HEPARINASES OBTAINED FROM SPHINGOBACTERIUM DAEJEONENSE, PREPARATION THEREFOR AND APPLICATION THEREOF

Abstract

Heparinases SDhep I and SDhep II obtained from a bacterium Sphingobacterium daejeonense are heparin enzymes that have not been reported. The enzymes were obtained by steps of bacterium fermentation, crude enzyme extraction, multi-step column chromatography and so on. A study in properties showed that the two enzymes are specific for enzymolysis of heparin and are expected to be used in low molecular weight heparins preparation or heparin quality testing.

Claims

1. A heparinases SDhep I and SDhep II obtained from Sphingobacterium daejeonense.

2. The heparinases SDhep I and SDhep II according to claim 1, wherein the molecular weights of heparinase SDhep I and SDhep II are 74692 Da and 94716 Da, respectively.

3. The heparinases SDhep I and SDhep II according to claim 1, wherein the isoelectric points of the heparinases SDhep I and SDhep II are 5.64 and 5.76, respectively.

4. A method for obtaining heparinases SDhep I and SDhep II from Sphingobacterium daejeonense bacterium, comprising the following steps: (1) the Sphingobacterium daejeonense is inoculated on the slant medium; (2) the strain obtained in step (1) is inoculated into the seed culture medium and cultured for 1-2 days, inoculated into a secondary liquid seed medium and cultured for 1-2 days, and then inoculated into 2 L fermentation medium and cultured for 1-5 days, thereafter cells are collected; (3) the cells obtained in step (2) are suspended in Tris-HCl buffer, disrupted by a high pressure homogenizer, then centrifuged for a supernatant and subjected to ammonium sulfate precipitation in an ice bath to collect a precipitated component with 35%-85% saturation, and then the precipitate was dissolved in Tris-HCl buffer and dialyzed to obtain an enzyme solution; (4) the enzyme solution obtained in step (3) is loaded onto a Q column pre-equilibrated with a Tris-HCl buffer, then equilibrated with the same buffer and eluted with a linear gradient of a 0-0.5 M NaCl in the same buffer; wherein the fraction with enzyme activity collected from the effluent and equilibrate solution is heparinase SDhep I, which is not bound to the Q column; and wherein the fraction with heparinase activity collected from the eluate is heparinase SDhep II, which is bound to the Q column, then the fraction with heparinase activity collected from the eluate is combined and dialyzed; (5) the SDhep I fraction obtained in step (4) is loaded onto the CS column pre-equilibrated with a Tris-HCl buffer, then equilibrated with the same buffer and then eluted with a linear gradient of 0-0.5 M NaCl in the same buffer; the fractions with heparinase activity are collected, dialyzed and loaded onto the SP column pre-equilibrated with Tris-HCl buffer, then equilibrated with the same buffer and then equilibrated with Tris-HCl buffer with a linear gradient of 0-0.5 M NaCl; the fractions are collected and concentrated to 0.2 mL by an ultrafiltration centrifuge tube intercepting molecular weight of 30 kD; the concentrated enzyme is loaded onto a Sephadex G-100 column pre-equilibrated with a Tris-HCl buffer and then eluted with the same buffer; the fractions with activity are collected, combined and concentrated to obtain purified SDhep I; (6) SDhep II obtained in step (4) is loaded on a CS column pre-equilibrated with Tris-HCl buffer, then equilibrated with the same buffer, and then eluted with a linear gradient 0.1-0.6 M NaCl in the same buffer; the fractions with heparinase activity in eluent solution are tested, collected, combined and dialyzed; the enzyme solution is loaded onto a Q-column pre-equilibrated with Tris-HCl buffer, and eluted with a linear gradient of 0.08 M NaCl in the same buffer; the fractions are tested, collected and loaded onto a CS column pre-equilibrated with a Tris-HCl buffer, then equilibrated with the same buffer and then eluted with a linear gradient of 0-1 M NaCl in the same buffer; the fractions with activity are collected, combined, and concentrated to obtain purified SDhep II.

5. The method for obtaining heparinases SDhep I and SDhep II from Sphingobacterium daejeonense according to claim 4, wherein the Tris-HCl buffer for the steps (3), (4), (5), and (6) is CaCl.sub.2-containing Tris-HCl, pH 6.5-8.0, wherein Tris-HCl concentration is 10-50 mM and CaCl.sub.2 concentration is 1-50 mM.

6. The method for obtaining heparinases SDhep I and SDhep II from Sphingobacterium daejeonense according to claim 4, wherein the Q-column described in each of steps (4) and (6) is Q-Sepharose Fast Flow strong anions-exchange column.

7. The method for obtaining heparinases SDhep I and SDhep II from Sphingobacterium daejeonense according to claim 4, wherein the CS column described in each of steps (5) and (6) is Cellufine Sulfate affinity column.

8. The method for obtaining heparinases SDhep I and SDhep II from Sphingobacterium daejeonense according to claim 4, wherein the SP column described in each of steps (4) and (6) is SP-Sepharose Fast Flow strong cation-exchange column.

9. The heparinases SDhep I and SDhep II according to claim 1, used for quality test of heparin, low molecular weight heparins and ultra-low molecular weight heparins.

10. The heparinases SDhep I and SDhep II according to claim 1, used for production of low molecular weight heparins and ultra-low molecular weight heparins.

Description

DESCRIPTION OF THE FIGURES

[0027] FIG. 1. SDS-PAGE maps of SDhep I and SDhep II enzymes.

[0028] FIG. 2. SAX-HPLC map of the product from enzymolysis of HEP by SDhep I.

[0029] FIG. 3. SAX-HPLC map of the product from enzymolysis of HEP by SDhep II

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention is further illustrated by the following examples, but is not to be construed as limiting the present invention.

Example 1: Preparation of SDhep I

[0031] a) Sphingobacterium daejeonense Strain Fermentation and Crude Enzyme Solution Preparation:
Bacterium bodies were taken twice from a plate or a slope with an inoculating loop and inoculated into 50 mL seed medium, shake-cultured at 30 C. and 150 rpm for 12 hours, and then inoculated into 200 mL secondary liquid seed medium with 10% inoculum mount and shake-cultured at 30 C. and 150 rpm for 24 hours and then fed into 2 L of the fermentation medium with 10% inoculum amount and shake-cultured at 30 C. and 150 rpm for 24 hours. 1 L of bacteria solution was taken out and centrifuged at 10,000 rpm for 30 min at 4 C. The precipitate was collected and suspended in 100 mL of 25 mM Tris-HCl buffer solution (containing 10 mM CaCl.sub.2, pH 7.0) and disrupted in three cycles using a high pressure homogenizer at 4 C. and 800 bar, then centrifuged at 10,000 rpm for 30 min at 4 C., the supernatant being crude enzyme solution. Ammonium sulfate precipitation was carried out to collect precipitates with 35% to 85% of ammonium sulfate saturation. The precipitate was dissolved in 100 mL of Tris-HCl buffer and dialyzed overnight in the same buffer.

b) Q Column Separation:

[0032] The enzyme solution obtained in step a) was loaded onto a Q column with a size of 2.530 cm pre-equilibrated with 25 mM Tris-HCl (containing 10 mM CaCl.sub.2, pH 7.0) buffer and then equilibrated with 3 column volumes in the same buffer. Loading effluent and equilibration effluent was examined for heparinase activity, and fractions with activity were collected and combined.

c) CS Column Purification:

[0033] The loading effluent and equilibration solution collected in step b) were loaded onto a CS column with a size of 2.530 cm pre-equilibrated with 25 mM Tris-HCl (containing 10 mM CaCl.sub.2, pH 7.0) buffer. The column was equilibrated with 3 column volumes in the same buffer and then eluted with a linear gradient of 0-1 M NaCl in the buffer. The active fractions were collected and dialyzed in 2 L of 25 mM Tris-HCl containing 10 mM CaCl.sub.2, pH 7.0 buffer overnight.

d) SP Column Purification:

[0034] The dialyzed enzyme solution obtained in step c) was loaded onto a SP column with a size of 2.530 cm pre-equilibrated with 25 mM Tris-HCl (containing 10 mM CaCl.sub.2, pH 7.0) buffer, then equilibrated with 3 column volumes in the same buffer and then eluted with a linear gradient of 0-0.5 M NaCl in the buffer. The active fractions were collected and concentrated to 200 L with ultrafiltration centrifuge tubes over 30 KD.

e) Sephadex G100 Column Purification:

[0035] The concentrated sample obtained in step d) was loaded onto a Sephadex G-100 column with a size of 1.0100 cm pre-equilibrated with 25 mM Tris-HCl containing 10 mM CaCl.sub.2, pH 7.0 buffer, and then eluted with the same buffer solution, wherein a peristaltic pump was used to control a flow rate of about 2 mL/h. The fractions were collected into tubes, 1 mL per tube, and determined the activity of the fractions. The fractions with enzyme activity were collected and concentrated to 200 L by 30 KD ultrafiltration centrifuge tub, and placed in a 1.5 mL EP tube. 300 L of a buffer of 25 mM Tris-HCl (containing 10 mM CaCl.sub.2, pH 7.0) was added and then 500 L of glycerol were added to a volume of 1 mL. The enzyme activity and protein content for each step of the purification procedure are shown in the following table.

TABLE-US-00001 TABLE 1 Enzyme activity and protein content for each step in SDhep I purification HEP Total Total enzyme Total Specific SDhep I purification volume protein activity activity activity purification steps (mL) (mg) (IU/mL) (IU) (IU/mg) multiple Yield (%) Crude enzyme 150 76.65 2.41 361.5 4.72 / / Thiamine 135 37.80 2.60 351.0 9.29 1.97 97.10 precipitation Q column 135 12.82 1.22 164.7 12.85 2.72 45.56 CS column 75 2.47 1.38 103.5 41.90 8.88 28.63 SP column 62 0.81 1.03 63.9 78.84 16.70 17.67 Sephadex G-100 1 0.278 50.6 50.6 182.01 38.56 14.00

Example 2: Study on the Properties of SDhep I

[0036] The molecular weight of SDhep I was about 74700 Da by SDS-PAGE, as shown in FIG. 1, and its exact molecular weight was 74692 Da by MALDI-TOF-MS mass spectrometry analysis.

[0037] The isoelectric point of SDhep I was 5.64 by isoelectric focusing electrophoresis.

[0038] Initial enzyme reaction rates were determined under various substrate concentrations to obtain Michaelis constant, the Michaelis constant of the SDhep I were 0.5738 and 0.0418 respectively as HEP or HS being the substrate.

[0039] Activity of heparinase SDhep I was determined as HEP or HS being the substrate respectively, and substrate pH being 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 respectively. The results showed that pH range with enzyme activity was 6.5-9.5 as HEP being the substrate, and the optimum pH was 8.0. As HS being the substrate, pH range with enzyme activity was 5.5-9.0, and the optimum pH was 8.0.

[0040] Activity of heparinase SDhep I was determined as HEP or HS being the substrate respectively, and substrate temperatures being 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 C., respectively. The results showed that the optimal temperature was 47 C. as HEP being the substrate, and the optimum temperature was 45 C. as HS being the substrate.

[0041] Effects of metal ion species on SDhep I: effect on SDhep I of adding 10 mM Mg.sup.2+, Mn.sup.2+ and Ca.sup.2+, Na.sup.+, K.sup.+, Mg.sup.2+, Cu.sup.2+, Fe.sup.2+, Fe.sup.3+, or Mn.sup.2+ respectively in the substrate HEP or HS, with 25 mM Tris-HCl (pH7.0) as a buffer, was tested, taking no addition of any metal ions as a blank control group. The results showed that K.sup.+, Mg.sup.2+, Na.sup.+, Mn.sup.2+ and Ca.sup.2+ improved enzyme activity, wherein Ca.sup.2+ had the best effects on the improvement of enzyme activity, largely promoted enzyme activity. Mg.sup.2+ and Na.sup.+ had the obviously improvement for enzyme activities. However, Cu.sup.2+, Fe.sup.2+ and Fe.sup.3+ had an inhibitory effect, which inactivated the enzyme.

[0042] Effects of metal ion concentrations on SDhep I: the effect of Mg.sup.2+, Na.sup.+ or Ca.sup.2+ at 0, 1, 10, 50, 100, 500 and 1000 mM, respectively on the enzyme activity was examined, as HEP or HS being the substrate. The results showed that the optimum concentration of Mg.sup.2+ for improving the activity of SDhep I was 100 mM as HEP or HS being the substrate. As HEP being the substrate, the optimum concentration of Na.sup.+ was 500 mM. As HS being the substrate, the optimum concentration of Na.sup.+ for SDhep I was 50 mM. The optimum concentration of Ca.sup.2+ was 100 mM as HEP or HS being the substrate.

[0043] Effects of denaturants on SDhep I: effect of denaturants on heparinase SDhep I activity was examined taking H.sub.2O.sub.2, acetonitrile, SDS, guanidine hydrochloride or urea as a denaturant respectively, the respective concentrations being 1 mM and 10 mM for H.sub.2O.sub.2, 1% and 10% for acetonitrile, 1% SDS, 1 M guanidine hydrochloride and 1 M urea, and setting a blank control group without any denaturing agent. In the presence of the above denaturing agent, 1% and 10% acetonitrile, and 1 M urea had a little effect on SDhep I, while 1 mM and 10 mM H.sub.2O.sub.2, 1% SDS, and 1M guanidine hydrochloride had significant inhibitory effect on SDhep I.

Example 3: Study on Specificity of Products from Enzymolysis of HEP by SDhep I

[0044] a) study on specificity of SDhep I substrate: activity of SDhep I was determined taking heparin (HEP), heparan sulfate (HS), chondroitin sulfate (CS) or dermatan sulfate (DS) as the substrate respectively. It is found that there was no enzyme activity with CS or DS being the substrate; and there was enzyme activity with HEP or HS being the substrate, and their activity ratio was about HEP:HS=1:1.3. [0045] b) disaccharide composition after enzymolysis of HEP by SDhep I: 3 IU of SDhep I (with heparin being substrate) was added to 50 mg of heparin, volume being balanced to 500 L with 25 mM Tris-HCl buffer containing 10 mM CaCl.sub.2, pH 7.0, and hydrolyzed at 37 C. for 24 h, then placed in a 100 C. water bath for 5 min for inactivation. The sample was subjected to liquid phase analysis for disaccharide composition, and the results were shown in FIG. 2. The main products after hydrolysis of HEP by SDhep I were IVA, IVS, IIA, IIS and IS, in which IIA and IIS accounted for 15.35% and 12.2% and peak area of IS only accounted for 5.29%. There were numerous types of tetrasaccharides components in addition to the above several disaccharides.

Example 4: Preparation of SDhep II

[0046] a) bacteria fermentation and crude enzyme preparation were the same as in Example 1. [0047] b) the step of crude enzyme separation by Q column was the same as in Example 1. After equilibrated with buffer of 3 column volumes, the sample was eluted with a linear gradient of 0-0.5 M NaCl in the buffer. The eluent active fractions were collected and dialyzed overnight. [0048] c) CS column Purification: the enzyme solution obtained in step b) was loaded onto a CS column pre-equilibrated with 25 mM Tris-HCl buffer (containing 10 mM CaCl.sub.2, pH 7.0), then equilibrated with the same buffer of 3 column volumes, and then eluted with a linear gradient of 0-1 M NaCl in the buffer, and the active fractions were collected and dialyzed overnight with 2 L volume of 25 mM Tris-HCl buffer (containing 10 mM CaCl.sub.2, pH 7.0). [0049] d) Q column Purification: the enzyme solution obtained in step c) was loaded onto a Q column pre-equilibrated with 25 mM Tris-HCl buffer (containing 10 mM CaCl.sub.2, pH 7.0), and then eluted with 800 mL of 0.080 M NaCl in the same buffer. The eluent active fractions were collected. [0050] e) CS column Purification: an equal volume of buffer was added to the fractions with enzyme activity collected in step d) and the mixture was loaded onto CS column pre-equilibrated with 25 mM Tris-HCl (containing 10 mM CaCl2, pH 7.0), then equilibrated with the same buffer of 3 column volumes, and then eluted with a linear gradient of 0-1 M NaCl in the same buffer. The active fractions were collected and subjected to a SDS-PAGE. The active fractions showing single band in electrophoresis was collected and concentrated to 200 L with 30 KD ultrafiltration centrifuge tube. The concentration was taken out and placed into a 1.5 mL EP tube, adding 300 L of 25 mM Tris-HCl buffer (containing 10 mM CaCl.sub.2, pH 7.0), then adding 500 L glycerol and then balancing volume to 1 mL. The enzyme activity and protein content for each step of the purification procedure are shown in the following table.

TABLE-US-00002 TABLE 2 The enzyme activity and protein content in SDhep II purification Total HEP Puri- SDhep II vol- Total enzyme Total Specific fica- purification ume protein activity vitality Activity tion Yield step (mL) (mg) (IU/mL) (IU) (IU/mg) factor (%) Crude 150 76.65 2.41 361.5 4.72 / / enzyme Thiamine 135 37.80 2.60 351.0 9.29 1.97 97.10 precipitation Q column 54 10.69 2.82 152.2 14.25 3.02 42.12 CS column 120 3.96 0.82 98.4 24.85 5.26 27.22 Q column II 300 2.10 0.18 54.0 25.71 5.45 14.94 CS column 1 0.29 29.6 29.6 102.07 21.62 8.19 II

Example 5: Study on Properties of SDhep II

[0051] The results were shown in FIG. 1, the molecular weight of SDhep II was about 94700 Da by SDS-PAGE analysis, and its exact molecular weight was 94716 Da according to MALDI-TOF-MS mass spectrometry analysis.

[0052] The isoelectric point of SDhep II was 5.76 by isoelectric focusing electrophoresis analysis.

[0053] Initial enzyme reaction rates were determined under various substrate concentrations to obtain Michaelis constant. The Michaelisemi constant of the SDhep II was determined to be 0.0052 and 1.6618 respectively for HEP or HS being the substrate.

[0054] Activity of heparinase SDhep II was determined as HEP or HS being the substrate and substrate pH being 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 respectively. The results showed that the pH range with activity was 6.5-9.0 as HEP being the substrate, and the optimum pH was 8.0. As HS being the substrate, the pH range was 6.5-9.0, and the optimum pH was 8.0.

[0055] Activity of heparinase SDhep II was determined as HEP or HS being the substrate and substrate temperature being 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49 C., respectively. The results showed that the optimal temperature was 47 C. as HEP being the substrate, and the optimum temperature was 43 C. as HS being the substrate.

[0056] Effects of metal ion species on SDhep II: effects on enzyme SDhep II of adding 10 mM Mg.sup.2+, Mn.sup.2+ and Ca.sup.2+, Na.sup.+, K.sup.+, Mg.sup.2+, Cu.sup.2+, Fe.sup.2+, Fe.sup.3+, and Mn.sup.2+ respectively to substrate HEP or HS respectively were examined, with 25 mM Tris-HCl (pH7.0) as a buffer, taking no addition of any metal ions as a blank control group. The results showed that K.sup.+, Mg.sup.2+, Na.sup.+, Mn.sup.2+ and Ca.sup.2+ improved the enzyme activity, wherein Ca.sup.2+ had the best effects for the improvement of enzyme activity, largely promoted the enzyme activity. Mg.sup.2+ and Na.sup.+ had the obviously improvement for enzyme activities. However, Cu.sup.2+, Fe.sup.2+ and Fe.sup.3+ had an inhibitory effect, which inactivated the enzyme.

[0057] Effects of metal ion concentration on SDhep II: effect of Mg.sup.2+, Na.sup.+ or Ca.sup.2+ at 0, 1, 10, 50, 100, 500 and 1000 mM, respectively on enzyme SDhep II activity was examined, with HEP or HS being the substrate. The results showed that the optimum concentration of Mg.sup.2+ for improving the activity of SDhep II was 100 mM as HEP or HS being the substrate. As HEP or HS being the substrate, the optimum concentration of Na.sup.+ for improving the activity of SDhep II was 100 Mm and 50 mM. The optimum concentration of Ca.sup.2+ for improving the activity of SDhep II was 100 mM or 50 mM.

[0058] Effects of denaturants on SDhep II: effect of denaturants on heparinase SDhep II activity was examined taking H.sub.2O.sub.2, acetonitrile, SDS, guanidine hydrochloride or urea as a denaturant respectively, the respective concentrations being 1 mM and 10 mM for H.sub.2O.sub.2, 1% and 10% for acetonitrile, 1% SDS, 1 M guanidine hydrochloride and 1 M urea, and setting a blank control group without any denaturing agent. In the presence of the above denaturing agent, 1% and 10% acetonitrile, and 1 M urea had a little effect on SDhep II, while 1 mM and 10 mM H.sub.2O.sub.2, 1% SDS, and 1M guanidine hydrochloride had significant inhibitory effect on SDhep II.

Example 6: Study on Specificity of Products from Enzymolysis of HEP by SDhep II

[0059] a) study on specificity of SDhep II substrate: activity of SDhep II was determined taking heparin (HEP), heparan sulfate (HS), chondroitin sulfate (CS) or dermatan sulfate (DS) as the substrate respectively. The results showed that there was no enzyme activity with CS or DS being the substrate; and there was enzyme activity with HEP or HS being the substrate, and their activity ratio was about HEP:HS=1:3.5. [0060] b) disaccharide composition analysis after enzymolysis of HEP by SDhep II: 3 IU of SDhep II (with heparin being substrate) was added to 50 mg of heparin, volume being balanced to 500 L with 25 mM Tris-HCl buffer (containing 10 mM CaCl.sub.2, pH 7.0), hydrolyzed at 37 C. for 24 h, then placed in a 100 C. water bath for 5 min for inactivation. The sample was subjected to liquid phase analysis for disaccharide composition; the results were shown in FIG. 3. The main products after hydrolysis of HEP by SDhep II were IIS and IS, in which IS was of majority and accounted for 56.39%. Compared with SDhep I, enzymatic products of SDhep II was less and its enzymatic hydrolysis was more completely, very different from the products by SDhep I. Therefore, SDhep I and SDhep II were two heparin enzymes with very different enzymatic properties, which have potential application value in heparin quality detection and low molecular weight heparins preparation.