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Hyaluronic acid lyase as well as gene expression and application thereof

A technology of hyaluronic acid and lyase, applied in lyase, carbon-oxygen lyase, application, etc., can solve the problems of narrow temperature range and pH range of enzyme, uneven molecular weight of enzymatic hydrolysis products, cumbersome product separation steps, etc., to achieve The effect of wide temperature and pH range, small molecular weight and high product purity

Inactive Publication Date: 2022-05-20
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, hyaluronidase has problems such as uneven molecular weight of enzymatic hydrolysis products, low enzymatic hydrolysis conversion efficiency, narrow temperature range and pH range required for enzyme activity maintenance, specific buffer system is required, and product separation steps are cumbersome, etc.

Method used

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  • Hyaluronic acid lyase as well as gene expression and application thereof
  • Hyaluronic acid lyase as well as gene expression and application thereof
  • Hyaluronic acid lyase as well as gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A hyaluronan lyase, the amino acid sequence of which is shown in SEQ ID NO.1, the hyaluronan lyase is specifically obtained from a strain of Streptomyces alfalae screened in a laboratory.

[0033] Obtaining the coding gene of the above-mentioned hyaluronan lyase:

[0034] Using the genome of Streptomyces alfalae screened above as a template (DNA template), design upstream and downstream primers and predict the annealing temperature of the primers, add a BamHI restriction enzyme site and a protective base at the 5' end of the upstream primer, The 5′ end of the downstream primer introduces an XhoI restriction enzyme site and a protective base, and the primer sequence is as follows:

[0035] Sahyal-F: (BamHI) 5'-TTAAGAATTCGCCAGGGCCGCCGAGG-3' (SEQ ID NO.3);

[0036] Sahyal-R: (XhoI) 5'-CCGCTCGAGGCCCCGCAGGGTCACC-3' (SEQ ID NO. 4).

[0037] A large amount of amplification was performed using a PCR instrument. The DNA polymerase kit was purchased from Beijing Quanshijin Biot...

Embodiment 2

[0043] Construction of the recombinant expression vector of the above-mentioned hyaluronan lyase:

[0044] 1) Double enzyme digestion of the hyaluronan lyase-encoding gene and vector pET28a-SUMO amplified and purified in Example 1.

[0045] The coding gene of hyaluronan lyase and the vector pET28a-SUMO were respectively double-digested with restriction endonucleases BamHI and XhoI. The double-digestion system is shown in Table 3. Place the double enzyme digestion system in a 37°C water bath for 3 hours. After the end, add the above enzyme digestion system to 0.8% agarose gel for detection, and carry out the enzyme digestion gene fragment and pET28a-SUMO carrier band of the correct size. Cut gel recovery and purification.

[0046] Table 3 enzyme digestion system

[0047]

[0048] 2) Enzyme ligation of the digested gene fragment with the linearized pET28a-SUMO vector.

[0049]The purified enzyme-cut gene fragment and the linearized vector pET28a-SUMO were ligated by T4 DNA...

Embodiment 3

[0059] Acquisition of expression cells and expression of hyaluronan lyase.

[0060] The plasmid pACYC Duet-1-ulp (used to express the SUMO protease Ubiquitin-Like protein-specific Protease Ulp without the His tag, which can specifically recognize and remove the SUMO tag, pACYC Duet-1-ulp was used for double-plasmid co-expression. The structure of the plasmid is shown in figure 1 As shown, it was obtained by inserting the gene sequence expressing Ulp in the pACYC Duet plasmid) and the recombinant plasmid pET28a-SUMO-Sahyal were simultaneously transformed into competent cells BL21 (DE3) by the heat shock method, and 400 μL of anti-LB liquid medium was added, Incubate at 37°C, 180rpm for 60min. Take 80 μL of the above-mentioned bacterial solution and evenly spread it on a double-antibody (containing both kan and Cm resistance) solid LB plate, and culture it upside down at 37°C overnight. Pick a single colony in 2 mL of LB liquid medium (both containing kan and Cm resistance). ...

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Abstract

The invention relates to the technical field of biochemical engineering, and particularly discloses hyaluronate lyase as well as gene expression and application thereof. The amino acid sequence of the hyaluronate lyase is as shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2. The invention also provides an expression vector and an expression thallus containing the coding gene, and application of the hyaluronate lyase and the coding gene thereof in preparation of unsaturated hyaluronate disaccharide. The hyaluronic acid lyase provided by the invention has the advantages of high enzyme activity, high catalytic efficiency, good temperature and pH stability, wide temperature and pH range required for maintaining enzymatic hydrolysis activity, direct conversion in pure water and small and uniform molecular weight of enzymatic hydrolysis products; the method is suitable for large-scale industrial enzymolysis of hyaluronic acid to produce unsaturated hyaluronan disaccharide.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to a hyaluronic acid lyase and its gene expression and application. Background technique [0002] Hyaluronic acid is a high-molecular-weight polysaccharide widely present in the extracellular matrix of higher animals and lower animals. Its basic structure is a polysaccharide composed of D-glucuronic acid and N-acetylglucosamine as disaccharide units. Widely used in cosmetics, food and medicine fields. The roles and functions of different molecular weights are different. High molecular weight hyaluronic acid (>1.6 million Da) forms a dense protective film on the skin surface, which plays a long-term moisturizing and good repairing effect; medium molecular weight hyaluronic acid (200,000 Da ~ 1.6 million Da) plays moisturizing and lubricating properties , slow-release and stable emulsification; low-molecular-weight hyaluronic acid (10,000 Da to 200,000 Da) plays a role in no...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P19/26C12R1/19
CPCC12N9/88C12N15/70C12P19/26C12Y402/02001C12R2001/19Y02A50/30
Inventor 赵国刚李玲聪姚伟胡少峰刘妍池
Owner HEBEI AGRICULTURAL UNIV.
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