Immobilized glutathione synthetase, preparation thereof and applications thereof

A technology for glutathione, synthase, applied in immobilized enzymes, biochemical equipment and methods, immobilized on/in organic carriers, etc.

Active Publication Date: 2014-03-12
风火轮(上海)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the above reasons, there is no immobilized glutathione synthase in the art at present, so it is urgent to break through the bottleneck of enzymatic production of GSH and develop corresponding immobilized enzymes for industrial production of GSH

Method used

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  • Immobilized glutathione synthetase, preparation thereof and applications thereof
  • Immobilized glutathione synthetase, preparation thereof and applications thereof
  • Immobilized glutathione synthetase, preparation thereof and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0072] According to the glutathione synthase gene sequence, those skilled in the art can conveniently use various known methods to prepare the encoding nucleic acid of the present invention. These methods are for example but not limited to: PCR, artificial DNA synthesis, etc. For specific methods, please refer to J. Sambrook, "Molecular Cloning Experiment Guide." As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by the PCR method.

[0073] The gene sequence containing the coding glutathione synthase can be operably linked to the expression control sequence. The term "operably connected" or "operably connected to" refers to a situation in which certain parts of a linear DNA sequence can regulate or control the activity of other parts of the same linear DNA sequence. For example, if the promoter controls the transcription of the sequence, then it is operably linked to the coding sequence.

[0074] Those skilled...

Embodiment 1

[0120] Immobilization of glutathione bifunctional enzyme (Sa-GSH) derived from Streptococcus agalactiae

[0121] 1.1 Preparation of crude enzyme solution of glutathione bifunctional enzyme derived from Streptococcus agalactiae

[0122] 1.1.1 Preparation

[0123] Refer to Blythe E. Janowiak and Owen W. Griffith. Glutathione synthesis in Streptococcus agalactiae. Journal of Biological Chemistry. 2005.280:11829-39 for the expression of glutathione bifunctional enzyme (Sa-GSH) derived from Streptococcus agalactiae. The method is constructed, cultured and induced expression.

[0124] The fermented bacterial cells are centrifuged and weighed, suspended in a 1:4 (mass to volume ratio) 0.02M potassium phosphate buffer (pH 8.0±0.2), squeezed to break the cells, and centrifuged to obtain a crude enzyme solution.

[0125] 1.1.2 Activity determination of crude enzyme solution

[0126] 5ml reaction system: 0.1M PBS (pH7.4), 40mM Mg 2+ , 40mM ATP, 120mM glycine, 80mM L-cysteine, 120mM L-glutamic acid...

Embodiment 2

[0147] Immobilization of glutathione bifunctional enzyme (STH) of Streptococcus thermophilus

[0148] The expression strain of the glutathione bifunctional enzyme of Streptococcus thermophilus refers to Li W, Li ZM, Yang JH, Ye Q. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli.Journal of Biotechnology. 2011.154:261-8. The method described is constructed, cultured and induced expression.

[0149] The preparation of the crude enzyme solution, the HPLC detection and the pretreatment method of the immobilized carrier are the same as in Example 1.

[0150] In addition, the enzyme activity detection method of the glutathione bifunctional enzyme crude enzyme solution of Streptococcus thermophilus is the same as the glutathione bifunctional enzyme crude enzyme solution derived from Streptococcus agalactiae in Example 1 except that the pH is adjusted to 7.5 Determination of enzyme activity.

[0151] The prepa...

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PUM

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Abstract

The invention relates to an immobilized glutathione synthetase, preparation thereof and applications thereof. In particular, the immobilized glutathione synthetase having largely improved stability and reactivity is obtained by immobilization of a glutathione synthetase and a carrier. In particular, a long connecting component between the glutathione synthetase and the immobilization carrier can achieve an excellent immobilization effect. The immobilized glutathione synthetase has a long half-life period so that the enzyme productivity per unit is effectively enhanced. The immobilized glutathione synthetase can be used as a GSH biosynthesis system, thus simplifying production processes, increasing the production efficiency and simplifying purification technology.

Description

Technical field [0001] The present invention belongs to the field of biotechnology; specifically, the present invention relates to an immobilized glutathione synthase and its preparation and application. Background technique [0002] Glutathione (abbreviated as GSH), also known as γ-L-glutamyl-L-cysteyl-glycine, is a small molecule sulfhydryl compound widely present in the cells of animals, plants and microorganisms. The function of maintaining intracellular redox balance. The important physiological functions of GSH make it widely used in medicine, food, cosmetics, etc. [Meister A and Anderson ME, Glutathione.Annu Rev Biochem, 1983.52: p.711-760; Wu, G.,. Fang YZ, Yang S., Lupton JR, and Turner NDGlutathione Metabolism and Its Implications for Health. J. Nutr., 2004.134: p.489-492.]. [0003] The current production methods of GSH are mainly chemical synthesis and biological methods. Although chemical synthesis and extraction of GSH from yeast cells rich in intracellular GSH hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/00C12N11/02C12N11/08C12P21/02
Inventor 杨晟陶荣盛洪秀娟沈正权朱傅赟孙梁栋陈成沈青郑云
Owner 风火轮(上海)生物科技有限公司
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