SU1477742A1 - Method of extracting endonuclease from serratia marcescens culture fluid - Google Patents
Method of extracting endonuclease from serratia marcescens culture fluid Download PDFInfo
- Publication number
- SU1477742A1 SU1477742A1 SU874306951A SU4306951A SU1477742A1 SU 1477742 A1 SU1477742 A1 SU 1477742A1 SU 874306951 A SU874306951 A SU 874306951A SU 4306951 A SU4306951 A SU 4306951A SU 1477742 A1 SU1477742 A1 SU 1477742A1
- Authority
- SU
- USSR - Soviet Union
- Prior art keywords
- endonuclease
- culture fluid
- enzyme
- serratia marcescens
- extracting
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 11
- 239000012531 culture fluid Substances 0.000 title claims abstract description 8
- 102000004533 Endonucleases Human genes 0.000 title claims abstract description 6
- 108010042407 Endonucleases Proteins 0.000 title claims abstract description 6
- 241000607715 Serratia marcescens Species 0.000 title claims abstract 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 10
- 239000011780 sodium chloride Substances 0.000 claims abstract description 5
- 238000001179 sorption measurement Methods 0.000 claims abstract description 5
- 238000003795 desorption Methods 0.000 claims abstract 3
- 239000000872 buffer Substances 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 238000004255 ion exchange chromatography Methods 0.000 claims 3
- 238000000746 purification Methods 0.000 claims 1
- 238000004904 shortening Methods 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 12
- 108090000790 Enzymes Proteins 0.000 abstract description 12
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 6
- 239000007788 liquid Substances 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 abstract 2
- 108010077544 Chromatin Proteins 0.000 abstract 1
- 241000257303 Hymenoptera Species 0.000 abstract 1
- 108091034117 Oligonucleotide Proteins 0.000 abstract 1
- 206010033799 Paralysis Diseases 0.000 abstract 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 abstract 1
- 238000005341 cation exchange Methods 0.000 abstract 1
- 210000003483 chromatin Anatomy 0.000 abstract 1
- 238000004587 chromatography analysis Methods 0.000 abstract 1
- 230000008876 conformational transition Effects 0.000 abstract 1
- 125000002228 disulfide group Chemical group 0.000 abstract 1
- 230000003301 hydrolyzing effect Effects 0.000 abstract 1
- 108020004707 nucleic acids Proteins 0.000 abstract 1
- 102000039446 nucleic acids Human genes 0.000 abstract 1
- 150000007523 nucleic acids Chemical class 0.000 abstract 1
- 235000017557 sodium bicarbonate Nutrition 0.000 abstract 1
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract 1
- 230000003612 virological effect Effects 0.000 abstract 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 238000010828 elution Methods 0.000 description 2
- 239000007974 sodium acetate buffer Substances 0.000 description 2
- 239000002594 sorbent Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Эндонуклеаза, выдел ема из культуральной жидкости SERRATIA MARCESCENS, гидролизующа нуклеиновые кислоты до ди-, три-, тетра-, и олигонуклеотидов, примен етс дл лечени и профилактики вирусного паралича пчел, дл изучени структуры хроматина, может служить моделью дл исследовани вли ни одновременного наличи сульфгидрильных и дисульфидных групп на конформационные переходы молекул. Цель изобретени - упрощение и ускорение процесса. Способ выделени эндонуклеазы из культуральной жидкости S.MARCESCENS включает катионообменную хроматографию на бикарбе с сорбцией фермента при рН 5,0-5,5 и десорбцией при рН 8,0-8,5 в присутствии 0,35-0,40 М хлористого натри . Способ позвол ет за 40-45 ч из 100 л культуральной жидкости выделить 70-110 г препарата, содержащего 7-14 г белка без уменьшени степени чистоты препарата с сокращением времени выделени фермента в 8-10 раз и упрощением процесса по сравнению с известным. 1 з.п. ф-лы.Endonuclease secreted from SERRATIA MARCESCENS culture fluid, hydrolyzing nucleic acids to di-, tri-, tetra-, and oligonucleotides, is used to treat and prevent viral paralysis of bees to study the structure of chromatin, can be used as a model for studying the effect of simultaneous sulfhydryl and disulfide groups on conformational transitions of molecules. The purpose of the invention is to simplify and speed up the process. The method for isolating the endonuclease from the S.MARCESCENS culture liquid includes cation-exchange bicarb chromatography with sorption of the enzyme at pH 5.0-5.5 and desorption at pH 8.0-8.5 in the presence of 0.35-0.40 M sodium chloride. The method allows for 40-45 hours from 100 l of culture liquid to extract 70-110 g of the preparation containing 7-14 g of protein without reducing the purity of the preparation with reducing the enzyme release time by 8-10 times and simplifying the process as compared to the known. 1 hp f-ly.
Description
30 л/ч) при соотношении белок:сорбент не более 700 ед. Agjp : 1 см3 био- карба. По завершении сорбции колонку промывают 25,5 л (15-кратный объем) - 0,05 М трис-HCl буфера, рН 8,5. За- . тем элюируют фермент 8,5 л (5-кратный объем) 0,10 М трис-HCt буфера рН 8,5, содержащего 0,40 И хлористого натри . Полученный элюат высушивают при дробном режиме. При этом выход фермента составл ет 65-70%, активность не менее 30000 ед.акт./мг белка. Из 100 л культуральной жидкости удаетс выделить более 100 г препарата, содержащего более.10% белка.30 l / h) with a protein: sorbent ratio of not more than 700 units. Agjp: 1 cm3 bio-carba. Upon completion of the sorption, the column is washed with 25.5 l (15-fold volume) - 0.05 M Tris-HCl buffer, pH 8.5. Behind- . The enzyme 8.5 eluted (5-fold volume), 0.10 M Tris-HCt buffer pH 8.5, containing 0.40 AND sodium chloride. The resulting eluate is dried in fractional mode. The yield of the enzyme is 65-70%, the activity is not less than 30,000 units of act. / Mg of protein. More than 100 g of a preparation containing more than 10% protein can be isolated from 100 liters of culture liquid.
Пример 2. Биокарб Д-241 уравновешивают 0,10 М натрий-ацетат- ным буфером, рН 5,0 и помещают в колонку как в примере 1. Культураль- ную жидкость подкисл ют концентрированной уксусной кислотой до рН 5,0 и помещают на колонку как в примере 1. Сорбци эндонуклеазы составл ет 90-95%. Промывку колонки и элюцию фермента провод т как в примере 1. При этом выход фермента составл ет 60-65%, активность около 25000 ед.акт./ /мг белка.Example 2. Biocarb D-241 is equilibrated with 0.10 M sodium acetate buffer, pH 5.0 and placed in a column as in Example 1. The culture fluid is acidified with concentrated acetic acid to pH 5.0 and placed on a column. as in Example 1. The sorption of the endonuclease is 90-95%. Column washing and elution of the enzyme is carried out as in Example 1. The yield of the enzyme is 60-65%, the activity is about 25,000 units of act. / Mg of protein.
П р и м е р 3, Биокарб Д-24 уравновешивают 0,10 М натрий-ацетатным буфером, рН 5,5 и помещают в колонку как в примере 1. Культуральную жидкость подкисл ют концентрированной уксусной кислотой до рН 5,5 и помещают на колонку как в примере 1. Сорбци эндонуклеазы составл ет 40-87%. Промывку колонки и элюцию фермента провод т как в примере 1. При этом выход фермента составл ет 35-65%, активность около 30000 ед„акт./мгExample 3, Biocarb D-24 equilibrated with 0.10 M sodium acetate buffer, pH 5.5, and placed in a column as in Example 1. The culture fluid was acidified with concentrated acetic acid to pH 5.5 and placed on a column as in Example 1. The endonuclease sorption is 40-87%. The column was washed and the elution of the enzyme was carried out as in Example 1. The yield of the enzyme is 35-65%, the activity is about 30,000 units „act./mg.
t белка.t protein.
Пример 4. Биокарб Д-24 уравновешивают и помещают в колонку как в примере 1. Культуральную жидкость нанос т на колонку как в примере 1.Example 4. Biocarb D-24 is equilibrated and placed in a column as in Example 1. The culture fluid is applied to the column as in Example 1.
Промывку колонки провод т как в примере 1. Фермент элюируют 0,1 М трис-HCl буфером, рН 8,0, содержащим 0,35 М хлористого натри . При этом выход фермента составл ет 60-65%, активность.более 30000 ед.акт./мг белка.The column was washed as in Example 1. The enzyme was eluted with 0.1 M Tris-HCl buffer, pH 8.0, containing 0.35 M sodium chloride. At the same time, the yield of the enzyme is 60-65%, the activity. More than 30,000 units of act. / Mg of protein.
Пример 5. Биокарб Д-24Example 5. Biocarb D-24
уравновешивают и помещают а колонку как в примере 1. Культуральную жидкость нанос т на колонку как в примере 1. Промывку колонки провод т как в примере 1. Фермент элюируют 0,1 Мbalance and place the column as in Example 1. The culture fluid is applied to the column as in Example 1. The column is washed as in Example 1. The enzyme is eluted with 0.1 M
трис-HCl буфером рН 8,5, содержащим 0,35 М хлористого натри . При этом выход фермента составл ет 65-70%. активность более 30000 ед.акт./мг белка,Tris-HCl buffer pH 8.5, containing 0.35 M sodium chloride. The yield of the enzyme is 65-70%. activity of more than 30,000 units of act. / mg of protein,
При осуществлении способа в услови х , отличающихс от описанных, выход целевого продукта снижаетс .By implementing the method under conditions different from those described, the yield of the desired product is reduced.
Реализаци способа обеспечивает упрощение и ускорение процесса.The implementation of the method provides a simplified and accelerated process.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SU874306951A SU1477742A1 (en) | 1987-06-26 | 1987-06-26 | Method of extracting endonuclease from serratia marcescens culture fluid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SU874306951A SU1477742A1 (en) | 1987-06-26 | 1987-06-26 | Method of extracting endonuclease from serratia marcescens culture fluid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| SU1477742A1 true SU1477742A1 (en) | 1989-05-07 |
Family
ID=21328080
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SU874306951A SU1477742A1 (en) | 1987-06-26 | 1987-06-26 | Method of extracting endonuclease from serratia marcescens culture fluid |
Country Status (1)
| Country | Link |
|---|---|
| SU (1) | SU1477742A1 (en) |
-
1987
- 1987-06-26 SU SU874306951A patent/SU1477742A1/en active
Non-Patent Citations (1)
| Title |
|---|
| Yonemura К., Matsumoto К., Maeda H. Isolation and Characterization of Nuclease from Clinical Isolate of Serratia marcescens. - J. Biochem., 1983, v. 93, № 5, P. 1287. Авторское свидетельство СССР N° 537512, кл. С 12 N 9/10, 1972. Изобретение относитс к биотехнологии и касаетс получени ферментов, в частности внеклеточной бактериальной эндонуклеазы. Целью изобретени вл етс упрощение и ускорение процесса. Способ выделени эндонуклеазы из культуральной жидкости S.marcescens основан на том, что катионообменную хроматографию провод т на катионо- обменнике. Биокарб с сорбцией * |
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