Engineering strain expressing acid-resistant high-temperature alpha-amylase gene mutants

A technology of engineering strains and amylases, applied in genetic engineering, plant genetic improvement, microorganism-based methods, etc., can solve the problems of low enzyme activity and single strain, and achieve the effect of strong acid resistance

Inactive Publication Date: 2014-06-04
BEIJING ZHONGKE XINGGUAN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As early as the 1970s, domestic scholars began to study acid amylase, and achieved some research results, but compared with foreign countries, the gap is far away. The strains of acid α-amylase produced in China are relatively single, and the enzyme activity lower

Method used

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  • Engineering strain expressing acid-resistant high-temperature alpha-amylase gene mutants
  • Engineering strain expressing acid-resistant high-temperature alpha-amylase gene mutants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1. Sodium nitrite mutagenesis to obtain acid-resistant high-temperature α-amylase mutant gene amyM

[0017] Genomic DNA of Bacillus licheniformis CICC10181 (purchased from China Industrial Microorganism Culture Collection Center) was extracted. (Extraction method refers to Bron, S (1990). Plasmids. In Molecular Biological Methods for Bacillus, pp75-174. Edited by C.R. Harwood & S.M. cutting. Chichester: Wiley).

[0018] Using Bacillus licheniformis (B.Licheniformis) CICC10181 genomic DNA as a template for PCR amplification of amy gene, the upstream primer is primer 1 (5'-ACCTGCCTGTACACTTGCG-3') and the downstream primer is primer 2 (5'-CTCTCTGCTCTTCTATCTTTG-3') .

[0019] Amplification conditions: pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 1 minute, annealing at 56°C for one minute, extension at 72°C for 1 minute and 30 seconds. After completing 30 cycles, it was incubated at 72° C. for 10 minutes.

[0020] The amy fragment of 1978b...

Embodiment 2

[0031] Example 2. Construction of the shuttle vector pAXOI-amyl carrying the LacA integration arm expressing the acid-resistant high-temperature α-amylase mutant gene amyM

[0032] The plasmid pEASY-T3-amyM was used as a template to amplify the acid-resistant and high-temperature α-amylase mutant gene amyM by PCR. The amplification primer is: upstream primer 5'-cg ggatcc ACCTGCCTGTACACTTGCG-3' (the underlined part is the BamHI restriction site) and downstream primer 5'tccat ccgcgg -CTCTCTGCTCTTCTATCTTTG-3' (the underlined part is SacII, restriction site).

[0033]The amyM fragment of 1978bp was obtained from the above amplification, and sequencing showed that the fragment included the promoter of the acid-resistant high-temperature alpha-amylase gene, the signal peptide coding sequence and the structural gene.

[0034] The PCR product amyM and the shuttle integration plasmid pAXOI (purchased from the Ohio State University Culture Collection Center, Bacillus Genetic Stock C...

Embodiment 3

[0035] Embodiment 3, the construction of engineering bacteria ZHWY expressing acid-resistant high-temperature α-amylase gene mutant

[0036] The expression shuttle integration plasmid pAXOI-amyM carrying the promoter, signal peptide and structural gene of acid-resistant high-temperature amylase obtained by mutagenesis in Example 2 was electroporated with protoplasts (Romero D, Journal of Microbiology Methods, 2006 Vol66p556-559 ) to transform Bacillus licheniformis CICC10181 (purchased from the China Industrial Microbiology Culture Collection Management Center, the preservation number is CICC10181) producing high-temperature starch, and the specific method is as follows:

[0037] After the Bacillus licheniformis CICC10181 protoplasts were obtained using the Romero D Bacillus licheniformis protoplast preparation method (Romero D, Journal of Microbiology Methods, 2006Vol66p556-559), take 5ul (2ug) of the purified plasmid pAXOI-amyM in a 1.5ml centrifuge Put it and the 0.2CM elec...

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Abstract

The invention discloses an engineering strain expressing acid-resistant high-temperature alpha-amylase gene mutants. The engineering strain is obtained by introducing a recombinant vector expressing acid-resistant high-temperature alpha-amylase genes into bacillus licheniformis, and performing screening to obtain the engineering strain with improved acid-resistant high-temperature alpha-amylase expression level; the acid-resistant high-temperature alpha-amylase has an amino acid sequence as shown in sequence 2 in the sequence table. The engineering strain has stronger capability of producing acid-resistant high-temperature alpha-amylase than current domestic production strains, has a unique identification label, and is a bacillus licheniformis production strain of acid-resistant high-temperature alpha-amylase that has great production and application value.

Description

technical field [0001] The invention relates to an engineering strain expressing an acid-resistant high-temperature alpha-amylase gene. Specifically, the high-temperature α-amylase gene amy is mutated by a chemical mutagen, and then a new acid-resistant high-temperature α-amylase gene mutant amyM is obtained by screening under acidic conditions, and then expressed and applied in Bacillus licheniformis engineering bacteria. Background technique [0002] High-temperature α-amylase, that is, the α-amylase that can hydrolyze the a-1,4 glucosidic bonds in starch, soluble dextrin and oligosaccharides, because its optimum reaction temperature is relatively high, generally above 60 degrees Celsius, so It is called high-temperature alpha-amylase. [0003] High-temperature α-amylase is widely used in the industrial production of wine, monosodium glutamate, and starch sugar. It replaces the traditional process, which not only saves labor and plant equipment, but also improves the yiel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/56C12N9/28C12R1/10
Inventor 孟青青王凤寰杨建国汪兵
Owner BEIJING ZHONGKE XINGGUAN BIOLOGICAL TECH
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