CN101701212A - Method for improving enzyme activity by utilizing atmospheric pressure discharge plasma - Google Patents
Method for improving enzyme activity by utilizing atmospheric pressure discharge plasma Download PDFInfo
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- CN101701212A CN101701212A CN200910237616A CN200910237616A CN101701212A CN 101701212 A CN101701212 A CN 101701212A CN 200910237616 A CN200910237616 A CN 200910237616A CN 200910237616 A CN200910237616 A CN 200910237616A CN 101701212 A CN101701212 A CN 101701212A
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- enzyme
- cold plasma
- enzyme solution
- atmospheric pressure
- discharge plasma
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 50
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 19
- 230000000694 effects Effects 0.000 title abstract description 9
- 230000005495 cold plasma Effects 0.000 claims abstract description 26
- 102000004882 Lipase Human genes 0.000 claims description 12
- 108090001060 Lipase Proteins 0.000 claims description 12
- 229910052751 metal Inorganic materials 0.000 claims description 11
- 239000002184 metal Substances 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 239000004367 Lipase Substances 0.000 claims description 10
- 235000019421 lipase Nutrition 0.000 claims description 10
- 239000007789 gas Substances 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- 239000001307 helium Substances 0.000 claims description 6
- 229910052734 helium Inorganic materials 0.000 claims description 6
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000007599 discharging Methods 0.000 claims description 4
- 229910001220 stainless steel Inorganic materials 0.000 claims description 4
- 239000010935 stainless steel Substances 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000005138 cryopreservation Methods 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 239000004568 cement Substances 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- -1 p-nitrophenyl cetylate Chemical compound 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 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 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 102000016559 DNA Primase Human genes 0.000 description 1
- 108010092681 DNA Primase Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003570 air Substances 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 235000019626 lipase activity Nutrition 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a method for improving the enzyme activity by utilizing an atmospheric pressure discharge plasma, belonging to the field of cold plasma technology application. The method is characterized by comprising the following steps in sequence: preparing enzyme solution, selecting the type of an atmospheric pressure discharge plasma generator and setting working parameters, and treating the enzyme solution by utilizing the atmospheric pressure discharge plasma. The invention has the advantages of high efficiency and convenient change of the enzyme activity.
Description
Technical field
The present invention relates to, the cold plasma technology relates in particular to the application of cold plasma in the control enzymatic activity in the application of microorganism field.
Background technology
Plasma body is the 4th attitude of material, and the system by particles such as a large amount of molecules, atom, ion and electronics are formed because positive corpusc(u)le and negative particle carried charge are equal, so presents electric neutrality on the macroscopic view.Cold plasma is a kind of nonequilibrium plasma, and active particle energy height can (less than 50 ℃) keep sufficiently high chemically reactive, thereby at biological chemical field boundless application prospect be arranged when low temperature.
Enzyme is a kind of have catalytic activity and highly narrow spectrum specific proteins, and environmental factors is very big to its stability and activity influence, and high temperature, high pressure, strong acid, highly basic or ultraviolet ray etc. all can make protein denaturation, thereby lose catalytic activity.Keep enzyme and live stablely, fundamental method is in the processing or catalytic process of enzyme, makes it be in optimum regime.Biologically improve method that enzyme lives normally by molecular biology method, the gene of codase is transformed or rite-directed mutagenesis at random, and then the enzyme of the different aminoacids sequence that gives expression to is screened, in the hope of obtaining the enzyme of high catalytic activity.
Enzyme is lived and is depended on its 3-D solid structure usually, and its intramolecular various chemical bonds and surface charge are very big to its space structure and even enzyme influence alive.Common some violent physical treatments (as ultraviolet ray, temperature, pH regulator or the like) process often makes enzyme denaturation, loss of activity.The present invention adopts this mild condition of atmospheric low-temperature plasma and the higher physical means of energy density that enzyme is handled, and can carry the enzyme of enzyme efficiently, easily and live.
Summary of the invention
The object of the present invention is to provide a kind of atmosphere cold plasma that utilizes to improve the method that enzyme is lived.This method has simple and convenient, characteristics of high efficiency.
The invention is characterized in: carry out according to the following steps successively:
Step (1) preparation enzyme solution: is the molecular weight of taking from candiyeast that the lipase of 5T KD is placed in the 10K molecular weight dialysis tubing, dialysis obtained stand-by enzyme solution, cryopreservation in-20 ℃～4 ℃ environment in 24 hours in the phosphoric acid buffer that be 7.0 at 50mM, PH, concentration is 4mg/mL;
Step (2) adopts the Atomospheric pressure glow discharge plasma producer of built-in two coaxial-type water-cooled bare metal electrodes, with helium as discharge gas, spacing between the described coaxial-type water-cooled bare metal electrode is 1mm～10mm, helium gas flow is 1slpm～50slpm, unit is the standard Liter Per Minute, the virtual value of impressed voltage is 0.1kV～100kV, and supply frequency is 0MHz～100MHz;
Step (3) is stated from the stainless steel slide glass to described stand-by enzyme solution, places the fluerics of described Atomospheric pressure glow discharge cold plasma generator, handles 10s～20min under 4 ℃ of envrionment temperatures.
The present invention has efficiently, convenient and advantage that can the primase sex change, and can reach the effect that improves lipase activity.
Description of drawings
Fig. 1. Atomospheric pressure glow discharge plasma producer treat enzyme synoptic diagram
Fig. 2. utilize atmosphere pressure discharging cold plasma generators to improve the active method flow diagram of enzyme
Embodiment
The step that realizes the inventive method is as follows:
1. enzyme solution is prepared
Enzyme is dissolved in the damping fluid (the damping fluid kind is selected according to the required condition of enzyme itself, crude enzyme liquid need through dialysis treatment).It is standby that stand-by enzyme liquid needs low temperature (20 ℃～4 ℃) to preserve.
2. select the kind of atmosphere pressure discharging cold plasma generators and set working parameter
The atmosphere cold plasma producer that the present invention uses is for adopting the Atomospheric pressure glow discharge cold plasma generator of two water-cooled bare metal electrodes. as required the atmosphere cold plasma generator also can adopt the single or multiple atmospheric dielectric barrier discharge cold plasma generators that all scribble the water-cooling metal electrode of insulation dielectric layer, adopt the atmospheric pressure resistive barrier discharge cold plasma generator of the single or multiple water-cooling metal electrodes that all scribble the very high resistive material of resistivity and adopt single needle-like metal electrode or the atmospheric pressure corona discharge cold plasma generator of needle-like metal electrod-array and single water-cooled flat metal electrode in a kind of replacement. When needing to produce the Atomospheric pressure glow discharge cold plasma, discharge gas can adopt a kind of in the mixed gas of helium, argon gas, nitrogen, oxygen, air and above-mentioned gas, water-cooled bare metal (plate or coaxial-type, material is copper, aluminium, stainless steel etc.) spacing between electrodes is (1～10) mm, airshed is (1～50) slpm (a standard Liter Per Minute), the impressed voltage virtual value is (0.1～100) kV, and supply frequency is (0～100) MHz.
3. atmosphere cold plasma producer treat enzyme
As shown in the figure, pending enzyme is stated from the stainless steel slide glass, places the fluerics of Atomospheric pressure glow discharge cold plasma generator to handle, the treatment time is between 10s～20min.Treating processes needs to carry out in 4 ℃ of environment.
4. the activity through enzyme after the Cement Composite Treated by Plasma detects
Use concentration and the activity of corresponding method measurement according to the characteristic of enzyme through enzyme after the Cement Composite Treated by Plasma.Handling back enzyme liquid to be detected needs to preserve in 4 ℃ of environment.
The experimentation of handling lipase below in conjunction with the Atomospheric pressure glow discharge cold plasma generator is described in detail specific implementation method of the present invention.
One. used material and preparation thereof:
Lipase (source: candiyeast, purchase company in Sigma), molecular weight needs to dialyse 24 hours in phosphoric acid buffer (50mmol/L, pH 7.0) with 10K molecular weight dialysis tubing before being about the 57KD use; Experimental concentration is about 4mg/mL.
Two. experimental procedure
1. preparation enzyme solution, about 10mL.
2. the working gas of setting the Atomospheric pressure glow discharge cold plasma generator as required is a helium, and working parameter is interelectrode distance 2mm, airshed 15slpm, power 120W, supply frequency 13.56MHz.
3. use the Atomospheric pressure glow discharge cold plasma generator that lipase is handled, the treatment time is respectively 0,10s, 20s, 30s, 40s, 50s.
4. the enzyme amount and the enzyme work of the lipase after the Atomospheric pressure glow discharge cold plasma generator being handled detect.
Detection method is as follows:
(1) enzyme mensuration alive
Adopt p-nitrophenyl cetylate method that the activity of lipase is measured.During measurement, add 50ul enzyme solution (enzyme concn is 50ug/mL) to substrate solution, substrate solution for Tris-HCl damping fluid preparation 0.5mmol/L to nitro cetylate (p-NPP).Use ultraviolet-visible spectrophotometer (Shimadzu Multispec-1501) to detect 1～3min under 348nm, the slope ratio of gained scanning curve is relative enzyme and lives.
(2) mensuration of enzyme amount (mensuration of protein concentration)
BCA method (test kit is purchased in green skies company).The concentration of testing protein should be diluted for about 0.5mg/mL
Lipase after contrast is handled through the Atomospheric pressure glow discharge cold plasma generator of different time with find with reference to lipase, lipase enzyme amount is through constant substantially after the Cement Composite Treated by Plasma (being 4.0mg/ml) before and after handling, but the enzyme work of lipase has obtained increasing significantly, the results are shown in Table 1, with handle before compare, handles the raising 21.8% of living of enzyme after 50 seconds.
Lipase enzyme after table 1 is handled through the different time atmosphere cold plasma is lived
| Time | ??0 | ??10s | ??20s | ??30s | ??40s | ??50s |
| Enzyme is lived relatively | ??1.000 | ??1.006 | ??1.067 | ??1.073 | ??1.101 | ??1.218 |
Claims (1)
1. utilize atmosphere pressure discharging cold plasma generators to improve the active method of enzyme, it is characterized in that, carry out according to the following steps successively:
Step (1) preparation enzyme solution: is the molecular weight of taking from candiyeast that the lipase of 5T KD is placed in the 10K molecular weight dialysis tubing, dialysis obtained stand-by enzyme solution, cryopreservation in-20 ℃～4 ℃ environment in 24 hours in the phosphoric acid buffer that be 7.0 at 50mM, PH, concentration is 4mg/mL;
Step (2) adopts the Atomospheric pressure glow discharge plasma producer of built-in two coaxial-type water-cooled bare metal electrodes, with helium as discharge gas, spacing between the described coaxial-type water-cooled bare metal electrode is 1mm～10mm, helium gas flow is 1slpm～50slpm, unit is the standard Liter Per Minute, the virtual value of impressed voltage is 0.1kV～100kV, and supply frequency is 0MHz～100MHz;
Step (3) is stated from the stainless steel slide glass to described stand-by enzyme solution, places the fluerics of described Atomospheric pressure glow discharge cold plasma generator, handles 10s～20min under 4 ℃ of envrionment temperatures.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910237616XA CN101701212B (en) | 2009-11-13 | 2009-11-13 | Method for improving enzyme activity by utilizing atmospheric pressure discharge plasma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910237616XA CN101701212B (en) | 2009-11-13 | 2009-11-13 | Method for improving enzyme activity by utilizing atmospheric pressure discharge plasma |
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| Publication Number | Publication Date |
|---|---|
| CN101701212A true CN101701212A (en) | 2010-05-05 |
| CN101701212B CN101701212B (en) | 2011-05-11 |
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|---|---|---|---|
| CN200910237616XA Expired - Fee Related CN101701212B (en) | 2009-11-13 | 2009-11-13 | Method for improving enzyme activity by utilizing atmospheric pressure discharge plasma |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754863A (en) * | 2016-12-26 | 2017-05-31 | 苏州大学 | Improve the dry processing method of xylanase activity |
| CN106893702A (en) * | 2016-12-26 | 2017-06-27 | 苏州大学 | Improve the Low Temperature Plasma Treating method of β Mannanase Activities |
| CN106929503A (en) * | 2016-12-26 | 2017-07-07 | 苏州大学 | The method for improving pectinase activity |
| CN107034526A (en) * | 2017-05-05 | 2017-08-11 | 盐城工业职业技术学院 | Cotton stalk handling process and cotton stalk bark fibre preparation technology |
| CN112689376A (en) * | 2021-03-15 | 2021-04-20 | 四川大学 | Microwave plasma jet excitation device adopting piezoelectric material |
| CN113367182A (en) * | 2021-05-25 | 2021-09-10 | 四川省食品发酵工业研究设计院有限公司 | Method for preserving and preserving cold fresh meat |
-
2009
- 2009-11-13 CN CN200910237616XA patent/CN101701212B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754863A (en) * | 2016-12-26 | 2017-05-31 | 苏州大学 | Improve the dry processing method of xylanase activity |
| CN106893702A (en) * | 2016-12-26 | 2017-06-27 | 苏州大学 | Improve the Low Temperature Plasma Treating method of β Mannanase Activities |
| CN106929503A (en) * | 2016-12-26 | 2017-07-07 | 苏州大学 | The method for improving pectinase activity |
| CN106754863B (en) * | 2016-12-26 | 2019-10-01 | 苏州大学 | Improve the dry processing method of xylanase activity |
| CN106893702B (en) * | 2016-12-26 | 2019-10-01 | 苏州大学 | Improve the active Low Temperature Plasma Treating method of 'beta '-mannase |
| CN106929503B (en) * | 2016-12-26 | 2020-02-18 | 苏州大学 | Method for improving pectinase activity |
| CN107034526A (en) * | 2017-05-05 | 2017-08-11 | 盐城工业职业技术学院 | Cotton stalk handling process and cotton stalk bark fibre preparation technology |
| CN112689376A (en) * | 2021-03-15 | 2021-04-20 | 四川大学 | Microwave plasma jet excitation device adopting piezoelectric material |
| CN113367182A (en) * | 2021-05-25 | 2021-09-10 | 四川省食品发酵工业研究设计院有限公司 | Method for preserving and preserving cold fresh meat |
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| Publication number | Publication date |
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| CN101701212B (en) | 2011-05-11 |
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Granted publication date: 20110511 |