CN103712934B - A kind of for monitor laccase conformation change exempt from mark ultraviolet absorption spectroscopy - Google Patents
A kind of for monitor laccase conformation change exempt from mark ultraviolet absorption spectroscopy Download PDFInfo
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- CN103712934B CN103712934B CN201410002215.7A CN201410002215A CN103712934B CN 103712934 B CN103712934 B CN 103712934B CN 201410002215 A CN201410002215 A CN 201410002215A CN 103712934 B CN103712934 B CN 103712934B
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- laccase
- urea
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- nitrine
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 60
- 230000008859 change Effects 0.000 title claims abstract description 31
- 238000004847 absorption spectroscopy Methods 0.000 title claims abstract description 14
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 44
- 239000004202 carbamide Substances 0.000 claims abstract description 44
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 238000000862 absorption spectrum Methods 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000010586 diagram Methods 0.000 claims abstract description 10
- 238000010521 absorption reaction Methods 0.000 claims description 7
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 238000004925 denaturation Methods 0.000 claims description 5
- 230000036425 denaturation Effects 0.000 claims description 5
- 239000008351 acetate buffer Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- CYWSDGUZWKUALI-UHFFFAOYSA-N 3-azidocoumarin Chemical class C1=CC=C2OC(=O)C(N=[N+]=[N-])=CC2=C1 CYWSDGUZWKUALI-UHFFFAOYSA-N 0.000 claims description 2
- 238000006736 Huisgen cycloaddition reaction Methods 0.000 claims description 2
- 125000002534 ethynyl group Chemical class [H]C#C* 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 abstract description 14
- 230000008569 process Effects 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 4
- 239000011259 mixed solution Substances 0.000 abstract description 4
- 238000002372 labelling Methods 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 229910052802 copper Inorganic materials 0.000 description 4
- 239000010949 copper Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- TZRXHJWUDPFEEY-UHFFFAOYSA-N Pentaerythritol Tetranitrate Chemical compound [O-][N+](=O)OCC(CO[N+]([O-])=O)(CO[N+]([O-])=O)CO[N+]([O-])=O TZRXHJWUDPFEEY-UHFFFAOYSA-N 0.000 description 3
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000001142 circular dichroism spectrum Methods 0.000 description 2
- 229910001431 copper ion Inorganic materials 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000233866 Fungi Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 238000004497 NIR spectroscopy Methods 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical class [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000006035 Tryptophane Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000002983 circular dichroism Methods 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910000474 mercury oxide Inorganic materials 0.000 description 1
- UKWHYYKOEPRTIC-UHFFFAOYSA-N mercury(ii) oxide Chemical compound [Hg]=O UKWHYYKOEPRTIC-UHFFFAOYSA-N 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 235000011091 sodium acetates Nutrition 0.000 description 1
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical class [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The invention discloses a kind of for monitor laccase conformation change exempt from mark ultraviolet absorption spectroscopy.First 3-nitrine-umbelliferone is joined in the urea liquid of nine kinds of variable concentrations together with laccase, after fully reacting, use ultraviolet spectrometer (UVS) 400 ~ 700 again? the absorption spectrum of the above-mentioned mixed solution containing 3-nitrine-umbelliferone, laccase and urea is gathered within the scope of nm, finally draw the ultra-violet absorption spectrum changing trend diagram of laccase under different urea concentration condition, just can monitor laccase conformation change according to this ultra-violet absorption spectrum changing trend diagram.The present invention only needs laccase, 3-nitrine-umbelliferone simply to mix in urea liquid in operation, without any need for labeling process just can reach the object of monitoring laccase conformation change, greatly reduce the complicacy of operation, decrease monitoring time.In addition, the present invention is used for achieving comparatively satisfied result to the monitoring of laccase conformation change, demonstrates efficient, single-minded, timely, simple advantage.
Description
Technical field
The invention belongs to analytical chemistry field, especially relate to a kind of for monitor laccase conformation change exempt from mark ultraviolet absorption spectroscopy.
Background technology
Laccase (EC 1.10.3.2) is mainly distributed in majority of plant and fungi.Because laccase can effective catalysis organic and mineral compound in a large number, be water by the hydrogen reduction of compound Middle molecule, such as phenolic compound and organic amine, be therefore widely used in the field such as synthesis and decolouring of immune detection, bio-sensing, dyestuff.In addition, laccase or a kind of many cupreins, comprise 4 copper ions, can be divided three classes: be respectively the I type copper (Cu1) being distributed in 3rd district, and be distributed in II type copper (Cu2) and the type III copper (Cu3a and Cu3b) of a district and three district's intersections.In laccase structure, these copper ions all combine with histidine and tryptophane, jointly as the catalytic center of laccase.
In physiological conditions, numerous protein all exists with naturally folding form itself, and wherein hydrogen bond plays very important effect in protein folding procedure.If protein is heated, or be exposed to acidity, alkaline environment, or with denaturant as the materials such as urea, guanidine hydrochloride and ethanol mix mutually time, the hydrogen bond in protein is all easily made to rupture, protein is caused to produce unfolding effect, cause protein conformation to change, such as three-dimensional structure is caved in, and becomes one dimension or two-dimensional structure.In order to understand the relation between protein function and its sequence, conformation better, researchers propose a lot of monitoring method such as, for the folding of Study on Protein and unfolding state and conformation change process etc., fluorescence method, circular dichroism detector, capillary electrophoresis, mass spectroscopy, electrochemical process, near infrared spectroscopy etc.Ultraviolet absorption spectroscopy is because cost is low, simple to operate, selectivity is high and be subject to extensive concern, but it is considerably less to adopt ultraviolet absorption spectroscopy to change the report of monitoring to protein conformation at present.Therefore, developing a kind of mark ultraviolet absorption spectroscopy of exempting from simply, efficiently goes the conformation change of monitoring protein to be very necessary.
summary of the invention:
The object of the present invention is to provide that a kind of method is simple, cost is low, specificity is good for monitor laccase conformation change exempt from mark ultraviolet absorption spectroscopy, the method only needs laccase, 3-nitrine-umbelliferone simply to mix in urea liquid in operation, without any need for labeling process, thus greatly reduce the complicacy of operation, decrease monitoring time.
The object of the invention is to realize in the following manner:
For monitor laccase conformation change exempt from mark a ultraviolet absorption spectroscopy, feature is: concrete steps are as follows:
(1) according to list of references (Sivakumar, K.; Xie, F.; Cash, B. M. et al., A Fluorogenic 1,3-Dipolar Cycloaddition Reaction of 3-Azidocoumarins and Acetylenes. Org. Lett., 2004,6,4603-4606.) shown in method, adopt 4-dihydroxy benzaldehyde, N-acetoglycocoll and sodium azide prepare 3-nitrine-umbelliferone;
(2) laccase of 5.0 μ L 10 mg/mL and 5.0 μ L 0.25 mM 3-nitrine-umbelliferones are added to nine kinds of variable concentrations respectively and are in the urea liquid of 190 μ L, the concrete concentration of nine kinds of urea liquids is respectively 0.0 M, 1.0 M, 2.0 M, 3.0 M, 4.0 M, 5.0 M, 6.0 M, 7.0 M, 8.0 M, 4
ocultivate 1.0 hours under C, stir after mixing, impel urea to produce denaturation to laccase, cause laccase conformation change;
(3) after step (2) reaction terminates, adopt ultraviolet spectrometer (UVS) within the scope of 400 ~ 700 nm, distinguish the absorption spectrum of nine kinds of reacted urea liquids in acquisition step (2);
(4) according to urea concentration and the uv absorption intensity of nine kinds of urea liquids, draw the ultra-violet absorption spectrum changing trend diagram of laccase under different urea concentration condition, just can monitor laccase conformation change according to this ultra-violet absorption spectrum changing trend diagram.
In step (2), urea liquid is dissolved in acetate buffer formulated by urea, and pH value is modulated to 6.0.
The azido group of 3-nitrine-umbelliferone is an electron donating group, causes the uv absorption of this compound more weak.When but 3-nitrine-umbelliferone mixes mutually with laccase, the histidine producing coordination in laccase with copper site is easy to be combined by hydrogen bond action with the nitrogen-atoms in azido group, form nitrine cumarin--this new complexes of laccase, this reduces the electron donation of azido group in 3-nitrine-umbelliferone, nitrine cumarin--laccase has the stronger ultraviolet absorption ability of generation.But laccase easily produces denaturation in urea liquid, the three-dimensional structure of laccase is destroyed, urea combines with histidine with stronger adhesion, destroy nitrine cumarin--the structure of laccase complex, mixed solution medium ultraviolet absorption intensity containing 3-nitrine-umbelliferone, laccase and urea in above-mentioned steps (2) is reduced gradually along with the increase of denaturation, reaches the object of monitoring laccase conformation change.
3-nitrine-umbelliferone joins in the urea liquid of nine kinds of variable concentrations by the present invention together with laccase, after fully reacting, use ultraviolet spectrometer (UVS) within the scope of 400 ~ 700 nm, gather the absorption spectrum of the above-mentioned mixed solution containing 3-nitrine-umbelliferone, laccase and urea again, finally draw the ultra-violet absorption spectrum changing trend diagram of laccase under different urea concentration condition, just can monitor laccase conformation change according to this ultra-violet absorption spectrum changing trend diagram.
Remarkable advantage of the present invention is:
(1) the present invention only needs laccase, 3-nitrine-umbelliferone simply to mix in urea liquid in operation, without any need for labeling process just can reach the object of monitoring laccase conformation change, greatly reduce the complicacy of operation, decrease monitoring time.
(2) the present invention is used for achieving comparatively satisfied result to the monitoring of laccase conformation change, demonstrates efficient, single-minded, timely, simple advantage.
(3) the present invention adopts simple ultraviolet spectrometer (UVS) image data, has easy and simple to handle, sensitive, that cost is low advantage.
accompanying drawing illustrates:
Fig. 1 is the principle schematic exempting to mark ultraviolet absorption spectroscopy for monitoring laccase conformation change of the present invention;
A in Fig. 2 is uv absorption spectra of the present invention, and urea concentration is respectively 0.0 M, 1.0 M, 2.0 M, 3.0 M, 4.0 M, 5.0 M, 6.0 M, 7.0 M, 8.0 M from a to i; B is the variation relation of wavelength between the absorption intensity and urea concentration at 493 nm places.The concentration of laccase is in 250 μ g/mL(steps (2) be 10 mg/mL, and be 5.0 μ L to containing in 5.0 μ L 3-nitrine-umbelliferones and 190 μ L urea liquids at the volume added, in conversion mixed solution, laccase concentration is 250 μ g/mL).
Fig. 3 is the unfolding rate adopting distinct methods to monitor laccase conformation change is the activity change of laccase in different urea concentration from the relation between laccase activity: a; B adopts mark ultraviolet absorption spectroscopy of exempting from of the present invention to the monitoring of laccase conformation change; C is for adopting traditional circular dichroism spectra (CD) method to the monitoring of laccase conformation change.
embodiment:
Contrast accompanying drawing below in conjunction with embodiment the present invention is described in further detail.
In order to understand content of the present invention better, further illustrating technical scheme of the present invention below by specific embodiment, specifically comprising synthesis, property testing etc.These embodiments, just to explanation of the present invention, do not limit the present invention.
For monitor laccase conformation change exempt from mark a ultraviolet absorption spectroscopy, concrete steps are as follows:
1. prepare 3-nitrine-7 hydroxy coumarin compound: take 1.38 g 2 respectively, in 4-4-dihydroxy benzaldehyde and 1.12 g N-acetoglycocoll to 50 mL anhydrous sodium acetates, and ultrasonic dissolution, then return stirring 4.0 hours, after having reacted, gained potpourri is poured in frozen water, obtain yellow mercury oxide, filtration extracting yellow precipitates, and with frozen water washing and precipitating thing, and then backflow 1 hour in the mixed liquor (2:1) of 30 mL hydrochloric acid and ethanol, after having reacted, adopt ice bath to be cooled by above-mentioned solution, and dilute above-mentioned solution with 20 mL frozen water; Add 20 mmol sodium nitrites, stir after 10 minutes, then add 30 mmol sodium azide, stir 20 minutes.After filtration, the brown solid obtained after drying under reduced pressure is 3-nitrine-7 hydroxy coumarin compound: (0.97 g, 47.8%).
1h NMR (DMSO, 400 MHz) δ 6.76 (d,
j=2.13 Hz, 1 H), 6.80 (dd,
j=8.47 Hz,
j=2.28 Hz, 1 H), 7.48 (d,
j=8.54 Hz, 1 H), 7.61 (s, 1 H), 5.23 (s, 1 H). IR (KBr, cm
-1): 3159 (s), 2117 (vs), 1688 (s), 1617 (m), 1310 (s). MS (EIS) m/z:calculated for C
9h
5n
3o
3:203.0, found 202.5 ([M]
-); 238.1 ([M+ Cl]
-).
2. first urea is dissolved in acetate buffer, the urea liquid of the preparation kind variable concentrations coldest days of the year end, the concrete concentration of nine kinds of urea liquids is respectively 0.0 M, 1.0 M, 2.0 M, 3.0 M, 4.0 M, 5.0 M, 6.0 M, 7.0 M, 8.0 M, and pH value is modulated to 6.0; Again the laccase of 5.0 μ L 10 mg/mL, 5.0 μ L 0.25 mM 3-nitrine-umbelliferones are added to above-mentioned nine kinds of variable concentrations respectively and are in the urea liquid of 190 μ L, then 4
okeep 1.0 hours under C, stir after mixing, impel urea to produce denaturation to laccase, cause laccase conformation change.
3., after step (2) reaction terminates, adopt ultraviolet spectrometer (UVS) within the scope of 400 ~ 700 nm, distinguish the absorption spectrum of nine kinds of reacted urea liquids in acquisition step (2);
4., according to urea concentration and the uv absorption intensity of nine kinds of urea liquids, draw the ultra-violet absorption spectrum changing trend diagram of laccase under different urea concentration condition, just can monitor laccase conformation change according to this ultra-violet absorption spectrum changing trend diagram.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (2)
1. for monitor laccase conformation change exempt from mark a ultraviolet absorption spectroscopy, feature is: concrete steps are as follows:
(1) according to list of references: Sivakumar, K.; Xie, F.; Cash, B. M. et al., A Fluorogenic 1,3-Dipolar Cycloaddition Reaction of 3-Azidocoumarins and Acetylenes. Org. Lett., 2004, method shown in 6,4603-4606., adopts 4-dihydroxy benzaldehyde, N-acetoglycocoll and sodium azide to prepare 3-nitrine-umbelliferone;
(2) laccase of 5.0 μ L 10 mg/mL and 5.0 μ L 0.25 mM 3-nitrine-umbelliferones are added to nine kinds of variable concentrations respectively and are in the urea liquid of 190 μ L, the concrete concentration of nine kinds of urea liquids is respectively 0.0 M, 1.0 M, 2.0 M, 3.0 M, 4.0 M, 5.0 M, 6.0 M, 7.0 M, 8.0 M, 4
ocultivate 1.0 hours under C, stir after mixing, impel urea to produce denaturation to laccase, cause laccase conformation change;
(3) after step (2) reaction terminates, adopt ultraviolet spectrometer (UVS) within the scope of 400 ~ 700 nm, distinguish the absorption spectrum of nine kinds of reacted urea liquids in acquisition step (2);
(4) according to urea concentration and the uv absorption intensity of nine kinds of urea liquids, draw the ultra-violet absorption spectrum changing trend diagram of laccase under different urea concentration condition, just can monitor laccase conformation change according to this ultra-violet absorption spectrum changing trend diagram.
2. according to claim 1 exempting from marks ultraviolet absorption spectroscopy, is characterised in that: the urea liquid described in step (2) is dissolved in acetate buffer formulated by urea, and pH value is modulated to 6.0.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0543722A1 (en) * | 1991-11-19 | 1993-05-26 | Bio Serae Laboratoires Sa | Method and device for the determination of laccase in musts by the syringaldazine test |
| CN101419162A (en) * | 2008-11-19 | 2009-04-29 | 河北大学 | Lysozyme detecting method in honey |
| WO2013032308A2 (en) * | 2011-09-02 | 2013-03-07 | 동국대학교 산학협력단 | Novel enzymatic-type time-temperature integrator using a laccase |
| CN103344576A (en) * | 2013-07-12 | 2013-10-09 | 福州大学 | Dual-output sensor for lysozyme detection and preparation method of lysozyme |
-
2014
- 2014-01-03 CN CN201410002215.7A patent/CN103712934B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0543722A1 (en) * | 1991-11-19 | 1993-05-26 | Bio Serae Laboratoires Sa | Method and device for the determination of laccase in musts by the syringaldazine test |
| CN101419162A (en) * | 2008-11-19 | 2009-04-29 | 河北大学 | Lysozyme detecting method in honey |
| WO2013032308A2 (en) * | 2011-09-02 | 2013-03-07 | 동국대학교 산학협력단 | Novel enzymatic-type time-temperature integrator using a laccase |
| CN103344576A (en) * | 2013-07-12 | 2013-10-09 | 福州大学 | Dual-output sensor for lysozyme detection and preparation method of lysozyme |
Non-Patent Citations (5)
| Title |
|---|
| "Laccase activity in soils：Considerations for the measurement of enzyme activity";Ivana Eichlerová et al.;《Chemosphere》;20121231;第1-7页 * |
| "Phenolic substrates for fluorometric detection of laccase activity";Greg Lonergan et al.;《FEMS Microbiology Letters》;19971231;第153卷;第485-490页 * |
| "一种改进的漆酶酶活检测方法";何为等;《华南理工大学学报(自然科学版)》;20031231;第31卷(第12期);第46-50页 * |
| "原子吸收光谱法测定漆酶的含量";肖海燕等;《化学与生物工程》;20061231;第23卷(第6期);第53-55页 * |
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