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A kind of acid-resistant high-temperature beta-amylase mutant and its application

An amylase and mutant technology, which is applied in the application field of producing high-purity maltose syrup, can solve the problem that β-amylase cannot take into account acid resistance and temperature resistance at the same time, and achieves the advantages of reducing production cost, reducing cost and simplifying process. Effect

Active Publication Date: 2015-09-16
南宁邦尔克生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The invention provides an acid-resistant and high-temperature β-amylase mutant, which solves the problem that the existing β-amylase cannot take into account both acid resistance and temperature resistance

Method used

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  • A kind of acid-resistant high-temperature beta-amylase mutant and its application
  • A kind of acid-resistant high-temperature beta-amylase mutant and its application
  • A kind of acid-resistant high-temperature beta-amylase mutant and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0018] This example illustrates the acquisition of the β-amylase gene ctba and the construction of a recombinant expression vector

[0019] 1) Extract Thermoanaerobacterium thermosulfurigenes, American Type Culture Collection No. ATCC33743, chromosomal DNA as a template;

[0020] 2) Use SEQ ID NO: 2 as the upstream primer (containing a Nco I restriction site) and SEQ ID NO: 3 (containing a Bam HI restriction site and introducing a 6x histidine tag coding sequence) as The downstream primers are used to amplify the mature peptide coding region of the β-amylase gene ctba, whose GenBank sequence number is M22471.1, by polymerase chain reaction PCR, corresponding to the 660-2219 fragment in the ctba gene, and the length of the obtained PCR target product is 1605bp ;

[0021] 3) The target product and the pSE380 vector plasmid were double-digested with Nco I and Bam HI enzymes respectively, after the gel was recovered, ligated with T4DNA ligase, and transformed into E. coli (E.coli...

Embodiment 2

[0024] This example illustrates the construction of mutant expression plasmids

[0025] Using the PCR primer-mediated site-directed mutagenesis method, the whole plasmid pSBA DNA was used as a template to construct a single-point mutant, and then the recombinant plasmid containing the single-point mutation was used as a template to introduce the second mutant with primers containing the second mutation site. Mutation, and so on to construct mutant plasmids containing multiple mutation sites. details as follows:

[0026] 1) Use pSBA plasmid DNA as a template, and use SEQ ID NO:4 and SEQ ID NO:5 as upstream and downstream primers respectively to construct a PCR reaction system. The 25 μL PCR reaction system was constructed as follows: 1 μL pSA7D plasmid DNA, 0.5 μL upstream primer Amy7-S (concentration: 10 mmol / L), 0.5 μL downstream primer Amy7-A (concentration: 10 mmol / L), 2 μL dNTPs (per dNTP2.5mmol / L), 5μL5x buffer, 0.25μL (2.5U / μL) DNA polymerase, add 16.75 μL ddH 2O ...

Embodiment 3

[0031] This example illustrates the induction, expression, purification and characterization of mutant enzymes

[0032] 1) Pick a single colony of Escherichia coli engineering bacteria containing the recombinant expression plasmid pSBA-S4, and inoculate it in 5ml LB culture medium containing 100μg / ml ampicillin (yeast extract 10g / L, peptone 5g / L, sodium chloride 10g / L , natural pH), cultured overnight at 37°C and 220r / min. The overnight cultured bacterial solution was transferred to 500ml LB culture solution containing 100μg / mL ampicillin according to the inoculum size of 1%. When the bacterial solution was cultured to OD 600 is about 0.6, add IPTG inducer with a final concentration of 1mmol / L, and continue to induce culture for 16h under the same conditions;

[0033] 2) Centrifuge at 9000r / min and 4°C to collect engineered bacteria that induce expression, wash the precipitate once with 0.05mol / L acetate buffer (pH6.0), resuspend in 100ml of the same buffer, and pass the bac...

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Abstract

The invention discloses an acid-resistant and high-temperature-resistant beta-amylase mutant and an application thereof. Small amino acid nearby a catalytic functional domain, including 115-site cysteine, 312-site alanine, 351-site threonine and 354-site cysteine is reconstructed into serine by taking thermoanaerobacterium thermosulfurigenes high-temperature beta-amylase as a parent and by adopting a molecular enzyme engineering technique, and the acid-resistant and high-temperature-resistant beta-amylase mutant particularly comprises an SEQ ID NO:1 (sequence identifier number 1) amino acid sequence. The obtained beta-amylase mutant keeps a temperature resistance characteristic, an optimum pH (power of hydrogen) value is lowered to 4.0 from 6.0 of parent enzyme, the activity is improved by 1.4 times, and the acid-resistant and high-temperature-resistant beta-amylase mutant more facilitates industrial production of the starch industry in comparison with a wild beta-amylase mutant.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an acid-resistant high-temperature beta-amylase mutant, and the application of the mutant enzyme in starch degradation and starch-containing material processing, especially for the production of high-purity maltose syrup Background technique [0002] β-amylase, also known as α-1,4-glucan-maltohydrolase (1,4-α-D-glucan maltohydrolase, EC3.2.1.2), can hydrolyze α- 1,4-glucosidic bonds generate β-type maltose, so it is widely used in the production of maltose, maltose, maltitol, maltodextrin and fermentation industries such as brewing beer, alcohol and vinegar (Wu Linde, Zheng Dapeng. Introducing a new enzyme preparation- β-amylase [J]. Journal of Microbiology, 1986, (3)). β-amylase can be produced by higher plants and some microorganisms. However, the β-amylase produced by bacteria has the ability to bind raw starch, and has high temperature tolerance and catalytic activity, so it is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12P19/14C12R1/01
CPCC12N9/2425C12P19/12C12P19/22C12Y302/01002
Inventor 王成华蒙健宗梁莲华程婷婷李晓明韦航陈发忠杜奇石周礼芹
Owner 南宁邦尔克生物技术有限责任公司
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