A mutant tba-h2 of acid-resistant high-temperature beta-amylase and its application

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: 2015-11-18
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

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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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  • A mutant tba-h2 of acid-resistant high-temperature beta-amylase and its application
  • A mutant tba-h2 of acid-resistant high-temperature beta-amylase and its application
  • A mutant tba-h2 of acid-resistant high-temperature beta-amylase and its application

Examples

Experimental program
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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] The chromosomal DNA of Clostridium thermothiobacterium (Thermoanaerobacterium thermosulfurigenes, American Type Culture Collection No. ATCC33743) was used as the template, SEQ ID NO: 2 was used as the upstream primer (containing a NcoI restriction site) and SEQ ID NO: 3 (containing a BamHI restriction site) was used as the template. site, and introduced a 6x histidine tag coding sequence) as the downstream primer, amplifying the mature peptide coding region of β-amylase by polymerase chain reaction (PCR), corresponding to the 660-2219 fragment in the ctba gene, the resulting The length of the target product is 1605bp; the target product and the pSE380 vector plasmid were double digested with NcoI and BamHI enzymes, respectively, and after the gel was recovered, ligated with T4 DNA ligase, and transformed into E. coli XL1-Blue competent cells...

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 / LpH4.0 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 at 60°C Warm in a water bath for 24 hours, heat-treat in a water bath at 100°C 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 to detect the enzymatic hydrolysis product by high performance liquid chromatography (HPLC). 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 under acidic condi...

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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 an acid-resistant high-temperature beta-amylase, and the application of the mutant enzyme in starch degradation and starch-containing material treatment, especially for producing high-purity maltose pulp 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 starch, dextrin and other polysaccharides by non-reducing The α-1,4-glycosidic 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. It 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 optimum pH value ...

Claims

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Application Information

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Patent Type & Authority Patents(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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