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Preparation method and application of recombinant trehalase

A technology of trehalase and trehalose, which is applied in the field of bioengineering, can solve the problems of high sample processing requirements and limitations, and achieve the effects of low equipment requirements, less time consumption, and simple strain culture conditions

Pending Publication Date: 2022-04-26
山东恒仁工贸有限公司
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  • Claims
  • Application Information

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Problems solved by technology

At present, the detection of trehalose is mainly carried out by HPLC, such as the method recommended by the national standard GB / T 23529-2009, but the analysis of trehalose by HPLC requires the use of acetonitrile reagent with certain toxicity, and the sample processing requirements are relatively high, and it is limited by specific equipment conditions

Method used

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  • Preparation method and application of recombinant trehalase
  • Preparation method and application of recombinant trehalase

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Embodiment 1

[0028] A preparation method for recombinant trehalase, comprising the steps of:

[0029] (1) Using the Escherichia coli (Escherichia coli) BL21 genome as a template, utilize the Shanghai Sangong SanPfuPCR Mix kit to PCR amplify the trehalase encoding gene trl to obtain the trl gene fragment; the nucleotide sequence of the trehalase encoding gene As shown in SEQ ID NO.1; the primers for the PCR amplification are shown in SEQ ID NO.2 and SEQ ID NO.3;

[0030] 19-trl-HindIIIF: CCCAAGCTTAAAGGAGGGAAATCATGCTCAATCAGAAAATTCAAAACC (SEQ ID NO. 2);

[0031] 19-trl-EcoRI: CCGGAATTCTTATGGTTCGCCGTACAAACC (SEQ ID NO. 3);

[0032] (2) The trl gene fragment and the pXMJ19 plasmid were cut and ligated with HindIII and EcoRI restriction enzymes (Shanghai Sangong), the ligated product was transformed into E.coli DH5α competent, the transformant was picked and the plasmid was extracted for verification, and the obtained Recombinant expression plasmid pXMJ19-trl;

[0033](3) The recombinant expr...

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Abstract

The invention relates to a preparation method of recombinant trehalase, and belongs to the technical field of bioengineering. According to the invention, a trehalase coding gene from escherichia coli BL21 is expressed in corynebacterium glutamicum ATCC13032, such that recombinant trehalase is obtained. The safe strain corynebacterium glutamicum is used for expressing the trehalase, the expression effect is good, the strain culture condition is simple, the culture time is short, the prepared recombinant trehalase is good in safety and wide in available range, the recombinant trehalase can specifically hydrolyze trehalose to generate glucose, and the yield is high. And the recombinant trehalase has good stability under low-temperature and freezing preservation conditions, and can be preserved for a long time. The prepared enzyme liquid containing the recombinant trehalase can be used for rapidly detecting the content of trehalose in a sample, the sample treatment is simple, the consumed time is short, the equipment requirement is low, and the detection accuracy is high.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant trehalase and its application in the detection of trehalose. Background technique [0002] Trehalose is a disaccharide composed of two molecules of glucose connected by α,α-1,1 glycosidic bonds, and has the characteristics of non-reducing and stable chemical properties. Trehalose is widely distributed in nature. Bacteria, fungi, plants and invertebrates such as insects, nematodes and shrimps all contain trehalose. Studies have shown that trehalose can protect biological macromolecules such as cell membranes and proteins, and help organisms withstand harsh environments such as high temperature, freezing, and high osmotic pressure. It is a stress synthetic metabolite in organisms. Trehalase can decompose trehalose into two molecules of glucose, which widely exists in animals, plants and microorganisms. In recent years, the application of trehalase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N9/24C12Q1/34C12Q1/54C12R1/15
CPCC12N9/2402C12N15/77C12Y302/01028C12Q1/34C12Q1/54G01N2333/924
Inventor 张建涛张建波吕宏涛马春艳满在伟崔慧慧
Owner 山东恒仁工贸有限公司
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