CN107189956A - A kind of heparinase superior strain and its selection - Google Patents

A kind of heparinase superior strain and its selection Download PDF

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CN107189956A
CN107189956A CN201710203311.1A CN201710203311A CN107189956A CN 107189956 A CN107189956 A CN 107189956A CN 201710203311 A CN201710203311 A CN 201710203311A CN 107189956 A CN107189956 A CN 107189956A
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CN107189956B (en
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赵丽青
刘文丽
蒋莹子
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Shenzhen Hepalink Pharmaceutical Group Co Ltd
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Abstract

The invention provides a kind of heparinase superior strain, bacterial strain is：Raoultella SP.NX‑TZ‑3,15；Depositary institution's preservation that the bacterial strain has been specified in State Intellectual Property Office, preservation date is on March 6th, 2017, depositary institution's title：China General Microbiological culture presevation administrative center, deposit number：CGMCC No.13723.The seed selection of the heparinase superior strain is that the precipitation produced by the use of nucleoprotamine and heparin reaction is used as indicant, the heparin enzymatic lysis heparin reaction that microorganism produces makes precipitation disappear, transparent circle is produced around thalline, so as to play a part of directly indicating heparinase source microbial strains, pass through secondary screening, 16S rDNA methods identification production heparinase bacterial strain Raoultella SP.NX TZ 3, 15, the present invention solves the original screening technique of heparinase source microorganism and taken, effort, the drawbacks of consumptive material, precipitation by the use of nucleoprotamine and heparin reaction generation is used as indicant, greatly save manpower, material resources and time.

Description

A kind of heparinase superior strain and its selection
[technical field]
The invention belongs to biological technical field, and in particular to a kind of heparinase superior strain Raoultella SP.NX-TZ- 3,15 and its selection.
[background technology]
Heparinase (Heparinase) or heparinlyase are that a class can crack heparin and heparitin sulfate in specific manner The protein of glycosidic bond.Heparinase finds that Flavobacterium heparinum is that one kind can utilize heparin from Flavobacterium heparinum earliest The microorganism of oligosaccharides is produced, is clinically mainly used to pre- preventing thrombosis or is used as external anticoagulation medicine.
At present, three kinds of heparinases have been identified from Flavobacterium heparinum：Heparinase I (heparinase, EC 4.2.2.7), heparinaseⅡ (heparinase II), and heparinase III (heparinase, EC 4.2.2.8).Heparinase I master Heparin is cracked, heparinase III major cleavage heparitin sulfate, heparinaseⅡ can crack heparin and heparitin sulfate simultaneously.Liver Plain Flavobacterium is mainly used in the production of LMWHs and ultra low molecular weight heparin, particularly Heparinase I and Heparinase I I.
In addition to isolating heparinase from Flavobacterium heparinum, domestic and foreign scholars are also successively from soil and food grinding waste In isolate sphingol bar category and bacillus produce heparinase.In addition, also having some scholars from human body intestinal canal, human body periodontal Intend bar, aspergillus flavus and acinetobacter calcoaceticus production heparinase with excrement is isolated in soil.
The screening technique of production heparinase microorganism mainly progressively replaces microbe to screen culture base using heparin at present The acclimation method of beginning carbon source, specific enrichment has the microbial strains of cracking ability to heparin, is then monitored with reddish black A methods The changes of contents of heparin in enrichment culture liquid, preliminary screening is each in sample to the sample containing the bacterial strain that can utilize heparin Bacterium colony is coated with, and picking single bacterium colony carries out secondary screening, and then secondary screening prepares acellular crude extract mainly by the strain culturing of primary dcreening operation, Crushed, determine whether it is the microorganism for producing heparinase using reddish black A methods measure enzyme activity, but this screening technique Need to attempt multiple formulations culture medium, waste substantial amounts of man power and material, therefore probe into a kind of simple and quick screening production heparin The method of enzyme microorganism is extremely important.
[content of the invention]
In view of the deficienciess of the prior art, the invention provides a kind of heparinase superior strain and its selection, profit Heparinase superior strain can be filtered out with the method.
On the one hand, the present invention provides a kind of heparinase superior strain, and bacterial strain is：Raoultella SP.NX-TZ-3,15； Depositary institution's preservation that the bacterial strain has been specified in State Intellectual Property Office, preservation date is on March 6th, 2017, depositary institution's name Claim：China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address：The Chaoyang District, Beijing City North Star No. 3 Institute of Microorganism, Academia Sinica of institute of West Road 1, deposit number：CGMCC No.13723.
On the other hand, the present invention provides a kind of selection of heparinase superior strain：It is anti-using nucleoprotamine and heparin The precipitation answered is as indicant, and the reaction of heparin enzymatic lysis heparin makes to produce transparent circle around thalline, so as to play to heparinase source The effect that microbial strains are directly indicated, passes through secondary screening, 16S rDNA methods identification production heparinase bacterial strain Raoultella SP.NX-TZ-3,15；
Specifically include following steps：
Step one, the enrichment culture of heparinase producing strains：The sample that 1g is gathered adds and fills 10mL sterile salines Test tube in shake up after, take 5mL suspension, or directly take 5mL water samples, add in the triangular flask for filling 25mL enriched mediums, 200r/min, 37 DEG C of Shaking cultures 2 days；
Step 2, the primary dcreening operation of heparinase producing strains：The bacterium solution of enriched culture is arrived with sterile saline gradient dilution Appropriate dilution factor, takes appropriate bacterium solution to be coated on primary dcreening operation flat board, cultivates 10~12h, treats that being born on flat board grows to suitable big It is small, bacterium colony is washed away with distilled water, 2% nucleoprotamine solution is added, 1h is placed in 37 DEG C, nucleoprotamine solution is outwelled, is used Distilled water is cleaned, and 12h is placed in room temperature, just has transparent circle in opportunistic pathogen strong point of being born and occurs, selection transparent circle and colony diameter In the larger bacterial strain transfer seed culture medium of ratio, 24h is cultivated；
Step 3, the secondary screening of heparinase producing strains：There is the bacterium colony access seed culture medium of transparent circle on picking primary dcreening operation flat board, 200r/min, 37 DEG C, Shaking culture 24h, then it is transferred to secondary screening culture medium, 200r/min, 37 DEG C, shaking flask training by 5% inoculum concentration 72h is supported, acellular crude extract is prepared, the vigor of enzyme is detected, the superior strain of heparinase is examined and identify.
Enriched medium composition described in above-mentioned steps one is (%)：Tryptone 1.0, liquaemin 0.5, NaCl 0.5, pH 6.5；Primary dcreening operation culture medium composition is (%)：Tryptone 1.0, (NH4)2SO4 0.1、KH2PO40.25；MgSO4·7H2O 0.05th, liquaemin 0.2, pH6.5；Seed culture medium composition is (%)：Tryptone 1.0, (NH4)2SO4 0.1、KH2PO4 0.25、 MgSO47H2O 0.05, liquaemin 0.2, pH 6.5；Primary dcreening operation culture medium is constituted：(%)：Yeast leaching cream 0.3, Tryptone 1.0, NaCl 0.5, liquaemin 0.2, agar 2.0, pH 7.0.
The superior strain thalli morphology authentication method of heparinase described in above-mentioned steps three uses leather blue decoration method：Adopt With quick gram staining liquid, observed under 100 times of oil mirrors, determine ne ar, arrangement and some result features, really It is set to gram-positive bacteria or Gram-negative bacteria.
The Producing Strain authentication method of heparinase described in above-mentioned steps three is with 16S rDNA identification production heparinase Producing Strains, side Method is as follows：1. the extraction of 16S rDNA genomic DNAs and electrophoresis detection：Utilize Omega D3350-00E.Z.N.A.TM bacteriums Genome DNA extracting reagent kit carries out agarose electrophoresis to extract the DNA of bacteriocin-producing lactic acid bacteria to extracting genome Detection and the detection of OD values, to determine the concentration and purity that reach needs；2. the PCR amplifications of 16S rDNA genes；③16S rDNA Sequencing and heparinase superior strain kind are determined：The 16S rDNA genomes amplified are reacted through agarose electrophoresis through PCR Detection, PCR primer are reclaimed, T- carriers are connected, Ke Longhou, are sent to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's sequencing, Sequencing result is inputted to the http of ncbi database:In //blast.ncbi.nlm.nih.gov/Blastn, received with NCBI The 16s rDNA sequences of record are compared, according to the result of sequence alignment, selection and production heparinase bacterial strain homology it is higher 20 Strain strain sequence, it is further true using bioinformatics software (Mega 4.0) by 20 plants of strain construction phylogenetic trees above Demonstrate,prove the kind of bacterial strain.
Acellular crude extract is prepared described in above-mentioned steps three, is by bacterium secondary screening medium centrifugal (6000rpm, 15min) Thalline is collected, with 30mL pH 7.0CaCl2- Tris-HCl buffer solutions are washed, and are centrifuged (6000rpm, 15min), are repeated twice, PH7.0CaCl is added in the ratio of every milliliter of 0.2g wet thallus2In-Tris-HCl buffer solutions, ultrasonic disruption 10 in ice bath Secondary, each 5s is spaced 5s, by broken liquid refrigerated centrifuge, 8000rpm, 20min, supernatant is acellular crude extract.
Above-mentioned CaCl2- Tris-HCl buffer methods：Take 5mL 10mM/LCaCl2It is slow with 25mL1M Tris-HCl Fliud flushing is mixed, and is settled to 1000mL, and regulation pH to 7.0 is stand-by；(1. 1M HCL solution：8.5mLHCl is measured in 100mL capacity Constant volume (being carried out in fume hood) in bottle；2. 1M Tris solution：12.2g Tris are weighed, make it fixed after being completely dissolved with distilled water Hold to 100mL；③10mM/L CaCl2：Weigh the anhydrous CaCl of 0.11099g2, it is completely dissolved with distilled water, is settled to 100mL；4. 1M Tris-HCl buffer solutions：45.7mL 1M HCL solution is taken, is mixed with 50mL 1M Tris solution stand-by.)
The method that enzyme activity is detected described in above-mentioned steps three is to take the 250 acellular crude extracts of μ L, adds 50 μ L 4% liver Plain sodium (150IU/mg) and 200 μ L 0.2mol/L pH 7.0CaCl2- Tris-HCl buffer solutions, 37 DEG C of water-bath insulations, survey enzyme It is living, 5 μ L reaction solutions are taken out, 5mL 0.002% reddish black solution A is added, A is surveyed505, determined according to standard curve in reaction solution not The concentration of degraded heparin is so as to draw the degradation amount of heparin, and enzyme-activity unit is defined as degrading per hour under conditions of 37 DEG C of pH7.0 Enzyme amount needed for l mg heparin is a unit；
Calculate：X=A × 10-3×0.4×106×1/T
Heparin enzyme activity in X-sample, U/L；
The amount of the heparin of degraded, μ L in A-unit interval；
The time of T-reagentia, h；
0.4×106- extension rate.
Above-mentioned standard curve formulating method：A certain amount of liquaemin is accurately weighed, 0.8% heparin is configured to distilled water Sodium solution, is carried out the dilution of different gradients, is made into 0.1~0.8% heparin solution；Take 9 test tubes, a 5 μ L of addition Distilled water makees blank control, and other test tubes sequentially add the heparin sodium standard solution of 5 μ L0.1%~0.8%；Finally, every examination Pipe adds the reddish black solution As of 5mL 0.002%, mixes after surveying A on ELIASA505, every test tube does three parallel laboratory tests.
The time of above-mentioned survey enzyme activity, it should stop from being put into 37 DEG C of water-bath insulations every a 30min enzyme activity of survey after 1.5h Only, calculated when calculating enzyme activity in units of the 1.5h times.
Heparinase superior strain and selection that the present invention is provided, solve the heparinase source original screening side of microorganism Method is time-consuming, effort, consumptive material the drawbacks of, by the use of nucleoprotamine and the precipitation of heparin reaction generation as indicant, greatly save Human and material resources and time, while improving the yield of heparinase.
[brief description of the drawings]
Fig. 1 is the flow chart of heparinase superior strain selection.
Fig. 2 occurs for bacterium colony transparent circle.1 is transparent circle in figure.
Fig. 3 is production heparinase bacterial strain 16SrDNA PCR amplification agarose electrophoresis figures.
Fig. 4 is Raoultella SP.NX-TZ-3,15 16SrDNA sequence NCBI comparison results.
Fig. 5 compares for the enzyme activity of different production heparinase bacterial strain fermentation liquors.
[embodiment]
In order that the technological means that the present invention is realized is clear, the present invention is further elucidated below.
Embodiment：The pedotheque that 1.0g is gathered is added shaken up in the test tube for filling 10mL sterile salines after, take 5mL suspension, adds and fills in the triangular flasks of 25mL enriched mediums, 200r/min, 37 DEG C of Shaking cultures 2 days.Will be enriched The bacterium solution of culture sterile saline gradient dilution to suitable dilution factor, takes appropriate bacterium solution to be coated on primary dcreening operation flat board. After the bacterial strain on flat board grows to a certain size, bacterium colony is washed away with distilled water, 2% nucleoprotamine solution is added, is put in 37 DEG C Put 1h.Nucleoprotamine solution is outwelled, cleaned with distilled water, a period of time is placed in room temperature, is just had in opportunistic pathogen strong point of being born Transparent circle occurs.Select in the transparent circle bacterial strain transfer seed culture medium larger with colony diameter ratio.On picking primary dcreening operation flat board Have a bacterium colony access seed culture medium of transparent circle, 200r/min, 37 DEG C, Shaking culture 24h, then by 5% inoculum concentration be transferred to it is multiple Sieve culture medium, 200r/min, 37 DEG C, Shaking culture 72h.Acellular crude extract is prepared, in A505Enzyme activity is surveyed, production heparinase is obtained 1 plant of bacterial strain, Raoultella SP.NX-TZ-3,15 are accredited as through 16SrDNA bacterial strains.
As shown in figure 5, compared by experimental data, in the enzyme activity of difference production heparinase bacterial strain fermentation liquor, Raoultella SP. NX-TZ-3,15 can produce 347.99IU/L, and XC-1 enzyme activity is 127.46IU/L, and 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, it can be seen that Raoultella SP.NX-TZ-3,15 bacterial strains Far above other production heparinase bacterial strains.
The preferred embodiment of the present invention is the foregoing is only, protection scope of the present invention is not limited in above-mentioned embodiment party Formula, every technical scheme for belonging to the principle of the invention belongs to protection scope of the present invention.For those skilled in the art Speech, some improvement carried out on the premise of the principle of the present invention is not departed from, these improvement also should be regarded as the protection model of the present invention Enclose.

Claims (10)

1. a kind of heparinase superior strain, it is characterised in that bacterial strain is：Raoultella SP.NX-TZ-3,15；The bacterial strain is The depositary institution's preservation specified in State Intellectual Property Office, preservation date is on March 6th, 2017, depositary institution's title：China is general Logical Microbiological Culture Collection administrative center, deposit number：CGMCC No.13723.
2. a kind of selection of heparinase superior strain, it is characterised in that produced using nucleoprotamine and heparin reaction Precipitation is as indicant, and the heparin enzymatic lysis heparin reaction that microorganism produces makes precipitation disappear, transparent circle is produced around thalline, from And play a part of directly indicating heparinase source microbial strains, pass through secondary screening, 16S rDNA methods identification production heparinase bacterium Strain Raoultella SP.NX-TZ-3,15；
Specifically include following steps：
Step one, the enrichment culture of heparinase producing strains：The sample that 1g is gathered adds the examination for filling 10mL sterile salines After being shaken up in pipe, 5mL suspension is taken, or directly takes 5mL water samples, is added in the triangular flask for filling 25mL enriched mediums, 200r/ Min, 37 DEG C of Shaking cultures 2 days；
Step 2, the primary dcreening operation of heparinase producing strains：By the bacterium solution of enriched culture with sterile saline gradient dilution to properly Dilution factor, take appropriate bacterium solution to be coated on primary dcreening operation flat board, cultivate 10~12h, treat that the bacterium colony on flat board is grown, use distilled water Bacterium colony is washed away, 2% fish-egg protamine solution is added, 1h is placed in 37 DEG C, fish-egg protamine solution is outwelled, is washed with distillation Only, 12h is placed in room temperature, strong point is born if any transparent circle appearance in opportunistic pathogen, you can the preliminary bacterial strain for regarding as producing heparinase, choosing Select and cultivated in the transparent circle bacterial strain transfer seed culture medium larger with colony diameter ratio；
Step 3, the secondary screening of heparinase producing strains：There is the bacterium colony access seed culture medium of transparent circle on picking primary dcreening operation flat board, 200r/min, 37 DEG C, Shaking culture 24h, then it is transferred to secondary screening culture medium, 200r/min, 37 DEG C, shaking flask training by 5% inoculum concentration 72h is supported, acellular crude extract is prepared, the superior strain of the vigor of enzyme, checking and identification heparinase is detected.
3. a kind of selection of heparinase superior strain as claimed in claim 2, it is characterised in that：Richness described in step one It is (%) to collect culture medium composition：Tryptone 1.0, liquaemin 0.5, NaCl 0.5, pH 6.5；Primary dcreening operation culture medium is constituted (%)：Tryptone 1.0, (NH4)2SO4 0.1、KH2PO40.25；MgSO4·7H2O 0.05, liquaemin 0.2, pH 6.5；Kind Sub- culture medium composition is (%)：Tryptone 1.0, (NH4)2SO4 0.1、KH2PO4 0.25、MgSO4·7H2O 0.05, heparin Sodium 0.2, pH 6.5；Primary dcreening operation culture medium composition is (%)：Yeast leaches cream 0.3, tryptone 1.0, NaCl 0.5, liquaemin 0.2nd, agar 2.0, pH 7.0.
4. a kind of selection of heparinase superior strain as claimed in claim 2, it is characterised in that：Liver is produced described in step 3 The authentication method of plain enzyme bacterial strain thalli morphology is using leather blue decoration method：Using quick gram staining liquid, under 100 times of oil mirrors Observed, determine ne ar, arrangement and some result features, be defined as gram-positive bacteria or Gram-negative bacteria.
5. a kind of selection of heparinase superior strain as claimed in claim 2, it is characterised in that：Heparin described in step 3 The Producing Strain authentication method of enzyme is to produce heparinase bacterial strain with 16S rDNA identifications, and method is as follows：1. 16S rDNA genomic DNAs Extract and electrophoresis detection：Production is extracted using Omega D3350-00E.Z.N.A.TM bacterial genomes DNA extraction kits thin The DNA of rhzomorph lactic acid bacteria, and genome carries out agarose electrophoresis detection and OD values are detected to extracting, to determine to reach needs Concentration and purity；2. the PCR amplifications of 16S rDNA genes；3. 16S rDNA sequencings and heparinase superior strain kind are true It is fixed：React that the 16S rDNA genomes that amplify are detected through agarose electrophoresis, PCR primer is reclaimed through PCR, the connection of T- carriers, gram After grand, Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's sequencing is sent to, sequencing result is inputted into ncbi database http:Retrieved in //blast.ncbi.nlm.nih.gov/Blastn, the 16s rDNA sequences included with NCBI are compared Right, according to the result of sequence alignment, selection and 20 plants of higher strain sequences of production heparinase bacterial strain homology utilize biological information The structure that software (Mega 4.0) 20 plants of production heparinase bacterial strains by more than carry out phylogenetic tree is learned, the kind of bacterial strain is further determined that Category.
6. a kind of selection of heparinase superior strain as claimed in claim 2, it is characterised in that：Prepared described in step 3 Acellular crude extract, is that bacterium secondary screening medium centrifugal (6000rpm, 15min) is collected into thalline, uses 30mL pH 7.0CaCl2- Tris-HCl buffer solutions are washed, and are centrifuged (6000rpm, 15min), are repeated twice, by every milliliter of 0.2g wet thallus Ratio adds pH7.0CaCl2- Tris-HCl buffer solutions, ultrasonic disruption 10 times in ice bath, each 5s is spaced 5s, will be broken Liquid refrigerated centrifuge, 8000rm, 20min, supernatant are acellular crude extract.
7. a kind of selection of heparinase superior strain as claimed in claim 6, it is characterised in that：The CaCl2-Tris- HCl buffer methods：Take 5mL 10mM/L CaCl2Mixed with 25mL1M Tris-HCl buffer solutions, be settled to 1000mL, PH is adjusted to 7.0, it is stand-by；(1. 1M HCL solution：8.5mL HCl constant volumes in 100mL volumetric flasks are measured (in fume hood to enter OK)；2. 1M Tris solution：12.2g Tris are weighed, it is settled to 100mL after being completely dissolved with distilled water；③10mM/L CaCl2：Weigh the anhydrous CaCl of 0.11099g2, it is completely dissolved with distilled water, 100mL is settled to；4. 1M Tris-HCl are buffered Liquid：45.7mL1M HCL solution is taken, is mixed with 50mL 1M Tris solution stand-by.
8. a kind of selection of heparinase superior strain as claimed in claim 2, it is characterised in that：Detected described in step 3 The method of enzyme activity is to take the 250 acellular crude extracts of μ L, adds 50 μ L 4% liquaemin (150UI/mg) and 200 μ L 0.2mol/L pH 7.0CaCl2- Tris-HCl buffer solutions, 37 DEG C of water-bath insulations, survey enzyme activity, take out 5 μ L reaction solutions, add 5mL 0.002% reddish black solution A, surveys A505, determine the concentration of undegraded heparin in reaction solution to draw heparin according to standard curve Degradation amount, the enzyme amount that enzyme-activity unit is defined as degrading per hour under conditions of 37 DEG C of pH7.0 needed for l mg heparin is a list Position；
Calculate：X=A × 10-3×0.4×106×1/T
Heparin enzyme activity in X-sample, U/L；
The amount of the heparin of degraded, μ L in A-unit interval；
The time of T-reagentia, h；
0.4×106- extension rate.
9. a kind of selection of heparinase superior strain as claimed in claim 8, it is characterised in that：Described standard curve Formulating method：A certain amount of liquaemin is accurately weighed, 0.8% heparin sodium aqua is configured to distilled water, difference is carried out The dilution of gradient, be made into 0.1~0.8% heparin solution；9 test tubes are taken, one adds 5 μ L distilled water and makees blank control, its Its test tube sequentially adds 5 μ L 0.1%-0.8% heparin sodium standard solution；Finally, every test tube adds 5mL 0.002% day Blue or green solution A, is mixed after surveying A on ELIASA505, every test tube does three parallel laboratory tests.
10. a kind of selection of heparinase superior strain as claimed in claim 8, it is characterised in that：The survey enzyme activity Time, it should stop from being put into 37 DEG C of water-bath insulations every a 30min enzyme activity of survey after 1.5h, with 1.5h during calculating enzyme activity Time is unit to calculate.
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CN108823139B (en) * 2018-07-27 2021-02-26 深圳大学 Escherichia coli for producing heparinase and construction method and application thereof
CN111218466A (en) * 2019-11-25 2020-06-02 深圳大学 Fusion gene MBP-H1 for expressing heparinase and recombinant plasmid and application thereof

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