Thermostable mutant of pyrroloquiniline quinine-dependent glucose dehydrogenase and high-throughput screening method thereof

A thermal stability and mutant technology, applied in the field of genetic engineering of enzymes, can solve problems such as poor thermal stability

Inactive Publication Date: 2012-07-11
ZHEJIANG DEQING HUINING BIOTECH
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Problems solved by technology

[0009] The purpose of the present invention is to solve the problem of poor thermal stability of soluble glucose dehydrogenase on the market, and the task of the invention is to provide a new type of s that has significantly improved thermal stability compared with wild-type enzymes through directed evolution technology and high-throughput screening methods. - GDH mutant or variant

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  • Thermostable mutant of pyrroloquiniline quinine-dependent glucose dehydrogenase and high-throughput screening method thereof
  • Thermostable mutant of pyrroloquiniline quinine-dependent glucose dehydrogenase and high-throughput screening method thereof
  • Thermostable mutant of pyrroloquiniline quinine-dependent glucose dehydrogenase and high-throughput screening method thereof

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Embodiment Construction

[0018] Two distinct types of quinone proteases with glucose dehydrogenase activity (membrane-bound and soluble) are grouped together under EC 1.1.5.2, but these two types of enzymes are not related.

[0019] For the present invention only the soluble glucose dehydrogenase s-GDH is relevant, improved variants of which are discussed below.

[0020] It is known in the art that the wild-type DNA sequence of s-GDH can be isolated from strains of Acinetobacter. Most preferentially was isolated from Acinetobacter calcoaceticus strain LMD79.41. The DNA sequence and polypeptide sequence of the wild-type s-GDH are given in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0021] The recombinant vector of the present invention is an expression vector, and appropriate vectors can be used according to the needs of expression and purification, such as pET series, pQE series, pMAL series, pGEX series and other expression vectors suitable for E. Expression vectors for other prokaryotic cells, ...

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Abstract

The invention relates to a molecular structure of a directed evolution soluble pyrroloquiniline quinine-dependent glucose dehydrogenase (s-GDH) and a method for screening the thermostable mutant by adopting the high-throughput technology. The method is characterized by comprising the following steps: a high-capacity mutation library is constructed through an error-prone polymerase chain reaction (PCR) of the s-GDH, the filter paper soaked with a colored substrate is used to perform in-situ screening on a colony plate, and the colony direct conversion method is combined to obtain the mutant ofthe s-GDH, of which thermostability is increased. Compared with the wild-type s-GDH, the thermostability of the obtained s-GDH mutant is obviously increased. The obtained soluble pyrroloquiniline quinine-dependent glucose dehydrogenase with high thermostability lays the foundation for the further expansion of the application range of glucose dehydrogenase.

Description

field of invention [0001] The invention relates to a novel mutant with improved thermostability of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase and a high-throughput screening method for screening the thermostability mutant, which belongs to the technical field of enzyme genetic engineering. Background technique [0002] Blood glucose concentration measurement is extremely important in the clinical diagnosis and management of diabetes. At present, there are about 97 million adult diabetic patients in my country, and 148 million pre-diabetic patients, which have surpassed Western developed countries and become the country with the largest number of diabetic patients in the world. Diabetes will be one of the serious public health problems in my country in the future, and blood glucose monitoring Gaining importance. Blood glucose testing accounts for nearly 16% of the market share in the self-diagnostic testing market. Statistics show that there are currentl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/63C12N1/21C12N1/19C12N1/15C12Q1/54C12Q1/32C12M1/34C12R1/01
Inventor 徐志南于怡黄磊朱建忠杨叶东
Owner ZHEJIANG DEQING HUINING BIOTECH
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