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Alpha-amylase truncated body and application thereof

An amylase and variant technology, which is applied in the field of truncation of α-amylase, can solve problems such as increasing cost, and achieves a technology that is beneficial to industrial production, improves thermal stability and specific activity, large catalytic efficiency and thermal stability. Effect

Inactive Publication Date: 2012-10-10
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The design of the above process is based on the application of high temperature resistant α-amylase Liquozyme and glucoamylase Dextrozyme GA. The optimum pH of Liquozyme is pH 5.5-pH 6.0, which can withstand high temperatures of 110°C, but the enzyme activity and stability require calcium ions (not less than 5ppm), and the optimum pH of Dextrozyme GA is about pH 4.5, and the optimum temperature is 60°C. The inconsistency between the pH value and temperature of the preceding and following processes has caused pH adjustment and temperature adjustment, increasing Costs due to chemical reagents and "excessive" energy consumption

Method used

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  • Alpha-amylase truncated body and application thereof
  • Alpha-amylase truncated body and application thereof
  • Alpha-amylase truncated body and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] This example illustrates the purification of the parent enzyme, the characterization of the purified enzyme and the analysis method of the enzymatic hydrolyzate

[0044] 1. Cultivation of Bacillus subtilis CN7

[0045]Bacillus subtilis CN7 was isolated from the soil sample of Guangxi University farm, and the strain was preserved in our laboratory, and submitted to the China Center for Type Culture Collection with the preservation number CCTCC M 2012061. The medium is liquid LB plus 1% soluble starch, and each liter of medium contains 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, and 10 g of soluble starch. The solid medium was the above liquid medium with 1.5% agar added. Take a tube of the original strains stored in the cryopreservation tube at -80°C, use an inoculation loop to get the bacteria to streak on the solid medium plate, cultivate overnight in a 37°C incubator, pick a single colony and inoculate it in the liquid medium , placed on a consta...

Embodiment 2

[0068] This example illustrates the cloning and analysis of the full-length α-amylase gene, the construction and expression of the recombinant plasmid, and the purification of the recombinant enzyme

[0069] 1. Cloning of full-length α-amylase gene and construction of recombinant plasmid

[0070] With reference to "Molecular Cloning Experiment Guide" (Sam Brook, Russell. Molecular Cloning Experiment Guide [M]. Beijing: Science Press, 2002.), extract the total DNA of Bacillus subtilis CN7, use this total DNA as a template, and use primers Amy7-S and Amy7-A, the complete α-amylase gene amy7 with a full length of 1980bp was amplified by PCR, and the primer sequences were as follows:

[0071] Amy7-S: 5′-GTA TCATGA TGTTTGAAAAACGATTCAAAAC-3′

[0072] Amy7-A: 5′-GCG AAGCTT AATCAATGCGGAAGATAACCATTC-3′

[0073] For convenience of operation, a PagI restriction site (underlined part) was introduced into the upstream primer Amy7-S, and a Hind III restriction site (underlined part) w...

Embodiment 3

[0082] This example illustrates the construction and purification of truncated α-amylase

[0083] 1. Construction of truncated α-amylase

[0084] Using Amy7M-S and Amy7M-A as primers, using the total DNA of Bacillus subtilis CN7 as a template, adopt the same method as in Example 1, 1, the cloning of the full-length α-amylase gene and the construction of the recombinant plasmid, to construct a gene containing the truncated Amy7M code. Gene recombinant plasmid pSA7M, the primer sequence is as follows:

[0085] Amy7M-S 5`-GAC TCATGA GCTCGGTCAAAAACGGGACCATC-3`

[0086] Amy7M-A 5`-GTAC AAGCTT ATGCGGAAGATAACCATTCAAA-3`

[0087] A Pag I restriction site (underlined part) was introduced into the upstream primer Amy7M-S, and a Hind III restriction site (underlined part) was introduced into the downstream primer Amy7M-A.

[0088] Using primers Amy7D-S and Amy7D-A as primers, and using the total DNA of Bacillus subtilis CN7 as a template, the same method as in Example 1, 1, the cl...

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Abstract

The invention provides an alpha-amylase truncated body and application thereof, and relates to meaningful alpha-amylase variant which simultaneously improves thermal endurance and catalytic activity, which is characterized in that 45-467 amino acid peptide fragment of bacillus subtilis Cn7alpha-amylase is cut off, and a fragment of artificially synthetic peptide is fused on C terminal. Obtained variant alpha-amylases respectively have temperature toleration increased by 2.7 DEG C, catalytic activity increased by 2.23 times compared with the parent alpha-amylase; and the variant alpha-amylases are more beneficial to industrialized production in the starch industry than the parent alpha-amylase.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a truncated body of α-amylase and its application. Background technique [0002] Starch is a homopolymer composed of D-glucose monomers and is the major storage form of sugar in plants. As one of the most abundant and important biomass resources, starch is not only used for food, but also widely used in industries such as textiles, papermaking, and pharmaceuticals. It is especially used as a carbon source in the fermentation industry to produce fuel ethanol and amylase. Sugar, etc. The key to the development and utilization of starchy biomass resources is to degrade them into fermentable sugars such as glucose and maltose, which can be used for microbial fermentation to produce high value-added chemical products including organic alcohols and amino acids, including bioethanol, Biomass energy including bio-butanol, and bio-based materials including L-lactic acid, etc. [0003] α-am...

Claims

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Application Information

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IPC IPC(8): C12N9/28C12N15/56C12N15/63C12N1/21C12P19/14C12P7/06D06L1/00C12R1/125
CPCY02E50/17Y02E50/10
Inventor 王成华黄日波王青艳申乃坤陈东黎贞崇黄志民
Owner GUANGXI ACAD OF SCI
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