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Engineering yeast strain with high yield of hyaluronidase and application of engineering yeast strain

A technology of hyaluronidase and yeast strain, applied in the field of genetic engineering, can solve the problems of high price and low level of recombinant expression, and achieve the effect of reducing cost

Pending Publication Date: 2022-04-15
BLOOMAGE BIOTECHNOLOGY CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently commercially available hyaluronidase is mainly BTH extracted from bovine testes, which is very expensive
Although there are reports of recombinant expression of human testis-derived (PH20) and some animal venom-derived hyaluronidases in microbial cells, the recombinant expression level is very low and cannot be used as a biocatalyst for the industrial production of hyaluronic acid oligosaccharides

Method used

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  • Engineering yeast strain with high yield of hyaluronidase and application of engineering yeast strain
  • Engineering yeast strain with high yield of hyaluronidase and application of engineering yeast strain
  • Engineering yeast strain with high yield of hyaluronidase and application of engineering yeast strain

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Experimental program
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Effect test

Embodiment 1

[0067] The construction of the engineering yeast strain of embodiment 1 high-yielding hyaluronidase

[0068] Based on the full-length gene sequence (Genbank: FX985505.1) of the hyaluronidase from the NCBI database, the codons were optimized according to the codon preference of Pichia pastoris. The gene sequence of the codon-optimized hyaluronidase from Pichia chinensis is shown in SEQ ID NO.2, which was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and cloned into EcoRI and NotI of Pichia pastoris expression vector pPIC9K Between the restriction sites, the recombinant expression vector pPIC9K-HYAL_OM was obtained opt .

[0069] Take pPIC9K-HYAL_OM opt The recombinant expression vector is used as a template,

[0070] Primers are designed to cut off a signal peptide sequence at the N-terminal of the full-length hyaluronan hydrolase gene to obtain a genetically engineered hyaluronan hydrolase gene fragment, the sequence of which is shown in SEQ ID NO.1.

[0071] Wh...

Embodiment 2

[0075] Embodiment 2 Recombinant Hyaluronidase Engineering Yeast Strain Shake Flask Fermentation

[0076] For the obtained recombinant engineering bacteria P.pastoris GS115 / pPIC9K-ΔN24HYAL_OM opt / pGAPZαA-ΔN24HYAL_OM opt Shake flask fermentation was carried out.

[0077] The pure glycerol culture and fermentation steps are as follows: pick a single clone and inoculate it in 40 mL of YPD medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L), and culture at 30°C and 200 rpm for 24 hours. Transfer to 40mL expression medium BMGYS (yeast extract 10g / L, peptone 20g / L, K 2 HPO 4 3g / L, KH 2 PO 4 11.8g / L, YNB 3.4g / L, ammonium sulfate 10g / L, biotin 4×10 -4 g / L, glycerol 40g / L), 30°C, 200rpm for 96h.

[0078] The methanol-induced fermentation steps are as follows: a single clone was picked and inoculated in 40 mL of YPD medium, and cultured at 30° C. and 200 rpm for 24 hours. Transfer to 40mL initial expression medium BMGY (yeast extract 10g / L, peptone 20g / L, K 2 HPO ...

Embodiment 3

[0081] Example 3 Recombinant Hyaluronidase Engineering Yeast Strain 5-L Fermentor High Density Cultivation (Using Methanol as Carbon Source)

[0082] The combined strain P. pastoris GS115 / pPIC9K-ΔN24HYAL_OM opt / pGAPZαA-ΔN24HYAL_OM opt Carry out high density culture in 5-L fermenter.

[0083] Inoculate a single colony on the YPD plate into 50mL YPD liquid medium, culture at 30°C 220rpm for 24h, inoculate the culture solution at 10% into 200mL BMGY medium, culture at 30°C 220rpm for 24h; inoculate 200mL seed liquid cultivated for 24h to containing 2L BSM fermentation medium (glycerol 40g / L, K 2 SO 4 18g / L, KOH 4.13g / L, 85%H 3 PO 4 26.7mL / L, CaSO 4 2H 2 O 0.93g / L, MgSO 4 ·7H 2 O 14.9g / L, 4.4mL / L filter-sterilized PTM1; PTM1 formulation: CuSO 4 ·5H 2 O 6g / L, KI 0.09g / L, MnSO 4 ·H 2 O 3g / L, H 3 BO 3 0.02g / L, MoNa 2 o 4 2H 2 O 0.2g / L, CoCl 2 ·6H 2 O 0.92g / L, ZnCl 2 20g / L, FeSO 4 ·7H 2 O 65g / L, biotin 0.2g / L, H 2 SO 4 5.0mL) in a 5-L fermenter.

[0084...

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Abstract

The invention provides an engineering yeast strain for high yield of hyaluronidase. The engineering yeast strain is obtained by respectively integrating hyaluronidase genes as shown in SEQ ID NO.1 into any two sites of a yeast genome by adopting a genome integrated vector. An engineering yeast strain for producing hyaluronidase at high yield is constructed through an integrated plasmid mediated recombinant strain construction method, the highest enzyme activity of the engineering yeast strain in a culture medium taking methanol as a carbon source is 5.9 * 10 < 5 > U / mL, and the highest enzyme activity of the engineering yeast strain in a culture medium taking glycerol as a carbon source is 3.6 * 10 < 5 > U / mL. Therefore, the method is beneficial to further reducing the cost of industrial production of the Hongyonia macrophylla hyaluronidase, so that the application range of the hyaluronidase in the fields of medicine, chemical industry and food is expanded.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an engineering yeast strain with high hyaluronidase production and application thereof. Background technique [0002] Hyaluronic acid (HA for short), also known as hyaluronic acid, is a repeating disaccharide unit composed of D-glucuronic acid and N-acetylglucosamine, through β(1-3) and β(1-4) It is a macromolecular viscous polysaccharide formed by alternating glycosidic bonds. It was first extracted from bovine glass globe eyes by Meyer et al. in 1934. Literature research shows that the biological activity of HA depends on its molecular weight, and HA with different molecular weight ranges exhibit different physiological functions. Due to its good viscoelasticity, moisture retention, anti-inflammatory, lubricating and other functions, high molecular weight HA can be used in the cosmetic industry, ophthalmic surgery viscoelastic agent and intra-articular ...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N9/26C12R1/84
CPCC12N9/2408C12N15/81C12R2001/84
Inventor 王浩徐荣荣张天萌张由恒冯建伟郭学平
Owner BLOOMAGE BIOTECHNOLOGY CORP LTD
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