Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof

A technology of uric acid oxidase and polyethylene glycol, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve problems such as long half-life, high cost of preparations, and increased risk of medication, and achieve production costs Low, low immunogenicity, long half-life effect

Inactive Publication Date: 2010-12-29
杭州北斗生物技术有限公司
View PDF6 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Chinese patent CN101327327A discloses the patent of polyethylene glycol-modified Aspergillus flavus uricase and its preparation process. Polyethylene glycol with a molecular weight of 20kd is used to modify cysteine. The product activity has not been reported.
[0009] Chinese patent CN101302501A discloses N-terminal modified PEG-uric acid oxidase, wherein urate oxidase is a recombinant urate oxidase derived from Aspergillus flavus, PEG is N-terminal selective PEG with a molecular weight of 20KD and 40KD, and the efficiency of the modification reaction reaches 61 %, 39% of the prototype protein has not been modified, so the yield of modified products is low and the preparation cost is high
[0010] In the prior art, some PEG-modified urate oxidases have low activity, so the dosage needs to be increased. As urate oxidase is a heterologous protein of the human body, a higher dosage means that the risk of medication is increased; Technology believes that the half-life of the modified product with large molecular weight PEG is longer and / or the activity is better maintained, but there are problems such as high cost of high molecular weight PEG; some modification methods have low modification efficiency and / or product stability is not ideal

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof
  • Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof
  • Polyethylene glycol modifier of aspergillus flavus urate oxidase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] The present invention will be further described below in conjunction with specific examples, but the present invention is not limited. Embodiment 1, preparation and activity determination of Aspergillus flavus uric acid oxidase

[0079] 1. Preparation of Aspergillus flavus uric acid oxidase

[0080] The preparation method of recombinant Aspergillus flavus urate oxidase comprises the following steps: (1), the acquisition of Aspergillus flavus urate oxidase gene; (2) construction of expression vector and transformation of Escherichia coli engineering bacteria: the synthetic Aspergillus flavus urate oxidase cDNA The fragment is inserted into the restriction site of the expression vector plasmid, and then directly transformed into E. coli host bacteria; (3), screening, culturing, and induced expression of positive clones: the positive clones are selected and cultured on the medium, and the target protein is induced to Expression; (4), separation and purification of fusion ...

Embodiment 2

[0093] Embodiment two, different molecular weight PEG modified uric acid oxidase

[0094] 1. Preparation of modified products

[0095] Purified Escherichia coli expressing Aspergillus flavus uric acid oxidase has a purity greater than 95%, a concentration of 2.0mg / ml, exchanged the buffer with a G25 column, and prepared an equilibrium buffer of 0.1mol / L Na 2 PO 4 -20mmol / L NaCl, flow rate 10ml / min, load the sample after the column effluent reaches equilibrium, and collect the protein peak. Monomethoxy PEG propionate with different molecular weights was added to urate oxidase in a different buffer system at a mass ratio of 7:1, and the reaction was terminated after stirring at room temperature for 2 hours. Use a 50KD ultrafiltration membrane to ultrafilter the urate oxidase PEG reaction mixture against 20mmo / L PB, pH 8.0 buffer solution to remove free PEG, which is the urate oxidase PEG modified stock solution.

[0096] The modified product was analyzed by SDS-PAGE, and the ...

Embodiment 2

[0126] Samples 1-4 prepared in Example 2 and Samples 5-8 prepared in Comparative Example 1 were placed at 37° C. for one day to conduct an accelerated test for stability investigation. The activity retention of the samples is shown in Table 6.

[0127] Table 6. PEG urate oxidase stability accelerated test results

[0128] Numbering

[0129] The results of accelerated stability test showed that the enzyme activity of Aspergillus flavus uricase modified by single-point PEG lost quickly at 37°C, while the 5K molecular weight PEG-modified uricase of Aspergillus flavus with higher enzyme activity in multi-point modified samples could maintain better Stability, 92.7% of the activity can be maintained at 37°C for one day.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a polyethylene glycol modifier of aspergillus flavus urate oxidase, wherein polyethylene glycol and the aspergillus flavus urate oxidase are connected through a covalent bond, the subunit of each aspergillus flavus urate oxidase molecule is averagely combined with 2-10 polyethylene glycol molecules, the average molecular weight of the polyethylene glycol molecule is 4-9kd, and the enzymatic specific activity of the polyethylene glycol modified aspergillus flavus urate oxidase is 5.0-10.0U/mg. A preparation method thereof is realized by adopting the following technical scheme: recombinant aspergillus flavus urate oxidase protein with the purity of larger than 95 percent is modified by adopting the activated polyethylene glycol molecule with the average molecular weight of 4-9kd for 0.1-10 hours at the pH value of 5-10 and the temperature of 4-37 DEG C, and the mass ratio of the polyethylene glycol molecule to the aspergillus flavus urate oxidase is 3:1-20:1. The polyethylene glycol modified aspergillus flavus urate oxidase realizes the balance in the aspects of high specific activity, long half life, low immunogenicity, high stability, low cost and the like and has industrialization value.

Description

technical field [0001] The invention relates to a modification of Aspergillus flavus urate oxidase for treating gout and hyperuricemia, in particular to a polyethylene glycol modification of Aspergillus flavus urate oxidase with high activity and a preparation method thereof. Background technique [0002] The final product of purine metabolism in the human body is uric acid. Unlike other species that express active urate oxidase, uric acid cannot be further oxidized and decomposed into allantoin that is easily excreted from the body. Under the influence of factors such as diseases, genetics, or diet, uric acid will accumulate in blood, urine, and interstitial fluid, and its concentration will gradually increase. When it exceeds its solubility, it will increase the burden on the kidneys and cause damage to kidney function, especially Long-term elevated uric acid levels will lead to the deposition of urate crystals in the joints and subcutaneous tissue where the blood flow is ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/08A61P19/06C12R1/67
Inventor 刘国安沈立民刘翔胡芬芬张弨张加慧刘沐荣
Owner 杭州北斗生物技术有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap