Bifunctional enzyme preparation method and applications of bifunctional enzymes in production of trehalose

A technology for trehalose and trehalose synthase, which is applied in the directions of biochemical equipment and methods, chemical instruments and methods, enzymes, etc., can solve the problems such as the conversion rate can not be further improved, the enzyme yield is low, etc. cost, improve conversion rate, simplify cost effectiveness

Inactive Publication Date: 2018-06-05
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Among the above methods, using starch as a substrate to produce trehalose with dual enzymes is a more economical way, but the current production method has problems such as low enzyme yield and no further improvement in conversion rate.

Method used

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  • Bifunctional enzyme preparation method and applications of bifunctional enzymes in production of trehalose
  • Bifunctional enzyme preparation method and applications of bifunctional enzymes in production of trehalose
  • Bifunctional enzyme preparation method and applications of bifunctional enzymes in production of trehalose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1, the construction of enzyme-producing engineering bacteria

[0083] In this example, Escherichia coli is used to express maltooligosaccharide-based trehalose synthase and maltooligosaccharide-based trehalose hydrolase through the fusion of linker peptides F or R of different lengths and rigidities, and obtain a series of maltooligosaccharide-based trehalose A bifunctional enzyme with synthetase and maltooligosaccharide-based trehalose hydrolase activities. Specific steps are as follows:

[0084] 1. Construction and identification of expression vectors

[0085] According to the difference of connecting peptide and its length, the following 12 different expression vectors expressing maltooligosaccharyl trehalose synthase and maltooligosaccharyl trehalose hydrolase were constructed. The names of each expression vector and the fragments connected into the expression vector are shown in Table 1.

[0086] Table 1. Composition of expression vectors

[0087] ...

Embodiment 2

[0146] Example 2, the application of fusion enzyme in the production of trehalose

[0147] 1. Comparison of conversion rate between free enzyme and fusion enzyme

[0148] With the pullulanase-treated DE11 obtained in 1 of step 4 of Example 1 as a substrate, it was divided into the following control group and experimental group according to the difference of added enzymes:

[0149] 1. Control group treatment (CK)

[0150] Regulate the pH of the above-mentioned pullulanase-treated DE11 to about 7.0 with sodium phosphate buffer, then add 0.14 mg MTSase pure enzyme (the purified MTS free enzyme obtained in step 3 of Example 1) and 0.11 mg MTHase ​​pure enzyme ( The purified MTH-free enzyme obtained in step 3 of Example 1). Among them, the ratio of DE11 substrate, MTSase pure enzyme and MTHase ​​pure enzyme is 1g: 0.14mg: 0.11mg.

[0151] 2. Experimental group treatment

[0152] The pH of the above-mentioned pullulanase-treated DE11 was adjusted to about 7.0 with sodium phospha...

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Abstract

The invention discloses a bifunctional enzyme preparation method and applications of the bifunctional enzymes in production of trehalose. According to the present invention, maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose hydrolase (MTHase) are subjected to fusion expression through linking peptides with different lengths and different flexibility to obtain a series of the bifunctional enzymes integrating the activities of maltooligosyl trehalose synthase (MTSase) and maltooligosyl trehalose hydrolase (MTHase), and the obtained bifunctional enzymes are used for the production of trehalose so as to further increase the conversion rate of trehalose, reduce the number of the cultured strains, save the production process and the production cost, and establish the foundation for the economical and efficient production of trehalose.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method of a bifunctional enzyme and its application in the production of trehalose. Background technique [0002] Trehalose is a non-reducing sugar composed of two glucose molecules bonded by α-1,1 glycosidic bonds, the molecular formula is C 12 h 22 0 11 , and its relative molecular weight is 342.30. Its structural formula is as shown in formula 1: [0003] [0004] Trehalose widely exists in bacteria, yeast, algae, fungi, insects and some stress-resistant plants. It has a non-specific protective effect on biological macromolecules. This protective effect is reflected in: when biological cells are in harsh environments such as starvation, dryness, high temperature, and high osmotic pressure, the content of trehalose in the cells increases rapidly. It produces a protective effect, thereby maintaining the life characteristics of organisms, so trehalos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N9/24C12N15/62C12P19/24C12P19/14C12P19/12
CPCC07K2319/00C12N9/2402C12N9/90C12N15/62C12P19/12C12P19/14C12P19/24C12Y302/01141C12Y504/99015
Inventor 林白雪袁慎键陶勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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