the high recovery efficiency and a better purification effect are achieved; and the fused acid thermophilic alpha-amylase ST0926-27 has higher optimum reaction temperature and thermal stability and a lower optimum pH value, and has good industrial application prospect.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an engineered bacterium (Escherichia coli) JDA001 that fuses acidic thermophilic alpha-amylase genes and produces fused acidic thermophilic alpha-amylase, using the genetically engineered bacteria A fusion acid thermophilic alpha-amylase produced by fermentation and the application of the amylase in starch deep processing. Background technique [0002] Amylase is a general term for a class of enzymes that can degrade glycosidic bonds of starch. According to the different hydrolysis methods, it is mainly divided into α-amylase, β-amylase, glucoamylase, isoamylase and pullulanase. [0003] The α-amylase family includes hydrolases and transferases. According to the similarity of sequences, they are divided into three sub-families, 13, 70, and 77. The members are all multi-domain proteins with many structural and catalytic mechanisms. Similarity, if there is one (α / β) 8 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/56C12N9/28C12N1/21C12N15/70C12P19/14C12R1/19C12R1/01
Inventor 高仁钧解桂秋常琳潘冬曹淑桂
Owner 欣雅利华生物技术(上海)有限公司