SAO

SAO

Sarcosine oxidase, referred to as SOX, is the third enzyme in the creatinine degradation metabolic pathway, which can specifically catalyze the production of glycine from the substrate sarcosine. Microorganisms are the most important source of creatine oxidase, which has a wide range of applications in clinical diagnosis. The determination of creatinine in serum by creatine oxidase coupled with creatinase, which is an important factor in judging kidney function. Sarcosine oxidase belongs to flavin protein and can be coupled to creatinase and creatinase to degrade creatinine into glycine formaldehyde and hydrogen peroxide. As an important diagnostic enzyme preparation, Sox is widely used in human serum creatinine level detection, used to judge the health of kidney function.

Figure 1. Protein structure of sarcosine oxidase.

Introduction

Sarcosine oxidase testing has become a routine testing item in medical institutions, and SOX has a large clinical need. At present, the use of genetic engineering to achieve heterologous high-efficiency expression of SOX is an effective means to meet its clinical diagnostic applications. E. coli, as a commonly used expression host, has many advantages such as low fermentation cost, high yield, easy operation, clear genetic background, short reproduction cycle and easy control. It is generally regarded as the preferred system for recombinant protein production for several months. In particular, due to the large differences in sox enzymological properties of different biological sources, it will directly affect its application effects, such as the relative molecular mass, subunit composition and protein composition. Therefore, it is very important for deep people to explore the properties of SOX enzymes. In addition, the generally poor stability of diagnostic enzyme reagents is also an important factor restricting the practical application of such enzyme preparations 02. And the detailed enzyme molecule inactivation machine.

How to improve the stability of sarcosine oxidase?

Since sarcosine oxidase is easily inactivated during storage and transportation, improving its stability is the basis for the large-scale application of this enzyme. At present, there are mainly methods for improving enzyme stability, such as protein engineering, immobilization, addition of protective agents, chemical modification, etc. Among them, immobilization is a more commonly used method. Glutaraldehyde is a bifunctional reagent widely used in enzyme immobilization. However, considering the complex operation and the toxicity of the reagent, carbodiimide method can be used to fix sarcosine oxidase, 1-ethyl- (3-dimethyl Ethylaminopropyl) carbodiimide (EDC) is a condensing agent that couples an amino group to a carboxyl group. The carboxyl group of the enzyme molecule is activated by carbodiimide to form an intermediate product, which then reacts with the amino carrier to form a conjugate.

Applications

The electrochemical detection method for sarcosine includes: connecting sarcosine oxidase to the first nucleic acid sequence by physical adsorption or chemical coupling; connecting the peroxidase to the second nucleic acid sequence; providing the first Nucleic acid nanostructures protruding from the first recognition sequence; providing a second nucleic acid nanostructures protruding from the second recognition sequence; the first nucleic acid sequence modification enzyme hybridizes with the first recognition sequence to obtain sarcosine oxidase and nucleic acid The first complex of nanostructures, the second nucleic acid sequence modifying enzyme hybridizes with the second recognition sequence to obtain a second complex of peroxidase and nucleic acid nanostructures; the first complex and the second complex are assembled into electrochemistry The working electrode surface of the device is subjected to electrochemical detection. The electrochemical detection method according to the present invention combines structural nucleic acid nanotechnology, enzyme-catalyzed reaction and electrochemical biosensor to realize quantitative detection of small sarcosine molecules.

Reference

1. Suzuki M. Purification and some properties of sarcosine oxidase from Corynebacterium sp. U-96. Journal of Biochemistry, 1981,89(2) :599-607.