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CN-122012538-A - Novel xanthine oxidase, gene cluster, recombinant expression vector, recombinant strain and application thereof

CN122012538ACN 122012538 ACN122012538 ACN 122012538ACN-122012538-A

Abstract

The invention belongs to the technical field of enzyme engineering, and discloses a novel Xanthine Oxidase (XOD) gene cluster, a recombinant expression vector, a recombinant strain and application thereof. The gene cluster is a novel XOD gene cluster which is discovered in Pseudomonas putida KT2440 genome for the first time through bioinformatics comparison and analysis, the gene composition mode of the novel XOD gene cluster is obviously different from that of the known XOD gene cluster, and a brand-new gene resource and technical path are provided for developing high-performance and low-cost recombinant XOD. The recombinant expression vector can realize the high-efficiency expression of the novel xanthine oxidase at Pseudomonas putida KT2440 by replacing the recombinant expression vector with a strong promoter.

Inventors

  • GUO XIAOYAN
  • WANG JIANJUN
  • LI TIANYI

Assignees

  • 北京石油化工学院

Dates

Publication Date
20260512
Application Date
20260206

Claims (10)

  1. 1. A novel xanthine oxidase gene cluster is characterized by being derived from Pseudomonas putida KT2440 genome, and the nucleotide sequence of the novel xanthine oxidase gene cluster is shown as SEQ ID NO. 1.
  2. 2. The novel xanthine oxidase gene cluster according to claim 1, wherein the novel xanthine oxidase gene cluster comprises 4 genes in total of Fe-S cluster binding protein gene, molybdenum binding protein gene, cytochrome C gene and XdhC gene.
  3. 3. A novel recombinant expression vector for xanthine oxidase comprising the novel cluster of xanthine oxidase genes of claim 1.
  4. 4. The recombinant expression vector of claim 3, wherein the recombinant expression vector is a recombinant expression vector of pPegP119,119, the novel xanthine oxidase gene cluster is cloned into a multiple cloning site, a strong promoter J23119 is introduced into the 5 'end, and a 6 XHis tag is introduced into the 3' end, and the recombinant expression vector is finally constructed and named pPegP119,119, 119PpXod.
  5. 5. The recombinant expression vector of novel xanthine oxidase according to claim 4, wherein the recombinant plasmid pPegP119,119 is prepared by replacing ampicillin resistance cassette in basic skeleton plasmid pUCP18 with P119-RBS-MCS fragment to obtain intermediate plasmid pUCP18P119, replacing pBR322 replication origin sequence in intermediate plasmid pUCP18P119 with low copy number replication origin of pSC101 plasmid containing RepA protein sequence, and finally obtaining pPegP119,119 recombinant plasmid with nucleotide sequence shown in SEQ ID NO. 2.
  6. 6. A novel xanthine oxidase recombinant strain, characterized in that said recombinant strain is obtained by transforming a recombinant expression vector comprising the novel xanthine oxidase of claim 3 into a host bacterium; The host bacterium is Pseudomonas putida KT2440.
  7. 7. A novel xanthine oxidase which is expressed by the recombinant strain according to claim 6.
  8. 8. The novel xanthine oxidase according to claim 7, wherein the specific activity of the novel xanthine oxidase is not less than 10U/mg.
  9. 9. Use of a novel xanthine oxidase according to claim 7 for the preparation of a reagent for the detection of xanthine and/or hypoxanthine concentrations.
  10. 10. The use of a novel xanthine oxidase according to claim 9 for the preparation of a reagent for the detection of xanthine and/or hypoxanthine concentrations, characterized in that the detection environment is at a pH of 6.0-9.0, 30-45 ℃.

Description

Novel xanthine oxidase, gene cluster, recombinant expression vector, recombinant strain and application thereof Technical Field The invention belongs to the technical fields of genetic engineering and enzyme engineering, and in particular relates to novel xanthine oxidase, a gene cluster, a recombinant expression vector, a recombinant strain and application thereof. Background Xanthine oxidase (Xanthine Oxidase, XOD) is a molybdenum-containing flavin protease which catalyzes xanthine and hypoxanthine to generate uric acid, is used as a key speed-limiting enzyme of purine metabolic pathway, and has irreplaceable application value in the fields of medical diagnosis, food detection, industrial catalysis and the like. In medical diagnosis, the detection of the activity of the XOD and the concentration of a substrate (xanthine and hypoxanthine) is an important index for evaluating diseases such as gout, renal function injury, myocardial infarction and the like, the XOD can be used for monitoring the freshness of foods such as meat, aquatic products and the like (the accumulation of hypoxanthine is positively related to the food spoilage degree), and the XOD can also be used for the directional synthesis and conversion of purine medicines in industrial catalysis, so that the market demand is continuous and stable. However, the industrialization application of XOD in the prior art still faces a number of bottlenecks to be solved: (1) The current commercial XOD mainly depends on natural extraction, and the core source is milk or a few specific microorganisms (such as bovine small intestine mucous membrane, aspergillus flavus and the like). Natural extraction is limited by factors such as raw material supply, extraction efficiency and the like, so that not only is the yield difficult to scale up, but also the product purity fluctuation is large, and the strict requirement of diagnostic reagents on enzyme preparation consistency cannot be met. (2) The production cost is high, the natural extraction process is complex, the purification steps such as chromatography and dialysis are needed to be carried out for a plurality of times, the cost of manpower and material resources for unit yield is high, and meanwhile, the purchase cost of raw materials (such as high-quality milk and specific microorganism strains) further increases the price of terminal products, and the popularization of the terminal products in middle-low end markets is limited. (3) The recombinant expression technology has a remarkable bottleneck that in order to break through the limitation of natural extraction, the prior research attempts to recombinant express the XOD in a heterologous host by a genetic engineering technology. However, the reported key problems of the expression system are that ① takes escherichia coli (E.coli) as a host, XOD is used as a complex multi-subunit metalloproteinase, the correct folding of the complex multi-subunit metalloproteinase needs to depend on the assembly of Fe-S clusters and molybdenum cofactors and covalent connection of cytochrome C, the intracellular environment of the escherichia coli cannot efficiently finish the post-translational modification, so that the recombinase mostly exists in the form of inclusion bodies, the activity is extremely low, ② takes actinomycetes such as Rhodococcus sp as hosts, the folding problem can be partially solved, but the growth rate of the host is slow, the fermentation period is long (usually 48-72 hours), and expensive inducers (such as IPTG) are needed to start the expression, the production difficulty and the cost are further increased, the ③ reported XOD homologous genes are mostly from known strains, the sequence conservation is high, a new function breakthrough is not formed, and key performances such as enzyme activity and stability are not remarkably different from natural extracted products. In recent years, with the development of microbial genomics and bioinformatics, the selection of novel functional gene clusters from microbial genomes which have not been fully mined has become an important direction for breaking through the technical bottleneck. Meanwhile, the existence of unreported XOD homologous gene clusters in certain strains is also found, and the method has potential industrial application value. Disclosure of Invention Aiming at the technical problems of limited sources and difficult expression of the prior XOD, the first aim of the invention is to provide a novel xanthine oxidase gene cluster which is a novel XOD gene cluster discovered in Pseudomonas putida KT2440 genome (hereinafter collectively referred to as p. Putida KT 2440) for the first time through bioinformatics comparison and analysis, and the gene composition mode (comprising Fe-S cluster binding protein gene, molybdenum binding protein gene, cytochrome C gene and XdhC gene, and lacking XdhB and XdhA subunits in the traditional XOD gene cluster) is significantly different from the known XOD