CN107189956B - Heparinase high-yield strain and breeding method thereof - Google Patents
Heparinase high-yield strain and breeding method thereof Download PDFInfo
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- CN107189956B CN107189956B CN201710203311.1A CN201710203311A CN107189956B CN 107189956 B CN107189956 B CN 107189956B CN 201710203311 A CN201710203311 A CN 201710203311A CN 107189956 B CN107189956 B CN 107189956B
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Abstract
The invention provides a heparinase high-yield strain, which comprises the following strains: raoultella sp. nx-TZ-3, 15; the strain is preserved in a preservation unit appointed by the national intellectual property office, the preservation date is 3 and 6 days in 2017, and the name of the preservation unit is as follows: china general microbiological culture preservation management center, preservation number: CGMCC No. 13723. The breeding of the heparinase high-yield strain is characterized in that the sediment generated by protamine and heparin reaction is used as an indicator, the heparin enzyme generated by microorganisms cracks the heparin reaction to eliminate the sediment, and transparent rings are generated around the bacteria, so that the direct indication effect on the heparinase source microorganism strains is achieved, and the Raoultella SP, NX-TZ-3 and 15 are identified by re-screening and a 16S rDNA method.
Description
[ technical field ]
The invention belongs to the technical field of biology, and particularly relates to a heparinase high-yield strain Raoultella SP, NX-TZ-3,15 and a breeding method thereof.
[ background art ]
Heparinases (heparinases) or heparin lyases are a class of proteins that specifically cleave heparin and heparan sulfate glycosidic bonds. Heparinase was first discovered from Flavobacterium heparinum, a microorganism that can produce oligosaccharides from heparin, and has been used clinically mainly for the prevention of thrombosis or as an in vitro anticoagulant.
Currently, three heparinases have been identified from flavobacterium heparinum: heparinase I (heparinase, EC 4.2.2.7), heparinase II (heparinase II), and heparinase III (heparinase, EC 4.2.2.8). Heparinase I mainly cracks heparin, heparinase III mainly cracks heparan sulfate, and heparinase II can simultaneously crack heparin and heparan sulfate. Flavobacterium heparinum is mainly used for the production of low molecular heparin and ultra-low molecular heparin, particularly heparinase I and heparinase II.
Besides separating heparinase from Flavobacterium heparinum, scholars at home and abroad also separate heparinase produced by Sphingobacterium and Bacillus from soil and food grinding waste in turn. In addition, some researchers have isolated fecal bacteroides, aspergillus flavus, and acinetobacter heparinase from human intestinal tracts, human periodontal pockets, and soil.
At present, the screening method of heparinase-producing microorganisms mainly adopts a domestication method for gradually replacing original carbon sources of a microorganism screening culture medium by heparin, specifically enriches microbial strains with the heparin cracking capacity, then monitors the content change of the heparin in an enrichment culture solution by an azure A method, preliminarily screens a sample containing the strains capable of utilizing the heparin, coats each bacterial colony in the sample, picks up a single bacterial colony for secondary screening, mainly cultures the primarily screened bacterial colony, then prepares a cell-free crude extract, breaks the cell-free crude extract, and determines whether the microorganism is heparinase-producing by utilizing the azure A method to measure the enzyme activity.
[ summary of the invention ]
Aiming at the defects in the prior art, the invention provides a heparinase high-yield strain and a breeding method thereof, and the heparinase high-yield strain can be screened out by using the method.
In one aspect, the invention provides a heparinase high-producing strain, which comprises the following strains: raoultella sp. nx-TZ-3, 15; the strain is preserved in a preservation unit appointed by the national intellectual property office, the preservation date is 3 and 6 days in 2017, and the name of the preservation unit is as follows: general microbiological center of China Committee for culture Collection of microorganisms, the address of the Collection center: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC No. 13723.
On the other hand, the invention provides a breeding method of a heparinase high-yield strain, which comprises the following steps: by using protamine and heparin reaction sediment as an indicator, performing heparinase cracking reaction to generate transparent rings around thalli so as to directly indicate heparinase-derived microbial strains, and identifying heparinase-producing strains Raoultella SP, NX-TZ-3 and 15 by re-screening and 16S rDNA methods;
the method specifically comprises the following steps:
step one, enrichment culture of heparinase producing bacteria: adding 1g of the collected sample into a test tube containing 10mL of sterile physiological saline, shaking uniformly, taking 5mL of suspension, or directly taking 5mL of water sample, adding into a triangular flask containing 25mL of enrichment medium, and carrying out shake-flask culture at 37 ℃ for 2 days at 200 r/min;
step two, primary screening of heparinase producing bacteria: diluting the enriched and cultured bacterial liquid to a proper dilution degree by using sterile normal saline in a gradient manner, coating a proper amount of bacterial liquid on a primary screen flat plate, culturing for 10-12 h, when the colony on the flat plate grows to a proper size, washing the colony by using distilled water, adding 2% of protamine solution, placing for 1h at 37 ℃, pouring the protamine solution, cleaning by using distilled water, placing for 12h at room temperature, allowing a transparent ring to appear at the growth position of the original colony, selecting a strain with a larger ratio of the transparent ring to the diameter of the colony to transfer to a seed culture medium, and culturing for 24 h;
step three, re-screening the heparinase producing strain: and (3) selecting a bacterial colony with a transparent ring on the primary screening flat plate, inoculating the bacterial colony into a seed culture medium, culturing at 37 ℃ for 24h in a shaking way, transferring the bacterial colony into a secondary screening culture medium according to the inoculation amount of 5%, culturing at 37 ℃ for 72h in a shaking way at 200r/min, preparing a cell-free crude extract, detecting the activity of enzyme, and inspecting and identifying the high-yield bacterial strain of the heparinase.
The enrichment medium in the first step consists of (%): tryptone 1.0, heparin sodium 0.5, NaCl 0.5, pH 6.5; the composition of the primary screening culture medium is (%): tryptone 1.0, (NH)4)2SO4 0.1、KH2PO4 0.25；MgSO4·7H2O0.05, heparin sodium 0.2, pH6.5; the seed medium composition (%) was: tryptone 1.0, (NH)4)2SO4 0.1、KH2PO40.25、 MgSO47H2O 0.05, heparin sodium 0.2, pH 6.5; the primary screening culture medium comprises the following components: (%): yeast extract 0.3, tryptone 1.0, NaCl 0.5, heparin sodium 0.2, agar 2.0, pH 7.0.
The method for identifying the shape of the high-yield strain thallus of the heparinase adopts a blue staining method: the fast gram staining solution is adopted to observe under a 100-fold oil lens, and the shape, arrangement and certain result characteristics of the bacteria are determined, so that the bacteria are determined to be gram-positive bacteria or gram-negative bacteria.
The method for identifying the high-yield bacteria of the heparinase in the step three is to identify the high-yield bacteria of the heparinase by using 16S rDNA, and the method comprises the following steps: extracting 16S rDNA genome DNA and detecting by electrophoresis: extracting DNA of the bacteriocin-producing lactic acid bacteria by using an Omega D3350-00E.Z.N.A.TM bacterial genome DNA extraction kit, and performing agarose electrophoresis detection and OD value detection on the extracted genome to determine the required concentration and purity; PCR amplification of 16S rDNA gene; thirdly, 16S rDNA sequence determination and heparinase high-yield strain species determination: the 16S rDNA genome amplified by PCR reaction is sent to Shanghai biological engineering technology service limited company for sequencing after agarose electrophoresis detection, PCR product recovery, T-vector connection and cloning, the sequencing result is input into http:// blast.ncbi.nlm.nih.gov/blast of NCBI database, and is compared with the 16S rDNA sequence recorded by NCBI, 20 strain sequences with higher homology with the strain producing heparinase are selected according to the sequence comparison result, and the 20 strains are constructed into a phylogenetic tree by utilizing bioinformatics software (Mega 4.0) to further confirm the strain species.
The step three is to prepare the cell-free crude extract by centrifuging the bacterial re-screening culture solution (6000rpm, 15min) to collect the bacteria, and using 30mL of CaCl with the pH value of 7.02Washing with Tris-HCl buffer, centrifuging (6000rpm, 15min), repeating twice, and adding 0.2g wet cells per ml of CaCl at pH7.02And (2) in Tris-HCl buffer solution, carrying out ultrasonic crushing for 10 times in ice bath at intervals of 5s for each time, freezing and centrifuging the crushed solution at 8000rpm for 20min, and obtaining supernatant which is cell-free crude extract.
Above CaCl2-Tris-HCl buffer preparation method: 5mL of 10mM/LCaCl was taken2Mixing with 25mL of 1M Tris-HCl buffer solution, diluting to 1000mL, and adjusting pH to 7.0 for later use; (1M HCL solution: measuring 8.5mLHCl in a 100mL volumetric flask to constant volume (in a fume hood), 1M Tris solution: measuring 12.2g Tris, completely dissolving with distilled water and then measuring to constant volume of 100mL, 10mM/L CaCl2: 0.11099g of anhydrous CaCl was weighed2By steamingDistilling water to completely dissolve the mixture, and fixing the volume to 100 mL; 1M Tris-HCl buffer: 45.7mL of 1M HCl solution was taken and mixed with 50mL of 1M Tris solution for use. )
The method for detecting the enzyme activity in the third step is to take 250 mu L of the cell-free crude extract, add 50 mu L of 4 percent heparin sodium (150IU/mg) and 200 mu L of 0.2mol/L of CaCl with the pH value of 7.02Tris-HCl buffer solution, water bath heat preservation at 37 ℃, enzyme activity measurement, 5 mu L reaction solution taking out, 5mL of 0.002% azure A solution adding, A measurement505Determining the concentration of undegraded heparin in the reaction solution according to a standard curve so as to obtain the degradation amount of heparin, wherein the enzyme activity unit is defined as the enzyme amount required for degrading l mg of heparin per hour at 37 ℃ under the condition of pH7.0;
and (3) calculating: x is Ax 10-3×0.4×106×1/T
X-heparinase activity in the sample, U/L;
a-amount of degraded heparin per unit time, μ L;
t-time of reaction, h;
0.4×106-dilution factor.
The standard curve making method comprises the following steps: accurately weighing a certain amount of heparin sodium, preparing a 0.8% heparin sodium solution by using distilled water, and diluting the heparin sodium solution with different gradients to prepare a 0.1-0.8% heparin solution; taking 9 test tubes, adding 5 mu L of distilled water into one test tube as a blank control, and sequentially adding 5 mu L of 0.1-0.8% heparin sodium standard solution into the other test tubes; finally, 5mL of 0.002% azure A solution is added into each test tube, and the mixture is uniformly mixed and then is measured on an enzyme-linked immunosorbent assay (ELISA) instrument505Three parallel experiments were performed for each test tube.
The enzyme activity measuring time is measured every 30min from the time of putting the enzyme into a 37 ℃ water bath for heat preservation, and is stopped after 1.5h, and the enzyme activity is calculated by taking 1.5h as a unit when the enzyme activity is calculated.
The heparinase high-yield strain and the breeding method provided by the invention solve the defects of time consumption, labor consumption and material consumption of the original screening method of heparinase-derived microorganisms, and the precipitate generated by the reaction of protamine and heparin is used as an indicator, so that the labor, physical strength and time are greatly saved, and the yield of the heparinase is improved.
[ description of the drawings ]
FIG. 1 is a flow chart of a breeding method of a heparinase high-producing strain.
FIG. 2 shows the appearance of a transparent circle of colonies. In the figure 1 is a transparent ring.
FIG. 3 is a photograph of agarose electrophoresis of the heparinase-producing strain 16SrDNA by PCR amplification.
Figure 4 is an alignment of Raoultella sp. nx-TZ-3,1516 SrDNA sequence NCBI.
FIG. 5 shows the enzyme activity comparison of fermentation broths of different heparinase-producing strains.
[ detailed description of the invention ]
In order to make the technical means for implementing the invention clear, the invention is further explained below.
Example (b): adding 1.0g of collected soil sample into a test tube containing 10mL of sterile normal saline, shaking uniformly, taking 5mL of suspension, adding into a triangular flask containing 25mL of enrichment medium, and performing shake-flask culture at 37 ℃ for 2 days at 200 r/min. Diluting the enriched culture bacterial liquid with sterile physiological saline to appropriate dilution, and coating appropriate amount of bacterial liquid on a primary sieve plate. After the strain on the plate grows to a certain size, the bacterial colony is washed by distilled water, 2% protamine solution is added, and the mixture is placed for 1h at 37 ℃. The protamine solution was poured off, washed with distilled water, and left at room temperature for a period of time, so that a transparent ring appeared on the original colony growth. And selecting the strain with larger ratio of the transparent ring to the colony diameter to transfer to a seed culture medium. And (3) selecting a bacterial colony with a transparent ring on the primary screening flat plate, inoculating the bacterial colony into a seed culture medium, performing shake-flask culture at 37 ℃ for 24 hours, transferring the bacterial colony into a secondary screening culture medium according to the inoculation amount of 5%, performing shake-flask culture at 200r/min and 37 ℃ for 72 hours. Preparation of a crude cell-free extract in A505Enzyme activity is measured to obtain 1 strain of heparinase, and the strain is identified as Raoultella SP. NX-TZ-3,15 by 16SrDNA strain.
As shown in figure 5, through experimental data comparison, among the enzyme activities of different heparinase strain fermentation liquids, Raoultella SP. NX-TZ-3,15 can generate 347.99IU/L, XC-1 enzyme activity is 127.46IU/L, XC-2 enzyme activity is 233.24IU/L, XC-3 enzyme activity is 49.17IU/L, and XC-4 enzyme activity is 25.89IU/L, so that Raoultella SP, NX-TZ-3,15 strains are far higher than other heparinase strains.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiment, and all technical solutions belonging to the principle of the present invention belong to the protection scope of the present invention. Modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention.
Claims (1)
1. A heparinase high-producing strain is characterized by comprising the following strains: raoultella sp. nx-TZ-3, 15; the strain is preserved in a preservation unit appointed by the national intellectual property office, the preservation date is 3 and 6 days in 2017, and the name of the preservation unit is as follows: china general microbiological culture preservation management center, preservation number: CGMCC No. 13723.
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| WO2005026269A1 (en) * | 2003-09-04 | 2005-03-24 | Mcdaniel C Steven | Microorganism coating components, coatings, and coated surfaces |
| WO2015095241A2 (en) * | 2013-12-16 | 2015-06-25 | Seres Health, Inc. | Bacterial compositions and methods of use thereof for treatment of immune system disorders |
| WO2016086206A1 (en) * | 2014-11-25 | 2016-06-02 | Epiva Biosciences, Inc. | Probiotic compositions containing clostridials for inhibiting inflammation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2005026269A1 (en) * | 2003-09-04 | 2005-03-24 | Mcdaniel C Steven | Microorganism coating components, coatings, and coated surfaces |
| WO2015095241A2 (en) * | 2013-12-16 | 2015-06-25 | Seres Health, Inc. | Bacterial compositions and methods of use thereof for treatment of immune system disorders |
| WO2016086206A1 (en) * | 2014-11-25 | 2016-06-02 | Epiva Biosciences, Inc. | Probiotic compositions containing clostridials for inhibiting inflammation |
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Effective date of registration: 20221111 Address after: 518057 No. 21, Langshan Road, Songping mountain, Nanshan District, Shenzhen, Guangdong Province Patentee after: SHENZHEN HEPALINK PHARMACEUTICAL GROUP Co.,Ltd. Address before: 518000 No. 3688 Nanhai Road, Shenzhen, Guangdong, Nanshan District Patentee before: SHENZHEN University |
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