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Acid-resistant amylase mutant, and preparation method and application thereof

A technology of amylase and mutants, which is applied in the field of acid-resistant amylase mutants and their preparation, can solve the problems of difficulty in obtaining target strains and large blindness, and achieve the effects of shortening the transformation time, strong acid resistance, and efficient degradation

Inactive Publication Date: 2013-03-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The blindness of the screening is large, and it is not easy to obtain the target strain

Method used

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  • Acid-resistant amylase mutant, and preparation method and application thereof
  • Acid-resistant amylase mutant, and preparation method and application thereof
  • Acid-resistant amylase mutant, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Analysis and method of acid-resistant site-directed mutation of amylase

[0022] By analyzing the 3D spatial structure of amylase ( figure 2 ) analysis to determine the histidine residues (His222, His275, His293, His310) in the catalytic region that are unstable to acid resistance of the enzyme.

[0023] According to the amylase sequence of Bacillus subtilis, the amylase was fully synthesized by chemical total synthesis, and then cloned into the plasmid pET-20b(+) to construct the recombinant plasmid pAmyQ.

[0024] For the site-directed mutagenesis of different His sites, design the corresponding site-directed mutagenesis primers (Table 1). Using site-directed mutagenesis primers, amylase was used for site-directed mutagenesis. PCR enzyme was used to amplify the recombinant plasmid pAmyQ with mutant primers. The amplified fragments were recovered and purified using a gel recovery kit. The obtained purified fragments were phosphorylated at both ends of t...

Embodiment 2

[0025] Example 2: Analysis and method of acid-resistant site-directed mutation of amylase

[0026] Determination of Alkaline Amylase Activity by DNS Method

[0027] 1) DNS reagent configuration: Weigh 2.5g of 3,5-dinitrosalicylic acid and dissolve it in a small amount of water, add 0.5g of phenol, then dissolve 0.075g of sodium sulfite, 2.5g of sodium hydroxide, and 50g of potassium sodium tartrate, and transfer it to Put it into a 500mL volumetric flask, shake it up to volume, store it in a brown bottle and place it in a refrigerator at 4°C until use.

[0028] 2) Preparation of maltose standard curve: Prepare maltose solutions with different concentrations from 0.2g / L to 1.0g / L. Take 1mL of different concentrations of maltose and mix it with the same volume of DNS solution, put it in a boiling water bath, and keep the water bath for 10min. Cool with cold water, dilute to 10mL, A 540 Measure the absorbance. Take the concentration of maltose as the abscissa and the absorban...

Embodiment 3

[0033] Example 3: Determination and Analysis of Catalytic Efficiency of Amylase under Acid Resistance Conditions (pH4.5)

[0034] The recombinant amylase after acid-resistant site-directed mutation was tested for its catalytic efficiency at pH 4.5. Under the condition of citric acid buffer at pH 4.5, mix the buffer with soluble amylase, and use the method in 3) to measure the catalytic efficiency of amylase (k cat ). It was found by measuring that the catalytic efficiencies (k cat ) from the original 2.1×10 2 the s -1 respectively grow to 4.7×, 5.3× and 6.0×10 2 the s -1 ; Catalytic efficiency (k cat ) from the original 2.1×10 2 the s -1 respectively increased to 5.8×, 8.7×, 9.8× and 10.7×10 2 the s -1 . The amylase has strong acid resistance and high catalytic efficiency under acidic conditions.

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Abstract

The invention discloses an acid-resistant amylase mutant and a preparation method thereof, belonging to the field of genetic engineering. In the invention, by using Bacillus subtilis amylase as a female parent, the Bacillus subtilis amylase sequence is subjected to site-directed mutagenesis by a molecular biology technique, and the optimum reaction pH value of the Bacillus subtilis amylase is converted into 4.5 from 7.0 of the control (before mutagenesis) sample under such transformation conditions; and after the mutagenesis, the catalytic efficiency of the amylase is enhanced by 5 times under the pH value of 4.5. The scheme disclosed by the invention can greatly enhance the acid resistance of the amylase, and provides foundation for industrial production. The mutagenesis has important instruction meanings for transforming properties of other enzymes.

Description

technical field [0001] The invention relates to an acid-resistant amylase mutant and a preparation method thereof. Background technique [0002] Acid amylase is an enzyme that can hydrolyze starch under acidic conditions. It can be applied to the processing of starch raw materials under acidic conditions. The remarkable acid resistance makes it have great application potential and development prospects. It is widely used in silage and fermented beverages. , waste liquid treatment and other fields. Acid amylase-producing strains were obtained mainly through screening and mutation. The blindness of the screening is large, and it is not easy to obtain the target strain. Mutation includes: natural mutation and mutagenesis. The probability of natural mutation is quite small, while the workload of mutagenesis is large and the probability of uncontrollable negative mutation is relatively high. Contents of the invention [0003] The invention provides an acid-resistant amylase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/28C12N15/56C12N15/10C12N15/70C12R1/125
Inventor 陈坚堵国成刘龙李江华杨海泉
Owner JIANGNAN UNIV
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