Fructosyl peptidyl oxidase with high specificity as well as encoding gene and application

A technology of peptide oxidase and fructosyl, applied in the field of enzyme engineering, can solve the problem of low specificity of fructosyl peptide oxidase

Active Publication Date: 2017-03-29
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a highly specific enzyme mutant and its coding gene and related uses in view of the shortcomings of the existing fructosyl peptide oxidase with low specificity

Method used

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  • Fructosyl peptidyl oxidase with high specificity as well as encoding gene and application
  • Fructosyl peptidyl oxidase with high specificity as well as encoding gene and application
  • Fructosyl peptidyl oxidase with high specificity as well as encoding gene and application

Examples

Experimental program
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Embodiment approach

[0040] In a third aspect, the present invention provides a recombinant vector having the gene described in the second aspect. The recombinant vector can be either a recombinant cloning vector or a recombinant expression vector. According to one embodiment of the present invention, the recombinant vector can be a recombination of the gene inserted between the multiple cloning sites (such as BamHI and XhoI) of the pET22b vector or the multiple cloning sites (such as NdeI and XhoI) of the pET28a vector. carrier.

[0041] In a fourth aspect, the present invention provides a transformant containing the recombinant vector described in the third aspect. The transformant can be a bacterial strain containing the recombinant vector of the present invention, for example, can be transformed into a competent bacterial strain (such as Escherichia coli competent bacterial strain Top10, TG1, DH5a or BL21 (DE3)) by transferring the recombinant vector of the present invention. get.

[0042] ...

Embodiment 1

[0051] This example is used to illustrate the construction of the coding gene expressing fructosyl peptide oxidase and the transformant.

[0052] Using the pET22b vector plasmid carrying the coding sequence of fructosyl peptide oxidase (SEQ ID NO: 2) between the BamHI and XhoI restriction sites as a template, it was subjected to saturation mutation to obtain bases 184-186 (CGA) Mutants R62K, R62A, R62D, R62E substituted with AAG, GCA, GAT, GAG, AAT, CAG, TTC, TAT, TGG, GGA, GTT, ATT, CAT, CTT, AGT, ACA, TGT, ATG and CCT, respectively , R62N, R62Q, R62F, R62Y, R62W, R62G, R62V, R62I, R62H, R62L, R62S, R62T, R62C, R62M and R62P.

[0053] The QuikChange site-directed mutagenesis kit (Stratagene Company) was used to carry out PCR. The specific mutation and the primers used are shown in Table 1. The PCR reaction system is as follows:

[0054]

[0055]

[0056] The PCR conditions are as follows:

[0057] Pre-denaturation: 98°C 30sec; denaturation: 98°C 10sec, annealing: 60°C...

Embodiment 2

[0063] This example is used to illustrate the expression of fructosyl peptide oxidase.

[0064] The BL21 strain transformed with the correct mutant gene in Example 1 was inoculated into 100 ml of LB medium (100 μg / ml ampicillin) at a ratio of 1:1000, and cultured overnight at 37°C. Then inoculate the overnight cultured bacterial liquid into a large bottle containing 800ml LB medium (100μg / ml ampicillin) at a ratio of 1:100, and cultivate it at 37°C and 200rpm for 3 hours until the OD value reaches 0.8. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM, the expression was induced at 16°C for 18 hours, and the cells were collected by centrifugation at 4°C and 4000 rpm for 30 minutes.

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Abstract

The invention relates to the field of enzyme engineering, discloses fructosyl peptidyl oxidase and an encoding gene thereof. The amino acid sequence of fructosyl peptidyl oxidase is shown in SEQ ID No:1. The invention also discloses a recombinant vector with the gene and a transformant containing the recombinant vector. The invention also discloses applications of fructosyl peptidyl oxidase, the gene, the recombinant vector or the tansformant to detection of glycosylated hemoglobin by an enzyme method. In addition, the invention also discloses a method for enhancing specificity of fructosyl peptidyl oxidase. The method comprises a step for replacing arginine at the 62th position of fructosyl peptidyl oxidase derived from Eupenicillium terrenum into the other amino acid, and the other amino acid is lysine, alanine, asparagine, glutamine, phenylalanine, valine, isoleucine, histidine, leucine, serine, methionine or proline. Fructosyl peptidyl oxidase with obviously improved specificity of enzyme activity is obtained, so that the business application values of the enzyme are increased.

Description

technical field [0001] The invention relates to the field of enzyme engineering, in particular to a fructosyl peptide oxidase, its coding gene and its use. Background technique [0002] Diabetes is a chronic disease with a high incidence in China, with an average of 1 in 10 people suffering from diabetes. At present, China has become the country with the largest number of diabetic patients and the highest prevalence rate in the world. Diabetes seriously endangers the health of patients and affects their quality of life, so the treatment and monitoring of diabetes are very important. [0003] During the 120-day life cycle of human red blood cells, hemoglobin can be continuously glycosylated, and the formation rate of glycosylated hemoglobin is proportional to the blood sugar concentration, so the content of glycosylated hemoglobin reflects the situation of blood sugar control in 2-3 months. Studies have found that compared with simple blood sugar levels, the level of glycos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12Q1/37C12Q1/26
CPCC12N9/0032C12Q1/26C12Q1/37G01N2333/805
Inventor 龚为民甘伟琼刘海萍邢克克李亚峰
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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