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Acid amylase AMYA4 and gene and application thereof

An acid amylase, gene technology, applied in the field of genetic engineering, can solve the problem of not degrading starch and the like

Active Publication Date: 2010-11-17
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reported amylases derived from Alicyclobacillus do not have the ability to degrade raw starch

Method used

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  • Acid amylase AMYA4 and gene and application thereof
  • Acid amylase AMYA4 and gene and application thereof
  • Acid amylase AMYA4 and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Example 1 Cloning of Alicyclobacillus peridum A4 (CGMCC No.3147) amylase-encoding gene AMYA4

[0132] Acquisition of gene sequence

[0133] According to the amylase gene sequence reported by Alicyclobacillus, and with reference to the mass spectrum sequence (GNGQWNIDFFGGDLK, HWLALGADGW) of the above-mentioned purified amylase, specific primers AF and AR were directly designed to use Alicyclobacillus hesperidum A4 (CGMCC No. .3147) Total DNA was used as template for PCR amplification. The PCR reaction parameters were: denaturation at 94°C for 5 min, annealing at 60°C for 30 sec, and extension at 72°C for 1.5 min. Finally, keep warm at 72°C for 10 minutes. A fragment of about 480bp was obtained, which was recovered and connected with the pEASY-T3 vector and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0134] According to the nucleotide sequence obtained by sequencing, three TAIL-PCR specific nested primers were designed upstream and downstream respectively: ...

Embodiment 2

[0141] The activity analysis of embodiment 2 purifying amylase

[0142] The method for separating and purifying acid amylase AMYA4 from Alicyclobacillus hesperidum A4 comprises the following steps:

[0143] 1) After shaking the seed medium at 55°C for 48 hours, transfer 1% of the inoculum to the enzyme-producing medium, and after shaking at 55°C and 200rpm for 48 hours, centrifuge the bacterial solution, and take the supernatant for purification;

[0144] 2) The culture was centrifuged to take about 101 of the supernatant, and then passed through a 6K hollow fiber ultrafiltration membrane to about 600ml in an ice bath, and then further concentrated to 200ml with a 5K ultrafiltration membrane bag;

[0145] 3) Remove 10ml of the above-mentioned concentrated solution in buffer A (20mmol / L citric acid-Na 2 HPO 4 pH 3.5) for overnight dialysis, cation exchange chromatography: take 2.0mL of the concentrated solution and put it on the HiTrap SP XL cation column equilibrated with buff...

Embodiment 3

[0147] The property determination of embodiment 3 purifying amylase AMYA4

[0148] 1. The optimum pH and pH stability assay methods of purified amylase AMYA4 are as follows:

[0149] The amylase purified in Example 2 was subjected to enzymatic reactions at different pHs to determine its optimum pH. Substrate soluble starch with different pH buffers (0.1mol / L citric acid-sodium hydrogen phosphate pH 3.0-7.4; 0.1M Tris-HCl pH 7.8-8.6; 0.1M glycine-sodium hydroxide pH 9.0-11.0;), Amylase activity assays were performed at 75 °C. result( figure 2 ) shows that its optimum pH is 4.2, and it maintains more than 70% relative enzyme activity at pH 3.8-5.8. The most stable pH range of the enzyme is 3.0-10.0, and the enzyme activity is kept above 85% after acting for 2 hours at 37°C. For the experimental results of the optimum action temperature and thermal stability of amylase, see image 3 .

[0150] 2. The optimum temperature and thermostability determination method of amylase i...

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Abstract

The invention relates to the field of gene engineering, in particular to acid amylase AMYA4 and gene and application thereof. The amino acid sequence of the acid amylase AMYA4 is expressed as SEQ ID NO.1. The optimal pH value of the AmyA4 is 4.2, the optimal temperature is 75 DEG C, and the AmyA4 has high activity at the temperature of between 55 and 75 DEG C and has strong raw starch degrading capability at the same time. The zymology properties meet the process requirement for preparing sugar by a double-enzyme method, so the acid amylase has great application potential. In the process for preparing sugar by simulating the double-enzyme method, no matter paste starch or raw starch is used as a substrate, the starch can reach good hydrolysis rate under the action of matching commercial saccharifying enzyme, so the AmyA4 has huge application potential.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an acid amylase AMYA4 and its gene and application. Background technique [0002] α-amylase (α-amylase) is currently one of the most important industrial enzyme preparations, widely present in animals, plants and microorganisms, it is an endoglucosidase, according to the International Enzyme Commission (Enzyme Commission) The standard is EC3.2.1.1. When α-amylase acts on starch, it can arbitrarily cut α-1,4 bonds from the inside of starch molecules, so that starch molecules can be rapidly degraded to generate dextrin and reducing sugar. α-amylase acts on starch, which usually causes starch to lose its iodine coloring reaction. When hydrolyzing amylose, α-amylase randomly cuts α-1, 4 bonds, hydrolyzes starch into maltose oligosaccharides and oligosaccharides, and then hydrolyzes into maltose and glucose. When amylopectin is used as a substrate, α-amylas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/63C12N1/15C12N1/19C12N1/21C12P19/14
Inventor 姚斌柏映国石鹏君孟昆罗会颖王亚茹袁铁铮杨培龙黄火清
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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