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A kind of preparation method and application of oxalate decarboxylase

A technology of oxalate decarboxylase and acid induction, which is applied in the field of preparation of oxalate decarboxylase, can solve the problems such as the inability to efficiently secrete and express OXDC, and achieve the effects of simplified production and purification steps, low production cost and high expression level

Active Publication Date: 2020-09-18
WUHAN KANGFUDE BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the technical problem that fungal-derived OXDC cannot be efficiently secreted and expressed in the Bacillus expression system in the prior art, and the extracellular OXDC can be kept stable in the fermentation broth, the present invention provides a high-efficiency production of fungal-derived OXDC Oxalate decarboxylase method

Method used

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  • A kind of preparation method and application of oxalate decarboxylase
  • A kind of preparation method and application of oxalate decarboxylase
  • A kind of preparation method and application of oxalate decarboxylase

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Embodiment 1

[0034] The screening of embodiment 1 acid-resistant bacillus

[0035] The inventors have successively screened more than a hundred species of Bacillus species preserved in the laboratory and purchased by the strain preservation center, including Bacillus circulans CICC10353, CICC10403, CICC21250 and CICC23053, Geobacillus stearothermophilus CICC10142, CICC10267, CICC10362, CICC10392, CICC10425, CICC10782 and CICC10842, etc., Bacillus amyloliquefaciens CICC10025, CICC10035, CICC10036, CICC10063, CICC10074, CICC 10081 and CICC10079, CICC10163, etc. Several strains of Bacillus with strong acid resistance were screened. The screening method is as follows:

[0036] The seed medium is: peptone 10g / L, yeast powder 5g / L, NaCl 5g / L, KH 2 PO 4 2g / L, glucose 10g / L, pH 6.0; screening medium (1L): 20g corn flour, 5g corn steep liquor, 2g yeast powder, 1.0g ammonium chloride, 0.5g manganese chloride, MgSO 4 ·7H 2 O 0.91g, CaCl 2 0.5g, 500mL of citric acid-sodium citrate buffer solut...

Embodiment 2

[0038] Cloning of embodiment 2 oxalate decarboxylase gene

[0039] The fungal oxalate decarboxylase gene in the present invention is based on the fungal oxdc gene registered in the public database, optimized according to the codons of Bacillus amyloliquefaciens to obtain an optimized gene, and then synthesized through a commercial gene sequencing and synthesis company. The following are several examples of oxalate decarboxylase genes without the original signal peptide obtained through experimental verification based on the codon optimization of fungal-derived oxdc genes registered in the public database (microbial name: database name: original gene accession number: optimized The sequence number of the gene sequence in the sequence table) is as follows:

[0040] Hypsizygus marmoreus: GenBank: LUEZ01000002.1: SEQ ID NO.1;

[0041] Coprinopsis cinerea okayama: GenBank: XM_001836586.2: SEQ ID NO.2;

[0042] Trametes versicolor: GenBank: XM_008041462.1: SEQ ID NO.3;

[0043] H...

Embodiment 3

[0061] Cloning of Example 3 Acid Inducible Promoter

[0062] As the acid-inducible promoter, a promoter derived from Bacillus can be used in principle, as long as it is an acid-inducible promoter derived from Bacillus identified so far, in principle, an acid-inducible promoter derived from Bacillus subtilis is used as an example. Chromosomal DNA was first obtained from Bacillus subtilis Bs168 strain by conventional methods. Refer to the sequence before the Bacillus subtilis oxdc gene (Gene ID 938620) on the database, design primers F2 and R2 to specifically amplify the acid-induced promoter region, use the chromosomal DNA of the Bacillus subtilis Bs168 strain as a template, and use the designed Primers (F2 and R2) were used for PCR, thereby obtaining a DNA fragment of the acid-inducible promoter Poxdc, the sequence of which is shown in SEQ ID NO.6.

[0063] F2 (SEQ ID NO.13): 5'-AGCTTGACCACTTCATTTTTC-3'

[0064] R2 (SEQ ID NO.14): 5'-GAAATGTTTCTCTCCTTACAT-3'

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Abstract

The invention discloses a preparation method of oxalate decarboxylase. The method comprises the following steps: constructing a recombinant expression plasmid vector comprising Bacillus-containing acid induced promoter, a secretion signal peptide, and a fungus-derived oxalate decarboxylase gene prepared under the control of the secretion signal peptide, transforming bacilli normally growing in a pH value being not more than 4.5 by the vector to prepare oxalate decarboxylase recombinant bacteria; and carrying out culture and acid feeding on the recombinant bacteria in a fermentation medium, inducing the recombinant bacteria, and carrying out simple purification to recover generated extracellular oxalate decarboxylase. The extracellular oxalate decarboxylase prepared through the method is used in diagnosis reagent raw materials, medicinal compositions, healthcare foods or formulated foods with special medical uses. The mutated acid induced promoter is adopted to substitute an original acid induced promoter in the recombinant expression vector, so the transformed bacilli have a high expression level.

Description

technical field [0001] The invention relates to the technical field of microbial enzyme preparation, in particular to a preparation method and application of oxalate decarboxylase. Background technique [0002] Oxalic acid (Oxalic acid), also known as oxalic acid (Ethanedioic acid), is a metabolite of organisms, widely distributed in plants, animals and fungi, and exerts different functions in different organisms. Studies have found that at least 100 kinds of plants are rich in oxalic acid, especially in the leaves and seeds of plants such as spinach, amaranth, beet, purslane, taro, tea, cocoa, sweet potato and rhubarb, because oxalic acid can reduce the content of mineral elements. Bioavailability, in the human body, it is easy to form calcium oxalate with calcium ions to cause kidney stones, so oxalic acid is often considered as an antagonist for the absorption and utilization of mineral elements. Oxalic acid is not easy to be oxidized and decomposed in the human body. Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N15/75C12N9/88
CPCC12N9/88C12N15/75C12N2830/34C12Y401/01002
Inventor 汪小锋汪卫吴玉峰宋保平
Owner WUHAN KANGFUDE BIOTECH CO LTD
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