Immobilized Fusion Enzyme and Method for Using It to Prepare Glutathione

A glutathione and fusion enzyme technology, applied in biochemical equipment and methods, fusion polypeptides, immobilized on/in organic carriers, etc. Final product purification and other issues, to achieve the effect of high immobilization unit activity density, high target product concentration, and improved substrate conversion rate

Active Publication Date: 2021-08-17
SHENZHEN READLINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Most of the existing technologies use free, unpurified glutathione synthase and ATP regeneration enzyme to produce glutathione. The liquid enzyme is used for one-time use and cannot be recycled, which greatly increases the production cost of the enzyme; At the same time, the mixed enzymes in the mixed enzyme liquid lead to the occurrence of many by-products, and the enzyme liquid itself is mixed with the product, which is not conducive to the purification of the final product; the fluctuation of each batch during the preparation of the mixed enzyme liquid in the early stage will affect the quality of the final product. Quality stability
Some technologies also involve immobilized enzymes. Most of the commonly used enzyme immobilization methods use enzyme purification to obtain free enzymes, and then fix them by adsorption, embedding, covalent bonding, etc. The process is cumbersome and the recovery rate of enzyme activity is low.

Method used

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  • Immobilized Fusion Enzyme and Method for Using It to Prepare Glutathione
  • Immobilized Fusion Enzyme and Method for Using It to Prepare Glutathione
  • Immobilized Fusion Enzyme and Method for Using It to Prepare Glutathione

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The fusion protein expression gene (GshAB-P38058) of glutathione bifunctional enzyme (GshAB) EC 6.3.2.2 and chitin protein binding domain (ChitinBinding Domain, P38058 derived from Clostridium cellulovorans) was constructed. The protein sequence of GshAB-P38058 is shown in Table 1. After the gene was synthesized, it was subcloned into pColdIII (TaKaRa, Japan) plasmid by NdeI and XhoI (NEB Company) digestion.

[0062] Construct polyphosphate kinase (PPK, EC 2.7.4.1) and chitin protein binding domain (Chitin BindingDomain, A0A1R1E3J9 derived from Paenibacillus amylolyticus), PPK-A0A1R1E3J9 protein sequence is shown in Table 1, after gene synthesis by NdeI and XhoI (NEB company ) and subcloned into pColdIII (TaKaRa, Japan) plasmid.

[0063] Adenylate kinase (Adk, EC 2.7.4.3) and chitin protein binding domain (Chitin BindingDomain, A0A089MPB0 derived from Paenibacillus odorifer) were constructed. The protein sequence of Adk-A0A089MPB0 is shown in Table 1. ) and subcloned i...

Embodiment 2

[0075] The fusion protein expression gene (GshAB-4B9F) of glutathione bifunctional enzyme (GshAB) EC 6.3.2.2 and chitin protein binding domain (ChitinBinding Domain, Clostridium Thermocellum-derived 4B9F) was constructed. See attached table 1 for the protein sequence of GshAB-4B9F. After gene synthesis, it was subcloned into pColdIII (TaKaRa, Japan) plasmid by NdeI and XhoI (NEB Company) digestion.

[0076] Construct polyphosphate kinase (PPK, EC 2.7.4.1) and chitin protein binding domain (Chitin BindingDomain, A0A1R1E3J9 derived from Paenibacillus amylolyticus), PPK-A0A1R1E3J9 protein sequence is shown in Table 1, after gene synthesis by NdeI and XhoI (NEB company ) and subcloned into pColdIII (TaKaRa, Japan) plasmid.

[0077] GshAB-4B9F wet cells and PPK-A0A1R1E3J9 wet cells were prepared by inducing expression of the above plasmids according to the method of Example 1.

[0078] Weigh 100g GshAB-4B9F wet cells, resuspend in 1000ml solution (containing 20mM Tris pH7.5) and c...

Embodiment 3

[0082] Construct the fusion protein expression gene (GshA-A0A173MZQ9) of glutathione monofunctional synthetase GshA (EC 6.3.2.2) and chitin protein binding domain (source A0A173MZQ9). The protein sequence of GshA-A0A173MZQ9 is shown in Table 1. After the gene was synthesized, it was subcloned into pColdIII (TaKaRa, Japan) plasmid by NdeI and XhoI (NEB Company) digestion.

[0083] The fusion protein expression gene (GshB-A0A173MZQ9) of glutathione monofunctional synthetase GshB (EC 6.3.2.3) and chitin protein binding domain (source A0A173MZQ9) was constructed. See attached table 1 for the protein sequence of GshB-A0A173MZQ9. After the gene was synthesized, it was subcloned into pColdIII (TaKaRa, Japan) plasmid by NdeI and XhoI (NEB Company) digestion.

[0084]Construct polyphosphate kinase (PPK, EC 2.7.4.1) and chitin protein binding domain (Chitin BindingDomain, 4B9F derived from Clostridium Thermocellum). The sequence of PPK-4B9F protein is shown in Table 1. After gene synthe...

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Abstract

The invention relates to the field of polypeptide synthesis, in particular to an immobilized fusion enzyme and a method for preparing glutathione with it. In the present invention, the glutathione synthetase gene, the adenosine triphosphate regenerating enzyme gene and the chitin protein domain gene are respectively connected and expressed to prepare a fusion enzyme, and the chitin carrier can be used to specifically and firmly bind the chitin protein domain , realize one-step purification of immobilized glutathione synthesis fusion enzyme and adenosine triphosphate regeneration fusion enzyme, the process is simple, and the space density of immobilized enzyme is high. The immobilized fusion enzyme prepared by the present invention has high enzyme activity and can realize continuous production of glutathione. The present invention adopts the batch and continuous process route of the immobilized fusion enzyme to generate glutathione up to 31g / L. The target The product concentration is high, and there is no enzyme residue in the product solution, which greatly simplifies the later purification process.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to an immobilized fusion enzyme and a method for preparing glutathione with it. Background technique [0002] Glutathione (GSH) is a natural tripeptide compound formed by linking L-glutamic acid, L-cysteine ​​and glycine, and its molecular formula is C 10 h 17 N 3 o 6 S, the molecular weight is 307.3. GSH widely exists in animals and plants, and plays an important antioxidant function in organisms. Since the active thiol-SH in GSH is easily oxidized and dehydrogenated, it can remove free radicals in the body; at the same time, GSH can also improve human immunity, maintain health, anti-aging, and slow down the aging cells. ; Coupled with broad-spectrum detoxification, therefore, glutathione is not only used as a safe drug additive, but also widely used as a base material for functional foods, as well as functions such as delaying aging, enhancing immunity, and anti-tumor. i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N9/12C12N15/62C12N11/10C12N11/18C12P21/02
CPCC07K5/0215C07K2319/00C12N9/1205C12N9/1229C12N9/93C12N11/10C12N11/18C12Y207/04001C12Y207/04003C12Y603/02003
Inventor 于铁妹樊卫谭文静何遂平何平
Owner SHENZHEN READLINE BIOTECH CO LTD
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