CN117025467A - Rabdosia for producing cholesterol oxidoreductase dependent on FAD, application, culture medium and preservation method - Google Patents

Rabdosia for producing cholesterol oxidoreductase dependent on FAD, application, culture medium and preservation method Download PDF

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CN117025467A
CN117025467A CN202311009706.XA CN202311009706A CN117025467A CN 117025467 A CN117025467 A CN 117025467A CN 202311009706 A CN202311009706 A CN 202311009706A CN 117025467 A CN117025467 A CN 117025467A
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fad
strain
culture medium
cholesterol
dependent
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CN117025467A8 (en
CN117025467B (en
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沈张奇
王苗利
兰邹然
王瑶
刘婕
马立超
宫晓玮
应璐宇
姜晓彤
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Shandong Animal Disease Prevention And Control Center Shandong Zoonotic Disease Surveillance Center
China Agricultural University
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Shandong Animal Disease Prevention And Control Center Shandong Zoonotic Disease Surveillance Center
China Agricultural University
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Abstract

The application discloses a Rhizopus arrhizus producing cholesterol oxidoreductase dependent on FAD, application, a culture medium and a preservation method, wherein the Latin name of the Rhizopus arrhizus strain is Rothia nasimurium, the Rhizopus arrhizus strain is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of 26534 and the preservation time of 2023 for 2 months and 10 days, and the protein sequence of the Rhizopus arrhizus strain is shown as SEQ ID No:1, the homology with the known GMC family protein is 64%, and the strain of the genus Roche can generate FAD-dependent cholesterol oxidoreductase, so that the strain has a relatively broad prospect in subsequent application. The application also discloses a culture medium and a culture method thereof for the Rauwolfia muricata which are suitable for the application and can produce the cholesterol oxidoreductase dependent on FAD, and the culture medium is convenient for subsequent use in related scientific researches.

Description

Rabdosia for producing cholesterol oxidoreductase dependent on FAD, application, culture medium and preservation method
Technical Field
The application relates to the technical field of microorganisms, in particular to a Rabdosia crenata producing cholesterol oxidoreductase dependent on FAD, application, a culture medium and a preservation method.
Background
Cholesterol oxidase is a key enzyme in cholesterol metabolic process, and the clinical application potential of using cholesterol oxidase as a detection of serum cholesterol content is huge. Cholesterol is oxidized by cholesterol oxidase to produce delta 4-cholestenone and hydrogen peroxide. Cholesterol oxidase genes (cholesterol oxidase, cho), also known as second-generation insect-resistant genes, whose expressed products are a novel class of pesticides. Cholesterol is a major component of cell membranes, and, along with the above-described reaction, intestinal epidermal cells ingested by insects undergo lysis, thereby causing death of the insects. Cholesterol oxidase has been found only in bacteria such as Streptomyces (Streptomyces), brevibacterium (Brevibacterium), pseudomonas (Pseudomonas), rhodococcus (Rhodococcus).
Although the Rhizoctonia cerealis (Rothia nasimurium) is a symbiotic bacterium of the upper respiratory tract, intestinal tract and other parts of mammals such as pigs, humans and birds, in recent years, research has found that the bacterium also contains pathogenicity and is pathogenic to animals such as arctic hive goose embryo, dog skin, ducks and the like. The genus rogowski (Rothia) is a gram positive bacterium, a facultative anaerobic, non-spore forming, unpowered bacterium. Currently, the genus contains six species, rothia aeria, rothia amara, rothia dentocariosa, rothia mucicularosa, rothia nasimurium and Rothia terrae, respectively.
In the prior art, the application patent with the patent application number of CN202210716891.5 discloses a specific primer and a probe for detecting the Rhizoctonia cerealis and application thereof, wherein the specific primer and the probe are applied to preparation of a kit for detecting the Rhizoctonia cerealis, so that the specific detection of the Rhizoctonia cerealis is realized. The application provides a specific primer and a probe for detecting the Rhizoctonia cerealis for the first time, is used for preparing a kit and detecting the Rhizoctonia cerealis, has a relatively good linear relationship in a range from 102 to 107 cobase/mu L, has a simple detection method and high specificity, but has no mention about the related performance, culture and application of the Rhizoctonia cerealis.
Disclosure of Invention
In view of the problems mentioned in the background, the present application provides a Rhizoctonia cerealis producing FAD-dependent cholesterol oxidoreductase and its use.
The technical aim of the application is realized by the following technical scheme:
a Latin name of the strain of the Rauwolzia muricata is Rothia nasimurium, and the strain is preserved in China general microbiological culture Collection center (CGMCC) No.26534, and the preservation time is 2023, 2 and 10 days.
Preferably, the protein sequence of the strain of the murine rhinoroche is shown as a sequence table SEQ ID No:1 shows a homology of 64% with known GMC family proteins. The nucleotide of the strain of the Rauwolfia murill is shown in a sequence table SEQ ID No: 2.
The application also discloses application of the strain of the genus rochanterium in preparation of cholesterol oxidoreductase capable of generating FAD. FAD is flavin adenine dinucleotide.
Preferably, any of the above-described modes, the cholesterol oxidoreductase produced by the strain of Rauwolzia murill can be used for clinical diagnosis and detection, food processing, pharmaceutical, tool enzymes, and pesticides.
Preferably, any of the above aspects, the clinical diagnosis and detection includes at least the ability to assess arteriosclerosis, thrombosis, cardiovascular disease and lipid disease.
Preferably, any of the above aspects, the food processing includes at least a process for reducing and converting cholesterol levels in dairy, meat, egg based foods.
Preferably, any of the above schemes at least comprises preparing drugs for treating hepatitis, resisting obesity and preventing skin keratinization, anti-tumor drugs and synthesizing hormone drugs as precursor substances or synthesizing antibiotics drugs as novel action targets.
The application also provides a culture medium for culturing the Rabdosia that can produce the cholesterol oxidoreductase dependent on FAD, wherein the culture medium is a TSA culture medium, and the culture medium further comprises calf serum and FAD, the calf serum accounts for 4-6% of the volume of the culture medium, and the FAD accounts for 0.008-0.015% of the volume of the culture medium.
Preferably, the culture medium comprises 15g/L tryptone, 5g/L plant peptone, 30g/L sodium chloride, 15g/L agar, 1000mL distilled water, 4% -6% calf serum by volume of the culture medium and 0.008% -0.015% FAD by volume of the culture medium.
Preferably, in any of the above embodiments, the calf serum is used in an amount ranging from about 4% to about 6% by volume of the medium, such as about 4%, about 4.5%, about 5%, about 5.5%, about 6%; FAD accounts for any value in the range of 0.008% -0.015% of the volume of the culture medium, such as 0.008%,0.01% and 0.015%.
Preferably, in any of the above embodiments, the calf serum is present in an amount of 5% by volume of the medium and FAD is present in an amount of 0.01% by volume of the medium.
The application also discloses a culture method of the Rhizopus arrhizus producing the cholesterol oxidoreductase depending on FAD, which adopts broth to store at low temperature of-20 ℃ to-80 ℃ or prepare freeze-dried powder to store at low temperature of-80 ℃, wherein the broth is broth containing 20% -40% of glycerol or TSB broth, and the freeze-dried powder is prepared by directly putting the broth without glycerol into a freeze dryer, and the low Wen Chougan is powder.
Advantageous effects
The application discloses a Rauwolfia murill for producing cholesterol oxidoreductase dependent on FAD for the first time, and related application in the aspects of producing cholesterol oxidoreductase dependent on FAD factors and the like, and has wider prospect in subsequent application. The application also discloses a culture medium and a culture method thereof for the Rauwolfia muricata which are suitable for the application and can produce the cholesterol oxidoreductase dependent on FAD, and the culture medium is convenient for subsequent use in related scientific researches.
Drawings
FIG. 1 is a graph showing predicted signal peptide and transmembrane protein for cholesterol oxidase;
FIG. 2 is a protein structure model predictive diagram of cholesterol oxidase;
FIG. 3 shows NCBI protein sequence alignment;
FIG. 4 is a tree of sequence homology of cholesterol oxidoreductase protein of Rauwolfia murill;
FIG. 5 is a comparative diagram of a culture medium assay for culturing Rabdosia murine capable of producing FAD-dependent cholesterol oxidoreductase.
Detailed Description
The technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present application, and it is apparent that the described embodiments are merely all other embodiments that a person skilled in the art may obtain without making any inventive effort, and are all within the scope of protection of the present application. Unless otherwise specified, the relevant materials appearing in the subsequent examples were all prepared from the preceding examples.
Example 1
A Rhizopus arrhizus producing cholesterol oxidoreductase dependent on FAD is a bacteria of genus Rhizopus, latin name of the Rhizopus arrhizus strain is Rothia nasimurium, the Rhizopus arrhizus strain is preserved in China general microbiological culture Collection center (CGMCC) No.26534, and the preservation time is 2023, 2 and 10 days. The protein sequence of the strain of the Rauwolfia murill is shown in a sequence table SEQ ID No:1 shows a homology of 64% with known GMC family proteins.
(1) By predicting the signal peptide and transmembrane protein of this enzyme, the result was a TAT signal peptide (Tat/SPI) confidence of 0.9147. The prediction results are shown in the following table 1 and fig. 1.
TABLE 1
(2) By prediction of the protein structure model, the predicted structure is as follows: homology to known GMC family proteins is 64%, model confidence as shown in fig. 2: very high (pLDDT > 90) blue, confident (90 > pLDDT > 70) light blue, low (70 > pLDDT > 50) yellow, very low (pLDDT < 50) red. The theme colors in fig. 2 are all deep blue.
The AlphaFold yields a per residue confidence score pLDDT of greater than 90.
(3) The protein sequence is shown in SEQ ID No: 1.
(4) NCBI protein sequence alignment results are shown in FIG. 3.
(5) The tree of sequence homology of cholesterol oxidoreductase protein of Rauwolfia murinus (NJ algorithm) is shown in FIG. 4.
Example 2
The cholesterol oxidase that can produce FAD-dependent cholesterol oxidases as in example 1 can be applied to:
(1) Clinical diagnosis and detection
Cholesterol oxidase can oxidize cholesterol to cholest-4-en-3-one and H 2 O 2 By detecting the amount of the product, the amount of cholesterol in the sample can be calculated, and arteriosclerosis, thrombosis, cardiovascular diseases and other lipid diseases can be estimated [1,2] 。
Examples: analysis methods combining cholesterol oxidase, cholesterol lipase and catalase have been widely used in clinical diagnosis of total plasma cholesterol levels [3] . Manufacturing cholesterol biosensor and measuring content of free cholesterol in human serum sample [4] Can also be used for determining total cholesterol content in food samples such as egg and meat [5] 。
(2) Food processing
Cholesterol oxidase can reduce and convert cholesterol in foods such as milk, meat, egg, etc.
Examples: lv Chenfeng and the like optimize the process of converting cholesterol oxidase into yolk cholesterol, and can be used for producing yolk product with low cholesterol and health promoting effect [6] The method comprises the steps of carrying out a first treatment on the surface of the The genetically engineered strain for producing cholesterol oxidase can also be directly used for milk product production to control cholesterolContent of (3) [2] 。
(3) Pharmaceutical engineering
The cholesterol oxidase oxidized cholesterol product cholest-4-ene-3-ketone can be used as a medicament for treating hepatitis, resisting obesity and preventing skin keratinization, can be used as a precursor substance for synthesizing hormone medicaments, and can be a novel action target point of antibiotics.
Examples: genes encoding cholesterol oxidase trigger the synthesis of the antibiotic polymamycin [7] The method comprises the steps of carrying out a first treatment on the surface of the Cholesterol oxidase has wide substrate specificity, can be used for bioconversion of various 3 beta hydroxyl steroids, and can be further used for synthesizing steroid hormones and other pharmaceutical steroids in the presence of an organic solvent; many pathogenic bacteria (rhodococcus equi) [8] Mycobacterium tuberculosis [9] ) Cholesterol oxidase can be produced and is thought to be associated with bacterial pathogenicity and thus may be a potential target for antibiotic action.
(4) As anti-tumor therapeutic molecules
Studies have shown that tumor tissue accumulates more cholesterol than normal tissue, which contributes to proliferation, differentiation and migration of tumor cells, and depletion of plasma membrane cholesterol leads to protein kinase inactivation and cell death, thus it has been proposed that membrane cholesterol may be a therapeutic target for tumors [10] 。
Examples: cholesterol oxidase (COD-B) of bordetella induces apoptosis of lung cancer cells by catalyzing membrane cholesterol oxidation and increasing active oxygen levels [11] The method comprises the steps of carrying out a first treatment on the surface of the Compared with sensitive cancer cell membranes, the drug-resistant cancer cell membrane is rich in cholesterol and sphingolipid, has the advantages of enhanced 'rigidity', reduced mobility and difficult entry of drugs into cells, and the depletion of cholesterol can reduce the rigidity of the resistant cell membrane, so that the chemotherapeutics can be efficiently delivered into cells, and therefore, the cholesterol oxidase is taken as a component of a drug delivery system to help reverse the multi-drug resistance of cancer cells [12] 。
(5) Tool enzyme
Cholesterol oxidase can be used as a tool enzyme for detecting the spatial structure of 3 beta-hydroxysteroid and as a probe for cell membrane or other structural cholesterol.
Examples: gallbladder-securing deviceAlcohol oxidase has been used as a probe to study cholesterol interactions with phospholipids [13] And eukaryotic cell membrane structures [14] The method comprises the steps of carrying out a first treatment on the surface of the Cholesterol oxidase as a probe impermeable to the membrane, selectively acting on cholesterol of the outer mitochondrial membrane, is used to study the structure of cholesterol of the mitochondria of adrenal cells, so as to investigate the in vivo synthesis process of adrenocortical hormone in depth [15] 。
(6) Insecticide
Cholesterol oxidase can inhibit the growth and development of lepidopteran insects, and is a very effective insecticide.
Examples: the transgenic plant has insect resistance by transferring cholesterol oxidase (ChoM) gene into plant and generating cholesterol oxidase in plant body, and Corbin transfers Streptomyces cholesterol oxidase gene into tobacco cell to obtain new transgenic plant with lepidopteran resistance [16] . Cholesterol oxidase (chokm) has become an important insect-resistant gene.
Example 3
The application also provides a culture medium for culturing the Rabdosia that can produce the cholesterol oxidoreductase dependent on the FAD, wherein the culture medium is a TSA culture medium, and comprises 15g/L tryptone, 5g/L plant peptone, 30g/L sodium chloride, 15g/L agar, 1000mL distilled water, 5% of calf serum accounting for the volume of the culture medium and 0.01% of FAD accounting for the volume of the culture medium.
A large number of experiments are carried out in the preparation process of the culture medium, and MHA culture medium and TSA culture medium (trypticase soy peptone agar culture medium) are selected for culture, and the results prove that the TSA culture medium with 5% of calf serum and 0.01% of FAD has the best growth vigor. As shown in FIG. 5, three strains of DX8, DX9 and DX30 were selected, and calf serum accounting for 5% of the volume of the culture medium and FAD accounting for 0.01% of the volume of the culture medium were added to the left one row of the culture medium, whereas calf serum and FAD were not added to the right one row of the corresponding culture medium, as can be seen from the figure, the left one row of the culture medium grew better than the right one.
The constructed chox recombinant vector expression strain has negative CAMP result without adding FAD or calf serum, and the FAD is a component necessary for producing cholesterol oxidoreductase as the calf serum ensures the growth of the Roche.
The application also discloses a culture method of the Rhizopus arrhizus producing the cholesterol oxidoreductase depending on FAD, which adopts broth to store at low temperature of-20 ℃ to-80 ℃ or prepare freeze-dried powder to store at low temperature of-80 ℃, wherein the broth is broth containing 20% -40% of glycerol or TSB broth, and the freeze-dried powder is prepared by directly putting the broth without glycerol into a freeze dryer, and the low Wen Chougan is powder.
References:
[1] Ma Yuchao the progress of the investigation of microbial cholesterol oxidase.
[2] Development of microbial cholesterol oxidase has progressed.
[3].Doukyu,N.,Characteristics and biotechnological applications of microbial cholesterol oxidases.Appl Microbiol Biotechnol,2009.83(5):p.825-37.
[4].Gholivand,M.B.and M.Khodadadian,Amperometric cholesterol biosensor based on the direct electrochemistry of cholesterol oxidase and catalase on a graphene/ionic liquid-modified glassy carbon electrode.Biosensors and Bioelectronics,2014.53:p.472-478.
[5].Basu,A.K.,et al.,Development of cholesterol biosensor based on immobilized cholesterol esterase and cholesterol oxidase on oxygen electrode for the determination of total cholesterol in food samples.Bioelectrochemistry,2007.70(2):p.375-379.
[6] Lv Chenfeng Wang Longgang Yang Shengli Wang Wu optimization of the process for converting cholesterol in egg yolk by cholesterol oxidase, university of tin-free light industry, university of light industry, 2001 (06): pages 555-559.
[7].Mendes,M.V.,et al.,Cholesterol oxidases act as signaling proteins for the biosynthesis of the polyene macrolide pimaricin.Chem Biol,2007.14(3):p.279-90.
[8].Identification and Mutagenesis by Allelic Exchange of choE,Encoding a Cholesterol Oxidase from the Intracellular Pathogen Rhodococcus equi.
[9].Brzostek,A.,et al.,Cholesterol oxidase is required for virulence of Mycobacterium tuberculosis.FEMS Microbiol Lett,2007.275(1):p.106-12.
[10] Chromobacterium.
[11].Liu,J.,et al.,Cholesterol oxidase from Bordetella species promotes irreversible cell apoptosis in lung adenocarcinoma by cholesterol oxidation.Cell Death Dis,2014.5(8):p.e1372.
[12] Bionic nano-meter based on cholesterol depletion strategy the reactor reversed tumor multidrug resistance study Huangshu.
[13].Lange,Y.,J.Ye and T.L.Steck,Activation of Membrane Cholesterol by Displacement from Phospholipids.Journal of Biological Chemistry,2005.280(43):p.36126-36131.
[14].Rouquette-Jazdanian,A.K.,et al.,Revaluation of the role of cholesterol in stabilizing rafts implicated in T cell receptor signaling.Cellular Signalling,2006.18(1):p.105-122.
[15].Cholesterol pools in rat adrenal mitochondria use of cholesterol oxidase to infer a complex pool structure.
[16].CORBIN,D.R.,et al.,Cloning of an insecticidal cholesterol oxidase gene and its expression in bacteria and in plant protoplasts.Applied and Environmental Microbiology,1994.60(12):p.4239-4244.
Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.

Claims (10)

1. A ralstonia strain producing FAD-dependent cholesterol oxidoreductase, characterized in that: the Latin name of the strain of the Rauwolzia muricata is Rothia nasimurium, the strain is preserved in the China general microbiological culture Collection center (CGMCC) No.26534, and the preservation time is 2023, 2 and 10 days.
2. The strain of bordetella mulina according to claim 2, characterized in that: the protein sequence of the murine nasal rochanterium strain is shown as a sequence table SEQ ID No:1 shows a homology of 64% with known GMC family proteins.
3. Use of a strain of the genus roche according to claim 1 for the preparation of a cholesterol oxidoreductase capable of producing FAD dependent FADs.
4. A use according to claim 3, characterized in that: the cholesterol oxidoreductase produced by the strain of the Rauwolfia murill can be used for clinical diagnosis and detection, food processing, pharmacy, tool enzyme and pesticide.
5. The use according to claim 4, characterized in that: the clinical diagnosis and detection at least comprises the steps of assessing arteriosclerosis, thrombosis, heart vascular diseases and lipid diseases.
6. The FAD-dependent cholesterol oxidoreductase-producing bacteria of the genus rochnella as claimed in claim 4, wherein: the food processing includes at least for reducing and converting cholesterol content in milk, meat, egg-based food.
7. The FAD-dependent cholesterol oxidoreductase-producing bacteria of the genus rochnella as claimed in claim 4, wherein: the preparation at least comprises the preparation of medicaments for treating hepatitis, resisting obesity and preventing skin keratinization, antitumor medicaments and synthetic hormone medicaments serving as precursor substances or synthetic antibiotic medicaments serving as novel action targets.
8. A medium for culturing a strain of moraxella capable of producing FAD-dependent cholesterol oxidoreductase, characterized in that: the culture medium is a TSA culture medium and further comprises calf serum and FAD, wherein the calf serum accounts for 4% -6% of the volume of the culture medium, and the FAD accounts for 0.008% -0.015% of the volume of the culture medium.
9. The FAD-dependent cholesterol oxidoreductase-producing bacteria of the genus rochnella as claimed in claim 4, wherein: the culture medium comprises 15g/L tryptone, 5g/L plant peptone, 30g/L sodium chloride, 15g/L agar, 1000mL distilled water, 4% -6% calf serum by volume of the culture medium and 0.008% -0.015% FAD by volume of the culture medium.
10. A method of preserving a FAD-dependent cholesterol oxidoreductase in a strain of lactobacillus plantarum, comprising: preserving broth at-20deg.C to-80deg.C or preparing lyophilized powder at-80deg.C, wherein the broth is broth containing 20% -40% glycerol or TSB broth, and the lyophilized powder is prepared by directly adding broth without glycerol into a lyophilizing machine, and pulverizing into powder at low Wen Chougan.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN101215531A (en) * 2007-12-29 2008-07-09 四川师范大学 Method for separating extracellular cholesterol oxidase producing strain
WO2012019058A1 (en) * 2010-08-05 2012-02-09 Hera Pharmaceuticals, Inc. Expression of antibody or a fragment thereof in lactobacillus
CN114875163A (en) * 2022-06-23 2022-08-09 山东省动物疫病预防与控制中心(山东省人畜共患病流调监测中心) Specific primer and probe for detecting mouse rhinoceros and application of specific primer and probe

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101215531A (en) * 2007-12-29 2008-07-09 四川师范大学 Method for separating extracellular cholesterol oxidase producing strain
WO2012019058A1 (en) * 2010-08-05 2012-02-09 Hera Pharmaceuticals, Inc. Expression of antibody or a fragment thereof in lactobacillus
CN114875163A (en) * 2022-06-23 2022-08-09 山东省动物疫病预防与控制中心(山东省人畜共患病流调监测中心) Specific primer and probe for detecting mouse rhinoceros and application of specific primer and probe

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