Mutant TBA-H2 of acid and high temperature resistant beta-amylase and application thereof

A technology of TBA-H2 and amylase, applied in the direction of enzyme, hydrolase, glycosylase, etc., can solve the problem that β-amylase cannot take into account acid resistance and temperature resistance at the same time, so as to improve catalytic activity and wide application foreground effect

Active Publication Date: 2014-06-25
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 purpose of the present invention is to provide a mutant TBA-H2 of acid-resistant high-temperature β-amylase and its application, which solves the problem that the existing β-amylase cannot take into account acid resistance and temperature resistance at the same time

Method used

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  • Mutant TBA-H2 of acid and high temperature resistant beta-amylase and application thereof
  • Mutant TBA-H2 of acid and high temperature resistant beta-amylase and application thereof
  • Mutant TBA-H2 of acid and high temperature resistant beta-amylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] This example illustrates the method for obtaining the β-amylase mutant TBA-H2.

[0018] 1) Cloning of β-amylase gene ctba

[0019] Using Thermoanaerobacterium thermosulfurigenes (Thermoanaerobacterium thermosulfurigenes, American Type Culture Collection No. ATCC33743) chromosomal DNA as template, SEQ ID NO: 2 as upstream primer (containing a Nco I restriction site) and SEQ ID NO: 3 (contains a Bam HI restriction site and introduces a 6x histidine tag coding sequence) as a downstream primer to amplify the mature peptide coding region of β-amylase by polymerase chain reaction (PCR), corresponding to the ctba gene The 660-2219 fragment in the center, the length of the obtained target product is 1605bp; the target product and the pSE380 vector plasmid were double-digested with Nco I and Bam HI enzymes respectively, after the gel was recovered, they were ligated with T4 DNA ligase, and transformed into Escherichia coli (E. .coli) XL1-Blue competent cells; select transforman...

Embodiment 2

[0026] This example illustrates the application of mutant enzyme TBA-H2 in the production of high-purity maltose syrup by hydrolyzing starchy raw materials

[0027] Take 1g of potato soluble starch and add 0.05mol / L pH4.0 acetic acid buffer solution to prepare 1% (mass percentage) starch slurry, add purified enzyme solution according to the amount of 1.2mg enzyme / g dry matter starch, and then place it in 60 Warm in a water bath at ℃ for 24 hours, heat treatment in a water bath at 100 ℃ for 10 minutes, centrifuge at 12,000 r / min for 5 minutes, filter the supernatant through a 0.22 μm filter membrane, and take 20 μL of the filtrate to pass through an amino column for high performance liquid chromatography (HPLC) detection of the enzymatic hydrolysis product . The detection conditions of the amino column were RI temperature of 50°C, flow rate of 1mL / min, and mobile phase of acetonitrile:water (70:30). Amino column detection results such as image 3 shown. It can be seen that u...

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Abstract

The invention provides a mutant TBA-H2 of acid and high temperature resistant beta-amylase and an application thereof. The amino acid sequence of the mutant is shown in SEQ ID NO:1 in a sequence table. Relative to enzymes before mutation, the mutant has an optimum pH value of acid and a higher function of generating maltose. The mutant TBA-H2 of high temperature resistant beta-amylase can be used for degrading starchy materials and is especially suitable for degrading starch under acidic conditions to generate maltose.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant TBA-H2 of acid-resistant high-temperature β-amylase, and the application of the mutant enzyme in starch degradation and starch-containing material processing, especially for producing high-purity maltose slurry application. Background technique [0002] β-amylase, also known as 1,4-α-D-glucan maltohydrolase (EC3.2.1.2), is an exonuclease that catalyzes the degradation of non-reducing polysaccharides such as starch and dextrin The α-1,4-glucosidic bond at the sex terminal is used to continuously produce maltose. β-amylase does not contain α-amylase activity, and is used to produce high maltose syrup from liquefied starch slurry, and is also widely used in fermentation industries such as maltitol, maltodextrin, and brewing alcohol (Wu Linde, Zheng Dapeng. Introduce a new type of enzyme preparation - β-amylase [J]. Journal of Microbiology, 1986, (3)). The optimal pH value of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12P19/22
CPCC12N9/2425C12P19/12C12P19/22C12Y302/01002
Inventor 梁莲华王成华蒙健宗李晓明韦航
Owner 南宁邦尔克生物技术有限责任公司
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