The Enzyme activity assay system and way of iron and α-ketoglutaric acid dependency dioxygenaseTechnical field
The application belongs to field of biological technology detection, be specifically related to the universal test method of dioxygenase activity, more specifically, all universal test methods active using α-ketoglutaric acid and iron ion as the dioxygenase (i.e. iron and α-ketoglutaric acid dependency dioxygenase) of cofactor are related to.
Background technology
Dioxygenase is the oxidasic general name that in oxygen molecule two Sauerstoffatoms are all combined with substrate.Iron and α-ketoglutaric acid dependency dioxygenase [α-oxoglutarate/Fe (II)-dependentdioxygenases (α-ODDs)] belong to the nonheme-iron protein being distributed widely in occurring in nature, it is a kind of important enzyme participating in multiple redox reaction, its reaction substrate wide variety, can be protein, such as prolyl hydroxylase (PHD), hypoxia inducible factor (hypoxiainducedfactor) and procollagen prolyl hydroxylase (collagenprolylhydroxylase) etc.; Also can be micromolecular compound, such as nucleic acid, as AlkB, FTO, thus at the synthesis important role of the nascent and secondary metabolite of animal, plant and microorganism.Such as, the synthesis etc. of these enzyme involved in plant hormone ethylene, Plant hormones regulators,gibberellins and flavonoid compound etc.Nearest research shows, iron and α-ketoglutaric acid dependency dioxygenase are the most all-round and extremely important oxidising biocatalyst.
There is a common mechanism in the reaction in iron and α-ketoglutaric acid dependency dioxygenase, namely ferrous ion and α-ketoglutaric acid are as cofactor, two Sauerstoffatoms of oxygen molecule in the reaction, one enters substrate, and another participates in the reaction of α-ketoglutaric acid decarboxylation and generates succsinic acid.
The detection general isotopic labeling of this type of enzymic activity analyzes change of the corresponding substrate structure of (radioactiveassay), mass spectrum, nucleus magnetic resonance (NMR) and macromole product and physical property etc. at present.These detection methods are loaded down with trivial details, are not suitable for the screening of large drug flux.In addition, existing detection method is only applicable to a kind of specific iron and α-ketoglutaric acid dependency dioxygenase, does not possess versatility.
Summary of the invention
On the one hand, this application provides the Enzyme activity assay system of iron and α-ketoglutaric acid dependency dioxygenase, it comprises: iron ion, α-ketoglutaric acid, coenzyme A, NTP, succinic thiokinase.
In above-mentioned Enzyme activity assay system, also comprise the substrate specificity of iron for initial enzymatic reaction and ketoisocaproic dependency dioxygenase, and for the ammonium molybdate solution that stops enzymatic reaction and the malachite green solution for developing the color.
In one embodiment, coenzyme A in described Enzyme activity assay system, the concentration of NTP and succinic thiokinase three is excessive, such as, the concentration of succinic thiokinase is excessive, and coenzyme A is identical with the concentration of NTP, can be more than 5-10 times of the corresponding substrate Km values of succinic thiokinase.In preferred embodiments, the ammonium molybdate solution in described Enzyme activity assay system and malachite green solution are highly acid.
On the other hand, the test kit that the enzyme that this application provides detection iron and ketoisocaproic dependency dioxygenase is lived or microarray, it comprises iron ion, α-ketoglutaric acid, coenzyme A, NTP, succinic thiokinase.In the test kit provided in the application or microarray, also comprise for the iron of initial enzymatic reaction and the substrate specificity of α-ketoglutaric acid dependency dioxygenase with for the ammonium molybdate solution that stops enzymatic reaction and the malachite green solution for developing the color.
In one embodiment, coenzyme A in described test kit or microarray, the concentration of NTP and succinic thiokinase three is excessive, such as, the concentration of succinic thiokinase is excessive, and coenzyme A is identical with the concentration of NTP, can be more than 5-10 times of the corresponding substrate Km values of succinic thiokinase.
In preferred embodiments, the ammonium molybdate solution in described Enzyme activity assay system and malachite green solution are highly acid.
Another aspect, this application provides the Enzyme activity assay method of iron and α-ketoglutaric acid dependency dioxygenase.
In one embodiment, the Enzyme activity assay method of iron provided by the invention and α-ketoglutaric acid dependency dioxygenase comprises the following steps: a) in testing sample, adds α-ketoglutaric acid dependency dioxygenase substrate specificity, iron ion, α-ketoglutaric acid carry out initial action and carry out reaction I; B) step a) in reaction I stop after, then add coenzyme A, NTP, MgCl2reaction II is carried out with succinic thiokinase; C) add ammonium molybdate solution to stop step b) in reaction II, finally add malachite green solution to develop the color, and 620-640nm place read absorbancy, determine thus iron and ketoisocaproic dependency dioxygenase enzyme live.
In preferred embodiments, step a) in reaction I heat at 100 DEG C and stop, heat-up time is, such as 30 minutes.
In preferred embodiments, step b) in reaction II add the ammonium molybdate solution being dissolved in sulfuric acid and stop.
In another embodiment, the Enzyme activity assay method of iron provided by the invention and α-ketoglutaric acid dependency dioxygenase comprises the following steps: a) in testing sample, add α-ketoglutaric acid dependency dioxygenase substrate specificity, iron ion, α-ketoglutaric acid carry out initial action and carry out reaction I, add coenzyme A, NTP, MgCl simultaneously2reaction II is carried out with succinic thiokinase; B) add ammonium molybdate solution and carry out termination reaction, then add malachite green solution to develop the color, and read absorbancy at 620-640nm place, determine that the enzyme of iron and ketoisocaproic dependency dioxygenase is lived thus.In addition, present invention also provides above-mentioned Enzyme activity assay system, above-mentioned test kit or microarray, and the application of above-mentioned method in the enzyme detecting iron and α-ketoglutaric acid dependency dioxygenase is lived.
Visible, the application produces the characteristic of succsinic acid after utilizing iron and these quasi-enzyme catalytic differential responses of α-ketoglutaric acid dependency dioxygenase, the method of the amount of the succsinic acid produced by detection reaction, detects the activity of iron and α-ketoglutaric acid dependence dioxygenase.With the generation of the consumption and primary product that detect iron and α-ketoglutaric acid dioxygenase detection substrate in prior art, and it is different to produce the method needing dependence chromatography, mass spectrum or isotopic labeling etc. to take time and effort thus, the Enzyme activity assay system and way of the iron that the application provides and α-ketoglutaric acid dependency dioxygenase is lived to detect enzyme by means of only the generation detecting by product succsinic acid, measure and rely on the photoabsorption of simple instrument reading visible ray, thus time saving and energy saving.
Accompanying drawing explanation
The exemplary conceptual diagram of the Enzyme activity assay method of the iron that Fig. 1 provides for the application and α-ketoglutaric acid dependency dioxygenase.
Fig. 2 is the gene mapping of sucC and sucD gene of succinic thiokinase β, α chain of encoding in the embodiment of the present application 1.
Fig. 3 is the pcr amplification product electrophoresis result figure of succinic thiokinase in the embodiment of the present application 1.
Fig. 4 is the Plastid transformation electrophoresis the result figure of succinic thiokinase in the embodiment of the present application 1.
Fig. 5 is the protein purification electrophoresis result figure of succinic thiokinase in the embodiment of the present application 1.
Fig. 6 is the activity checking of succinic thiokinase in the embodiment of the present application 1, and wherein 1 for not having succsinic acid (yellow); 2 is 50 μMs of succsinic acids (not having enzyme, yellow); 3 is 500 μMs of succsinic acids (not having enzyme, yellow); 4 is 5 μMs of succinic thiokinases and 50 μMs of succsinic acids (light green); 5 is 0.5 μM of succinic thiokinase and 500 μMs of succsinic acids (green).
Fig. 7 is the phosphoric acid concentration typical curve made in the embodiment of the present application 1.
Fig. 8 is in the embodiment of the present application 1 under condition of different pH, and according to the succinate coenzyme A synthetic enzyme reaction normal curve that different succinic acid concentration (10-100 μM) is set up, the reaction times is 45min.
Embodiment
There is provided to give a definition with method in order to define the application better and instruct those of ordinary skill in the art in the application's practice.Unless otherwise mentioned, term is understood according to the common usage of person of ordinary skill in the relevant.
Used in this article " NTP ", it is the abbreviation of ribonucleoside triphosphote (nucleosidetriphosphate), refer to a kind of Nucleotide containing three phosphate groups, comprise adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate(CTP) (CTP), thymus gland guanosine triphosphate (TTP) and uridine triphosphate (UTP) etc., it can generate corresponding nucleoside diphosphate NDP through hydrolysis and discharge inorganic phosphate Pi.Ribonucleoside triphosphote kind used in a particular embodiment, depends on source and the kind of used succinate coenzyme A synthetic enzyme.
Used in this article " strongly-acid ", refer to that a kind of material can put forward protogenic ability to other material in a solvent, at 25 DEG C, as pH<<7, solution is strongly-acid.Use sulfuric acid to obtain strongly acidic solution as solvent in a particular embodiment, such as, prepare ammonium molybdate solution by 25mM ammonium molybdate is dissolved in 3.4M sulfuric acid, prepare malachite green solution by 1mM Victoria Green WPB is dissolved in 6mM sulfuric acid.
Used in this article " enzyme is lived ", the enzyme (mg) being defined as (1min) unit mass in the unit time transforms the amount generating product.With regard to the enzyme of iron and α-ketoglutaric acid dependency dioxygenase is lived, refer to that the dioxygenase (mg) of (1min) unit mass in the unit time transforms the amount (nmole or pmole) generating succsinic acid, i.e. nmoles succsinic acid min-1mg dioxygenase to be measured-1.
In one embodiment, the Enzyme activity assay method that the application provides comprises the following steps: the substrate specificity 1) adding iron and α-ketoglutaric acid dependency dioxygenase in testing sample, iron ion and α-ketoglutaric acid, make it through enzymic catalytic reaction, produces succsinic acid; 2) by step 1) reaction system be heated to 100 DEG C and carry out termination reaction in 3 minutes, then add coenzyme A wherein, NTP, MgCl2and succinic thiokinase, make the succsinic acid of generation through further enzymic catalytic reaction, produce inorganic phosphate; And 3) in step 2) reaction system in add the ammonium molybdate solution being dissolved in sulfuric acid and carry out termination reaction, add the malachite green solution being dissolved in sulfuric acid subsequently to develop the color, and the absorbancy of assaying reaction thing (such as, absorbancy is read at 620-640nm place), determine that the enzyme of iron and ketoisocaproic dependency dioxygenase is lived thus.
Selectively, in the process, according to the speed of reaction of different dioxygenases, also can carry out above-mentioned steps 1 simultaneously) and step 2).
Such as, in another embodiment, the Enzyme activity assay method that the application provides comprises the following steps: in testing sample, add coenzyme A, NTP, MgCl2succinic thiokinase, iron ion, α-ketoglutaric acid and substrate specificity react, add the ammonium molybdate solution being dissolved in sulfuric acid subsequently and carry out termination reaction, finally add the malachite green solution being dissolved in sulfuric acid and develop the color, and the absorbancy of assaying reaction thing (such as, absorbancy is read at 620-640nm place), determine that the enzyme of iron and α-ketoglutaric acid dependency dioxygenase is lived thus.
In yet another embodiment, the Enzyme activity assay method that the application provides comprises the following steps: in α-ketoglutaric acid dependency dioxygenase sample to be measured, add its substrate specificity, iron ion, α-ketoglutaric acid carry out initial action (reaction I), depend on the height of enzymic activity to be measured, coenzyme A can be added, NTP, MgCl in the mixed system of reaction I simultaneously2carry out reaction II with succinic thiokinase, or within 3 minutes, carry out termination reaction I by 100 DEG C of heating, then add the coenzyme A of reaction needed for II, NTP, MgCl2and succinic thiokinase, after 30 minutes, add the ammonium molybdate solution being dissolved in sulfuric acid and carry out termination reaction, finally add the malachite green solution being dissolved in sulfuric acid to develop the color, after 20 minutes, read absorbancy in 620-640nm place, determine that the enzyme of iron and α-ketoglutaric acid dependency dioxygenase is lived thus.
In another embodiment, the succinic thiokinase that the reaction conditions of the Enzyme activity assay method that the application provides is originated according to different plant species and determining, such as, when using the succinic thiokinase of Escherichia coli, temperature of reaction is 30 ~ 40 DEG C, such as 30 DEG C, 31 DEG C, 32 DEG C, 33 DEG C, 34 DEG C, 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C, 40 DEG C, preferably 37 DEG C.When using the succinic thiokinase in thermophile bacteria source, temperature of reaction is higher, for 50-65 DEG C, such as 50 DEG C, 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 55 DEG C, 56 DEG C, 57 DEG C, 58 DEG C, 59 DEG C, 60 DEG C, 61 DEG C, 62 DEG C, 63 DEG C, 64 DEG C, 65 DEG C.
In yet another embodiment, in order to improve signal to noise ratio, reduce ATP and be hydrolyzed the background caused, add excessive succinyl-coenzyme A with Reaction time shorten, because succinic thiokinase is excessive, according to the speed of reaction of dioxygenase, its activity can reach 50U, the sensing range of the succsinic acid produced, when 5-100 μM, can complete reaction in 30 minutes.
In yet another embodiment, the substrate specificity of the iron added in the Enzyme activity assay method that the application provides and α-ketoglutaric acid dependency dioxygenase changes according to the kind of surveyed dioxygenase, such as, the substrate of prolyl hydroxylase (PHD) is hypoxia inducible factor (HIF) or its polypeptide, the substrate of procollagen prolyl hydroxylase is collagen protein or its polypeptide, and the substrate of alkane hydroxylase (AlkB) or nucleic acid demethylase (FTO) is DNA or RNA oligomer etc.
In preferred embodiments, the coenzyme A added in the Enzyme activity assay method that the application provides or test kit, it is excessive that NTP and succinic thiokinase three concentration are.Such as, the concentration of succinic thiokinase is excessive, and the concentration of coenzyme A and NTP is more than 5-10 times of the corresponding substrate Km values of succinic thiokinase, such as 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.
In a more preferred embodiment, each constituent concentration in described Enzyme activity assay system is as follows: 0.5 μM of succinic thiokinase, 200 μMs of NTP and 200 μM coenzyme As.
In preferred embodiments, the ammonium molybdate solution added in aforesaid method or test kit and the pH of malachite green solution are strongly-acid, such as, and pH<<7.Such as, in a more preferred embodiment, described ammonium molybdate solution is by being dissolved in 3.4M sulfuric acid to prepare by 25mM ammonium molybdate; Described malachite green solution is by being dissolved in 6mM sulfuric acid to prepare by 1mM Victoria Green WPB.
In one embodiment, aforesaid method also comprises the step being beforehand with out typical curve.Particularly, described typical curve each sample using the SODIUM PHOSPHATE, MONOBASIC of different concns as typical curve, adds the mixing solutions of ammonium molybdate and Victoria Green WPB, in the absorbancy of 620-640nm place working sample.
Enzyme activity assay side's ratio juris of the iron that the application provides and α-ketoglutaric acid dependency dioxygenase such as, as shown in Figure 1.The application mainly uses enzyme linked reaction, utilizes succinic thiokinase (succinylCoAsynthetase) catalysis succsinic acid to be converted into succinyl-coenzyme A (abbreviation succinyl CoA).The chemical equation of this reaction is as follows:
In one embodiment, above-mentioned catalyzed reaction consumes ribonucleoside triphosphote and produces inorganic phosphate, and inorganic phosphate can be detected by the mixing solutions of ammonium molybdate and Victoria Green WPB, namely the mixing solutions of ammonium molybdate and Victoria Green WPB and phosphoric acid produce mixture, and it has maximum absorbancy at 620-640nm.
The application is applicable to the succinic thiokinase in different plant species source, such as intestinal bacteria (Escherichiacoli), thermus aquaticus (Thermusaquaticus), β cell mitochondrial etc., and the succinic thiokinase in different plant species source is in the use of recombinant protein under identical object of different expression system expression and purification, be also applicable to the iron and the α-ketoglutaric acid dependence dioxygenase that detect other.
The application sets up general easy iron and α-ketoglutaric acid and relies on the active universal test method of dioxygenase (irondependentalphaketoglutaratedioxygenase) and test kit thereof or microarray, be suitable for all iron and α-ketoglutaric acid dependence dioxygenase, such as, prolyl hydroxylase (PHD), procollagen prolyl hydroxylase (collagenprolylhydroxylase), alkane hydroxylase (AlkB) or nucleic acid demethylase (FTO) etc.
In some embodiments, the method that the application provides relates to absorbance detection, can carry out, thus allow the detection of large flux with ordinary light absorbing apparatus such as platereader or microplate reader hyperchannel.Thus, in some embodiments, the test kit of the application or system can also comprise device or the reagent of above-mentioned absorbance detection.
Further, also the detection method of the application and test kit thereof or microarray can be used for the screening that iron and α-ketoglutaric acid rely on the activity inhibitor (or promotor) of dioxygenase, namely filter out the small molecules with potential drug therapeutic action this dioxygenase being played to suppression or activation.
Following examples are only illustrative rather than definitive thereof the object of the application's scope.If do not specialize, embodiment all conveniently experiment condition is carried out.
embodiment 1
The present embodiment sets forth for prolyl hydroxylase (PHD) the Enzyme activity assay method that the application provides.
1. the acquisition of succinic thiokinase
Succinic thiokinase by commercially available, such as, purchased from Megazyme company (article No. E-SCOAS), or can be prepared by the following method:
(1) gene clone
A.PCR increases: to coding succinic thiokinase β, sucC and the sucD gene (as shown in Figure 2) of α chain carries out pcr amplification, and PCR reaction system and condition are respectively as shown in following table 1 and table 2, and the electrophoresis result that PCR primer detects is shown in Fig. 3.
Table 1
Wherein used primer sequence is as follows:
Primer-F:5 '-GGGAATTCCATATG (NdeI) AACTTACATGAATATCAGGC-3 '
Primer-R:5 '-CGCGAAGCTT (HindIII) TTATTTCAGAACAGTTTTCAGTGC-3 '
Table 2
B. enzyme is cut and is connected with enzyme: carry out enzyme by restriction enzyme NdeI, HindIII to goal gene sucCDPCR product D NA fragment and pET-28a and cut, use T subsequently4gene fragment is connected with pET28a carrier segments by DNA ligase, recombinant plasmid clone enters bacillus coli DH 5 alpha, with the recombinant plasmid that increases, then confirm with identical restriction endonuclease NdeI/HindIII successfully to build the recombinant plasmid containing sucCD gene, Fig. 4 gives electrophoresis result figure.
(2) expression and purification of albumen
A. protein expression: form new plasmid pET28a-sucCD and be converted and enter in e. coli bl21 and abduction delivering is carried out to goal gene.
I) from-80 DEG C of refrigerators, get 100 μ L competent cell suspensions, under room temperature, make it thaw on ice.
II) add pET-28a-sucCD plasmid DNA solution (~ 50ng, 1-10 μ L), shake up gently, place after 30 minutes on ice.
III) thermal shock 60 seconds in 42 DEG C of water-baths, is placed in rapidly cooled on ice 2 minutes after thermal shock.
IV) Xiang Guanzhong adds 1mLLB liquid nutrient medium (not containing microbiotic), 37 DEG C of shaking culture 1 hour,
Bacterium is restore normal growth state.
V) centrifugal 10000g × 1min, abandons supernatant liquor 900 μ L.
VI) above-mentioned residue bacterium liquid 100 μ L suspension is coated on the screening flat board containing kantlex (Kan, 100 μ g/mL), face up and place half an hour, after bacterium liquid is absorbed by substratum completely, be inverted culture dish, cultivate 10-16 hour for 37 DEG C.
VII) mono-clonal bacterium colony is cultured to OD in LB+Kan nutrient solution600=0.6-0.8, add 0.3mMIPTG T7 operon is controlled under sucCD goal gene abduction delivering 2 hours.Cell centrifugation is retained in-80 DEG C and waits for protein purification.
B. protein purification: a step affinity chromatography can succinic thiokinase holoenzyme α2β2protein purification is to the purity of >95%.
I) cytoclasis: above-mentioned cell lysis buffer (50mMNaH2pO4, 300mMNaCl, 10mMimidazole, 1mMPMSF, pH8.0) ultrasonic or high-pressure homogeneous instrument (FrenchPress) is broken, makes the abundant cracking of thalline.
II) Ni-NTA column chromatography: sample and Ni-NTA are rocked in conjunction with 60min at 4 DEG C.By all upper props of the sample (comprising protein lysate and resin) in conjunction with 60min, with appropriate lavation buffer solution (50mMNaH2pO4, 300mMNaCl, 20mMimidazole, pH8.0) carry out wash-out, with appropriate elution buffer (50mMNaH2pO4, 300mMNaCl, 250mMimidazole, pH8.0) carry out wash-out, gained purifying protein dialysis the phosphoric acid in damping fluid is replaced into 50mMTris damping fluid, pH8.0, then with SDS-PAGE analyze, result is as shown in Figure 5.
C. active checking: adopt Victoria Green WPB color reaction to verify the activity of succinic thiokinase, result as shown in Figure 6.
2. other components of enzyme reagent kit
Other components of enzyme reagent kit comprise:
1) CoA, C21h36n7o16p3s, CAS:85-61-0, can purchased from such as Sigma (article No. C4282).
2) ATP, C10h16n5o13p3, CAS:56-65-5, can purchased from such as Sigma (article No. A1852).
3) Victoria Green WPB (malachite), C23h25n2cl, CAS:569-64-2, can purchased from such as Sigma (article No. 38800).
The chemical structural formula of above-mentioned each component is as follows respectively:
3. enzyme measured reaction initial, stop and detect
By 200 μMs of CoA, 200 μMs of ATP and 0.5 μM of succinic thiokinase holoenzyme mixing, and added iron content and α-ketoglutaric acid dependence dioxygenase---in each reacting hole of prolyl hydroxylase (PHD), at 37 DEG C, add the sequences polypeptide initial action of the α subunit of substrate hypoxia inducible factor (HIF), carry out about 30 minutes, add the ammonium molybdate solution (25mM ammonium molybdate is dissolved in 3.4M sulfuric acid) being dissolved in sulfuric acid and carry out termination reaction, add the malachite green solution (1mM Victoria Green WPB is dissolved in 6mM sulfuric acid) being dissolved in sulfuric acid subsequently to develop the color.After 20 minutes, read A620nmabsorbancy.According to the volume of reaction system, spectrophotometer or various microplate reader can be used to measure absorbancy.
4. the calculating of enzymic activity
Production standard curve: each sample using the SODIUM PHOSPHATE, MONOBASIC of different concns as typical curve, add the ammonium molybdate solution (25mM ammonium molybdate is dissolved in 3.4M sulfuric acid) being dissolved in sulfuric acid and carry out termination reaction, add the malachite green solution (1mM Victoria Green WPB is dissolved in 6mM sulfuric acid) being dissolved in sulfuric acid subsequently and develop the color.Add the ingrain dye(stuff) that Victoria Green WPB mixes with ammonium molybdate, in the absorbancy of 620-640nm place working sample.Result as shown in Figure 7.
Based on above-mentioned phosphate standard curves, the total phosphoric acid concentration produced in enzyme measured reaction can be calculated according to the absorbancy in enzyme measured reaction, (its source is the phosphoric acid concentration that the hydrolysis of ATP produces to deduct the phosphoric acid concentration do not added in the control group of dioxygenase, under this experiment condition, its background value is very low, minute quantity ATP is only had to be hydrolyzed), namely obtain the phosphoric acid concentration that succinyl-coenzyme A catalysis produces.
The product phosphoric acid of the succinic thiokinase catalyzed reaction obtained and its mol ratio of product succsinic acid of dioxygenase effect are 1:1, so the amount of the phosphoric acid that can detect according to Victoria Green WPB color reaction extrapolates the initial content of succsinic acid in system.
Under condition of different pH, the equilibrium constant of succinic thiokinase catalyzed reaction is different.PH is lower, and its reaction equilibrium constant is higher, and the direction of reacting to being conducive to producing phosphoric acid is moved.Fig. 8 is (pH6.5,20MmMes under condition of different pH; PH8.0,100MmTris; PH8.5,100MmTris), the succsinic acid of succinic thiokinase catalysis different concns produces succinyl-coenzyme A and phosphoric acid, and detects the data of phosphoric acid by Victoria Green WPB color reaction.Result shows, under condition of different pH, and A620the phosphoric acid concentration detected and the equal display line sexual intercourse of succinic acid concentration (R2>95%), and pH is lower, and transformation efficiency is higher.When the reaction of pH6.5-8.5 interval reaches balance, the transformation efficiency of succsinic acid is 40-65%.
Visible, under specific pH condition, do typical curve with the succsinic acid of different concns, method of the present invention can be used for the succinic acid content detecting testing sample, thus calculate the activity of dioxygenase to be measured, wherein Mei Huo unit is nmoles succsinic acid min-1mg enzyme-1or pmoles succsinic acid min-1μ g enzyme-1deng.