Alpha-AmyL (Amylase) mutant with increased activity and coding gene and application thereof

An amylase and mutant technology, applied in the field of genetic engineering, can solve the problems of high fermentation cost and low production activity, and achieve the effect of huge application potential and reduction of fermentation production cost.

Active Publication Date: 2017-05-31
内蒙古溢多利生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although α-amylase AmyL has great application potential, the production...

Method used

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  • Alpha-AmyL (Amylase) mutant with increased activity and coding gene and application thereof
  • Alpha-AmyL (Amylase) mutant with increased activity and coding gene and application thereof
  • Alpha-AmyL (Amylase) mutant with increased activity and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Synthesis and cloning of the α-amylase AmyL gene of Bacillus salsus

[0042] The published amino acid sequence of Bacillus salsus α-amylase AmyL (Genebank: SDP85898) was synthesized according to the codon optimization of Pichia pastoris.

[0043] Design PCR primers containing EcoRI and NotI restriction enzyme sites at the 5' end and 3' end of the synthesized gene respectively, and the primer sequences are as follows:

[0044] 5' end primer amyl-F1: 5'-GTAGAATTC ATGAGACAGGTTAGAATTGCTTTTG-3'

[0045] 3' primer amyl-R1: 5'-ACTGCGGCCGCTTATTTTTGTACATAAACTGAAACT-3'

[0046] Using the synthetic gene as a template, PCR amplification was carried out with the above primers, and the amplified fragment was cloned into the vector pGAPzαA to obtain the recombinant vector pGAPzαA-AMYL.

Embodiment 2

[0047] Embodiment 2, gene error-prone PCR random mutation

[0048]Using the above pGAPzαA-AMYL as a template, carry out error-prone PCR random mutation amplification. The specific amplification method is:

[0049] The first round of amplification: use the vector promoter primers amyl-F1 and amyl-R1 as primers for PCR amplification, and the reaction system is as follows:

[0050]

[0051] The reaction procedure is as follows:

[0052]

[0053] The first-round PCR product was recovered, and 1uL was diluted 50-100 times to be used as a template for the second-round PCR. The second round of error-prone PCR also uses specific primers amyl-F1 and amyl-R1 for PCR reaction.

[0054] The product of the second round was double digested with EcoRI and NotI, and connected to the pGAPzαA vector. The ligation product was transformed into Pichia pastoris X33, and the mutant strains were screened on YPDZ agarose plate.

Embodiment 3

[0055] Embodiment 3, high-throughput screening high enzyme activity mutant strain

[0056] Pick mutant single colonies from the error-prone PCR plate in Example 2, pick the recombinant transformants one by one to a 24-well plate with a toothpick, add 1mL medium containing YPD to each well, culture at 30°C, 220rpm for about 48 hours, and take it by centrifugation. clear. 200 μL of the above supernatants were taken out to a 96-well plate for α-amylase activity assay. The detection of α-amylase activity was carried out with reference to the national standard "GB / T 24401-2009" of the People's Republic of China. Genomic DNA was extracted one by one from 8 positive mutant clones with improved enzyme activity, and the target gene was amplified by PCR to determine the mutation site.

[0057] The sequencing results determined the amino acid mutation site, the mutation site of clone 1 was +18N replaced by +18D; the mutation site of clone 2 was +39S replaced by +39N; the mutation sit...

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Abstract

The invention relates to the field of genetic engineering, and in particular to an alpha-AmyL (Amylase) mutant with increased activity and a coding gene and application thereof. An amino acid sequence of the mutant is as shown in SEQ ID NO. 2. The fermentation enzyme activity of an improved alpha-AmyL strain is up to 36900 U/mL, and compared with an alpha-AmyL producing strain before improvement, the fermentation enzyme activity is increased by 41.5 percent. According to mutant BsaAmy6 (Bacillus Salsus Alpha-Amylase 6) and recombinant engineering bacterium, the fermentation production cost is greatly reduced, and a foundation is laid for further industrial application.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an activity-improved α-amylase AmyL mutant and its coding gene and application. Background technique [0002] α-amylase, the system name is 1,4-α-D-glucan hydrolase, and its aliases are liquefied amylase, liquefied enzyme, and α-1,4-dextrinase. α-amylase is a kind of endohydrolase, its main function is to catalyze the 1,4-α-D-glucan of starch to generate reducing dextrin and sugars, in the fields of starch, detergent, beverage and textile has an important role. [0003] Since the activity of α-amylase produced by wild bacteria screened from nature is generally relatively low, it cannot be directly applied to industrialized fermentation production. In the prior art, the enzyme-producing ability of the strain is generally improved through technical means such as mutagenesis and cross-breeding of wild strains. With the rapid development of molecular biology, genetic engineering...

Claims

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Application Information

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IPC IPC(8): C12N9/28C12N15/56C12N15/81C12N1/19C12R1/84
Inventor 李阳源黄江王建荣聂金梅陈丽芝何小梅杨玲黄佳乐
Owner 内蒙古溢多利生物科技有限公司
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