CN-122012440-A - Heparin 3-O-sulfate transferase mutant with high enzyme activity and high thermal stability, and encoding gene and application thereof

Abstract

The invention relates to a heparin 3-O-sulfate transferase mutant with high enzyme activity and high thermal stability, and a coding gene and application thereof. The amino acid sequence of the 3-O-sulfuric acid transferase mutant is shown as SEQ ID NO.2, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1. The 3-O-sulfuric acid radical transferase mutant has remarkable superior performance in the enzymatic synthesis of heparin glycosaminoglycan. The performance measurement shows that the 3-O-sulfate transferase mutant SM1 realizes the cooperative improvement of catalytic activity and thermal stability. The total enzyme activity is improved by 1.5 times compared with the wild chicken source 3-O-sulfotransferase. More particularly, the thermostability is improved fundamentally, namely, the activity of the wild-type chicken-derived 3-O-sulfotransferase is rapidly attenuated at 37 ℃ in 24 hours, and the 3-O-sulfotransferase mutant SM1 can still retain about 50% of the initial activity after being incubated for 8 days under the same conditions, and the excellent operation stability is shown.

Inventors

• SHENG JUZHENG
• LI CHAOYUE

Assignees

• 山东大学

Dates

Publication Date

20260512

Application Date

20260228

Claims (9)

1. 1. The 3-O-sulfate transferase mutant SM1 is characterized in that the amino acid sequence is shown as SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1.
2. 2. A recombinant vector comprising a gene encoding the 3-O-sulfate transferase mutant SM1 of claim 1.
3. 3. The recombinant vector according to claim 1, wherein the recombinant vector is obtained by ligating a gene encoding 3-O-sulfate transferase mutant SM1 with a plasmid, wherein the plasmid is a pMAL-c5x expression vector.
4. 4. A recombinant cell comprising a gene encoding the 3-O-sulfate transferase mutant SM1 of claim 1.
5. 5. The recombinant cell according to claim 4, wherein the recombinant cell is obtained by transforming a gene encoding a 3-O-sulfate transferase mutant SM1 into a host cell which is E.coli OrigamiB (DE 3) harboring a chaperone pGro7 helper vector.
6. 6. A method for producing a 3-O-sulfate transferase mutant SM1 by fermentation is characterized in that the method comprises the steps of culturing the recombinant cells, and separating to obtain the 3-O-sulfate transferase mutant SM1.
7. 7. The method for producing the 3-O-sulfate transferase mutant SM1 by fermentation according to claim 6, which is characterized by comprising the specific steps of culturing recombinant cells containing the encoding gene of the 3-O-sulfate transferase mutant SM1 in LB liquid medium until OD600 is 0.6-0.8, adding IPTG to induce expression for 16-18 h, collecting thalli, performing ultrasonic crushing, performing centrifugal filtration, and purifying by MBP labels to obtain the 3-O-sulfate transferase mutant SM1.
8. 8. Use of the 3-O-sulfuric acid transferase mutant SM1 according to claim 1 for the synthesis of 3-O-sulfated heparin oligo-or polysaccharides.
9. 9. The use of claim 8, wherein the catalytic reaction is carried out with PAPS as donor and heparin oligosaccharides and polysaccharides having a acceptor substrate structure comprising GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP as starting acceptors to produce 3-O-sulfated heparin oligosaccharides or polysaccharides having a structure GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS 6S-GlcA-pNP.

Description

Heparin 3-O-sulfate transferase mutant with high enzyme activity and high thermal stability, and encoding gene and application thereof Technical Field The invention relates to the technical field of biological enzymes, in particular to a heparin 3-O-sulfate transferase mutant with high total enzyme activity and high thermal stability derived from red primary chicken (Gallus gallu), and a coding gene and application thereof. Background Heparin (HP) as a highly sulfated glycosaminoglycan is composed of D-beta-glucuronic acid (or L-alpha-iduronic acid) and N-acetylglucosamine to form repeating disaccharide units. Heparin is an indispensable anticoagulation medicine in the global scope, is a 'basic stone medicine' for clinical anticoagulation and antithrombotic treatment, is widely used in the scenes of acute coronary syndrome, deep venous thrombosis, pulmonary embolism prevention and treatment, hemodialysis, cardiac surgery extracorporeal circulation and the like, and simultaneously has the anti-inflammatory, antiviral, tumor metastasis inhibition and other non-anticoagulation activities, so that the clinical application boundary is further expanded. The global heparin market scale of 2026 breaks through the 80 hundred million dollars, china is used as the large country for heparin medicine production and consumption, the preparation market scale reaches 280 hundred million yuan, wherein the low molecular heparin (LMWH) accounts for over 70 percent, and main stream varieties such as enoxaparin, dalteparin and the like accelerate domestic substitution under the condition of collection and harvest normalization, so that the industry is promoted to change to a high added value direction. The core pharmacological activity of heparin, in particular the binding and activating capacity of antithrombin III (AT), is mainly determined by the critical 3-O-sulfate group in the pentasaccharide sequence of heparin. However, the conventional production method for extracting heparin from animal tissues such as pig intestinal mucosa has the inherent defects of strong raw material dependence, complex process, non-uniform quality among product batches, potential virus pollution, immunogenicity risk (such as 2008 heparin pollution event) and the like. Therefore, development of "synthetic heparin" having a definite structure, high purity and safety has become a strategic direction in the field of sugar medicines. Among the various synthetic routes, the chemoenzymatic synthetic strategy is considered as an optimal route for the mass production of structurally uniform, active controlled heparin oligosaccharides and the like, because of its ability to build complex sulfated sugar chain structures in a highly stereoselective, stepwise manner. The core of this strategy is the use of a series of specific regioselective sulfate transferases, whereas 3-O-sulfate transferase-1 (3-OST-1) is the last step modification enzyme that catalyzes the formation of critical sites for anticoagulant activity, whose properties directly determine the potency and quality of the final product, thus becoming the "rate limiting enzyme" and key bottleneck in the overall technology chain. In order to break through the problems of low catalytic efficiency, poor protein stability (especially easy to inactivate at reaction temperature), insufficient soluble expression quantity and the like commonly existing in a heterogeneous expression system of the natural 3-OST-1, researchers at home and abroad have tried various protein engineering strategies, but all have obvious limitations, and are difficult to meet the triple requirements of chemical enzymatic synthesis on the enzyme preparation of high activity, high thermal stability and high expression. Disclosure of Invention Aiming at the problems existing in the prior art, the invention provides a heparin 3-O-sulfate transferase mutant with high enzyme activity and high thermal stability, and a coding gene and application thereof. Specifically, 3OST truncations of corresponding sources are constructed by exploring 3OST of other sources and removing N-terminal signal peptide, so that 3OST truncations (hereinafter referred to as Gg-3 OST-1) of red-former chicken sources are successfully obtained, and protein engineering is performed on the Gg-3OST-1 to improve the total enzyme activity and the stability of the Gg-3 OST-1. Description of the terms GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP Chinese is fully referred to as p-nitrophenyl-glucuronic acid-N-sulfate-6-O-sulfate glucosamine-2-O-sulfate iduronate-N-sulfate-6-O-sulfate glucosamine-glucuronate-N-sulfate-6-O-sulfate glucosamine, which is a substrate for 3-O-sulfate transferase, catalyzes the production of GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S-GlcA-pNP. PAPS, chinese is fully called 3 '-phosphoadenosine-5' -phosphosulfate, which is a sulfate donor. The technical scheme of the invention is as follows: in a first aspect of the invention, a 3-O-sulfate transferase