CN104450865A - Method for screening and detecting phosphatidase C - Google Patents
Method for screening and detecting phosphatidase C Download PDFInfo
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- CN104450865A CN104450865A CN201310430394.XA CN201310430394A CN104450865A CN 104450865 A CN104450865 A CN 104450865A CN 201310430394 A CN201310430394 A CN 201310430394A CN 104450865 A CN104450865 A CN 104450865A
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Abstract
The invention provides a method for detecting the enzyme activity of phosphatidase C, and a product for detecting the enzyme activity of the phosphatidase C. The method or the product has the advantages of accurate quantification of the enzyme activity, low detection cost, convenient massive screening and detection, and convenient high-flux screening or detection.
Description
Technical field
The present invention relates to Phospholipase C detection field, in particular to a kind of screening, the method detecting Phospholipase C.
Background technology
Coming unstuck in refining raw food oil process is a difficult problem for refinery practice development always.Conventional hydration degum, the chemical degumming means such as acid degumming, all cannot remove the phosphatide in crude oil efficiently, low residue, and also can adsorb a large amount of edible oils while removing phosphatide, reduce refining productive rate.Enzymatic degumming is the focus of research in recent years.In view of biological enzyme reaction has specificity, the series of advantages such as high efficiency and controllability, this method for degumming can be removed effectively to the phosphatide in edible oil, and reduce the loss of edible oil in scouring processes, although also extensively do not adopt now, think that enzymatic degumming is better than traditional Degumming method in theory.Such as cut phosphatidyl group at sn-3 position enzyme, thus make the phospholipid breakdown in oil become triglyceride and water-soluble phosphoric acid composite (phosphatide head), this fermentoid is called as Phospholipase C (PLC).Phospholipase C roughly can be divided into phosphatidylcholine-specific phospholipase C (PC-PLC) by substrate type, phosphatidylinositol-specific phospholipase C (PI-PLC), phosphatidylethanolamine specific phospholipase C(PE-PLC) and phosphatidic acid specific phospholipase C(PA-PLC) etc.
At the bacterial classification of abundant microbe-derived middle searching output height vigor PLC, it is one of Main way of Phospholipid hydrolase research now.How in the microflora of bulky complex, to find target bacterial classification rapidly, be that in research, technology needs the place broken through, and in fact namely refers to the detection of PLC vigor and the method exploitation of high flux screening thereof.
The method that PLC enzyme activity detects has much now (high woods. the progress .Journal of Anhui Agri Sci2010 of Microbial phospholipases C detection method, 38 (23): 12412-12413): as
Egg plate method: according to the hydrolytic action of PLC to Yelkin TTS in the yolk in plate culture medium, can produce oyster white haloing on flat board, judges that enzyme is lived just according to haloing diameter.Because material is simple, easy to operate, be widely used when enzyme activity primary dcreening operation, but cannot be used for accurate quantification detection enzyme lives, and generally can only be used for qualitative detection.
Acid base titration: utilize the Acidic Hydrolysis Products that PLC hydrolyzed lecithin discharges, i.e. phosphorylcholine, then carrys out the amount of titration phosphorylcholine with NaOH, thus measures the vigor of PLC.The method feature reaction times is fast, but the Acidic Hydrolysis Products amount discharged is few, and titration error is large, complex operation, and working efficiency is too low.
Radioisotope method: utilize the phospholipid substrate that different labelled with radioisotope is different, be hydrolyzed after various substrate until PLC, utilize radic just can detect the hydrolysate of different labelled with radioisotope, thus not only can measure the vigor of this PLC, also can understand the Preference of its phospholipid substrate.The method specificity is strong, and result is accurate, but technical too high, substrate is expensive and have requirement to plant and instrument.
NPPC method: this method utilizes a kind of synthetic of Yelkin TTS similar structures, namely to nitro phosphorylcholine (NPPC), as the reaction substrate of PLC, create p-NP after hydrolysis, this material has maximum absorption wavelength at 410nm place.This method is reproducible, easy to operate, and the reaction times is short, but due to synthetic cost comparatively large, so there is certain limitation.
HPLC method: utilize HPLC to carry out ultra-violet analysis to the hydrolysate of PLC, this method accuracy is best, and base consumption speed can be grasped, the much informations such as specific substrate kind, but due to technical too high, have higher requirement to the instrumentation level etc. of technician, and cost is higher, detection speed slow, so be not widely adopted.
Fluorescent marker method, chemiluminescence detecting method and spectrophotometry: the strategy that these several class methods adopt is similar, utilizes fluorophor respectively, luminophores etc. marked phospholipid substrate, through PLC hydrolysis, respectively by fluoroscopic examination, the photosensitive or spectrophotometer of egative film detects product.These methods and strategies are Main way of present PLC detection method exploitation, and high specificity is highly sensitive, has good accuracy, but substrate synthesis difficulty, synthesis cost are higher, also have some limitations.
Molybdenum blue fixing phosphorus method: after PLC hydrolytic phosphatide substrate, the phosphoric acid composite discharged, through Phosphoric acid esterase effect, can hydrolysis inorganic phosphate.In acid condition, through reductive agent effect, inorganic phosphate can form molybdenum blue complex compound with molybdate, thus has absorption value at 680-800nm place, thus measures the vigor of PLC.
The initial thought of molybdenum blue fixing phosphorus method to come from biased sample for measuring the method for phosphorus content: by sample acidolysis or calcination, organophosphorus is wherein made all to be converted into inorganic phosphorus, thus detect total phosphorous by molybdenum blue fixing phosphorus method, be the fixing phosphorus method in national standard method.But be applied to the new direction that phospholipase activity detection is follow-up developments.But show in bibliographical information before, although the method has good repeatability, but in order to ensure that the reacted phosphoric acid composite of PLC farthest discharges inorganic phosphate, require very large to the consumption of Phosphoric acid esterase, and in order to avoid other factors generation P-Mo blue complex compound in reaction, need the impact that certain method removes these factors.
In some researchs, reported that the viability examination of Phospholipid hydrolase enters high flux screening pattern.Amplex Red phosphatidylcholine test kit [9] of Invitrogen company, can detect for the specific Phospholipase C of phosphatidylcholine for means with multi-biological enzyme reaction.After Phospholipase C cuts phosphorylcholine to phosphatidylcholine, utilize Phosphoric acid esterase to cut choline monomer, make it oxidized under the effect of E.C. 1.1.99.1, discharge hydrogen peroxide, thus utilize horseradish peroxidase to produce chemoluminescence.And the Amplex Red reagent in this test kit is a kind of fluorescent reagent, when chemoluminescence produces, the light of this wavelength can be subject to thus be inspired fluorescence, finally by fluoroscopic examination, can measure PLC vigor.The method high specificity, highly sensitive, but also there is a lot of limitation.Such as because the characteristic reagent in this test kit is connected with choline monomer, so can only detect the specific phospholipase activity of phosphatidylcholine, and substrate synthesis cost is comparatively large, is not suitable for high flux screening purposes; Multi-biological enzyme reaction, due to its complicacy, also has drawback, occurs to fluoroscopic examination from product, and wherein any one substance reaction goes wrong and result will be caused to analyze.
Summary of the invention
The present inventor finds, use Phospholipase C (such as phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine specific phospholipase C, phosphatidic acid specific phospholipase C) and/or dilution after Phospholipase C enzymolysis composite phospholipid (include but not limited to soybean phospholipid, peanut phosphatide, sesame phosphatide, castor-oil plant phosphatide), then by enzymolysis solution solid-liquid separation, the solution of acquisition is joined in phosphatase reaction system and reacts, again reaction solution and molybdic acid or molybdate (are such as included but not limited to ammonium molybdate, Sodium orthomolybdate, potassium molybdate) solution contact, detect light absorption value, under certain extent of dilution, the enzyme of PLC is lived and the positive correlation of molybdenum blue light absorption value, therefore, the method can be used to detect PLC enzyme live.Contriver also finds, the method can be directly used in the detection that in fermented liquid, PLC enzyme is lived.Contriver also finds, under suitable extent of dilution, only just can determine therefore, to can be used for the rapid screening of bacterial strain or the quick selection of fermentation condition by the relative height that the enzyme of fermented liquid is lived according to the size of light absorption value.Contriver also finds, with an organic solvent and/or water-soluble composite phospholipid, all can be used for the present invention, but when (including but not limited to trichloromethane) when with an organic solvent and dissolve composite phospholipid, its speed of response is obviously faster than the speed of response using water dissolution composite phospholipid.
Therefore, one object of the present invention is, provides a kind of method detecting Phospholipase C enzyme and live.
Method provided by the invention comprises the following steps:
1) Phospholipase C and/or its diluent enzymolysis composite phospholipid is used;
2) solid-liquid separation, after after using separation, solution contacts with Phosphoric acid esterase, contacts with molybdic acid or molybdate solution, detects light absorption value;
3) extent of dilution of the work of Phospholipase C enzyme and molybdenum blue light absorption value linear dependence is determined,
4) calculate described Phospholipase C enzyme to live.
In one embodiment of the invention, the composite phospholipid of use is: soybean phospholipid, peanut phosphatide, sesame phosphatide, one or more in castor-oil plant phosphatide.
In one embodiment of the invention, in the water-soluble and/or organic solvent of the composite phospholipid of use.
In one embodiment of the invention, the composite phospholipid of use is dissolved in organic solvent.
In one embodiment of the invention, the composite phospholipid of use is dissolved in trichloromethane.
In one embodiment of the invention, after described separation, solution contacts with Phosphoric acid esterase and is: after being separated, solution and Phosphoric acid esterase react more than 30min in 30-45 DEG C.
In one embodiment of the invention, after being separated, the temperature of reaction of solution and Phosphoric acid esterase is 35-42 DEG C.
In one embodiment of the invention, after being separated, the temperature of reaction of solution and Phosphoric acid esterase is 37-40 DEG C.
In one embodiment of the invention, after being separated, the reaction times of solution and Phosphoric acid esterase is 0.5h-2h.
In one embodiment of the invention, described detection light absorption value is the light absorption value detecting 600-810nm.
In one embodiment of the invention, described detection light absorption value is the light absorption value detecting 650-750nm.
In one embodiment of the invention, described Phospholipase C is phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine specific phospholipase C and/or phosphatidic acid specific phospholipase C.
Present invention also offers a kind of method of screening the bacterial strain of expressing Phospholipase C.
Method provided by the invention comprises the following steps:
1) Phospholipase C and/or its diluent enzymolysis composite phospholipid is used;
2) solid-liquid separation, after after using separation, solution contacts with Phosphoric acid esterase, contacts with molybdic acid or molybdate solution, detects light absorption value.
In one embodiment of the invention, the composite phospholipid of use is: soybean phospholipid, peanut phosphatide, sesame phosphatide, one or more in castor-oil plant phosphatide.
In one embodiment of the invention, in the water-soluble and/or organic solvent of the composite phospholipid of use.
In one embodiment of the invention, the composite phospholipid of use is dissolved in organic solvent.
In one embodiment of the invention, the composite phospholipid of use is dissolved in trichloromethane.
In one embodiment of the invention, after described separation, solution contacts with Phosphoric acid esterase and is: after being separated, solution and Phosphoric acid esterase react more than 30min in 30-45 DEG C.
In one embodiment of the invention, after being separated, the temperature of reaction of solution and Phosphoric acid esterase is 35-42 DEG C.
In one embodiment of the invention, after being separated, the temperature of reaction of solution and Phosphoric acid esterase is 37-40 DEG C.
In one embodiment of the invention, after being separated, the reaction times of solution and Phosphoric acid esterase is 0.5h-2h.
In one embodiment of the invention, described detection light absorption value is the light absorption value detecting 600-810nm.
In one embodiment of the invention, described detection light absorption value is the light absorption value detecting 650-750nm.
In one embodiment of the invention, described Phospholipase C is phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine specific phospholipase C and/or phosphatidic acid specific phospholipase C.
Present invention also offers a kind of method detecting Phospholipase C enzyme work in fermented liquid.
Method provided by the invention comprises the following steps:
1) fermented liquid and/or its diluent enzymolysis composite phospholipid is used;
2) solid-liquid separation, after after using separation, solution contacts with Phosphoric acid esterase, contacts with molybdic acid or molybdate solution, detects light absorption value;
3) extent of dilution of the work of Phospholipase C enzyme and molybdenum blue light absorption value linear dependence is determined;
4) calculate described Phospholipase C enzyme to live.
In one embodiment of the invention, the composite phospholipid of use is: soybean phospholipid, peanut phosphatide, sesame phosphatide, one or more in castor-oil plant phosphatide.
In one embodiment of the invention, in the water-soluble and/or organic solvent of the composite phospholipid of use.
In one embodiment of the invention, the composite phospholipid of use is dissolved in organic solvent.
In one embodiment of the invention, the composite phospholipid of use is dissolved in trichloromethane.
In one embodiment of the invention, after described separation, solution contacts with Phosphoric acid esterase and is: after being separated, solution and Phosphoric acid esterase react more than 30min in 30-45 DEG C.
In one embodiment of the invention, after being separated, the temperature of reaction of solution and Phosphoric acid esterase is 35-42 DEG C.
In one embodiment of the invention, after being separated, the temperature of reaction of solution and Phosphoric acid esterase is 37-40 DEG C.
In one embodiment of the invention, after being separated, the reaction times of solution and Phosphoric acid esterase is 0.5h-2h.
In one embodiment of the invention, described detection light absorption value is the light absorption value detecting 600-810nm.
In one embodiment of the invention, described detection light absorption value is the light absorption value detecting 650-750nm.
In one embodiment of the invention, described Phospholipase C is phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine specific phospholipase C and/or phosphatidic acid specific phospholipase C.
Present invention also offers a kind of product detecting Phospholipase C enzyme and live.
Product provided by the invention comprises: composite phospholipid or composite phospholipid solution, Phosphoric acid esterase storage system solution, phosphatase reaction system solution, and molybdenum blue color reaction thing solution.
In one embodiment of the invention, described product is detection kit.
In one embodiment of the invention, described product also comprises Phospholipid hydrolase reaction system solution.
In one embodiment of the invention, described Phospholipid hydrolase is phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine specific phospholipase C and/or phosphatidic acid specific phospholipase C.
In this patent, molybdenum blue fixing phosphorus method is applied to the viability examination of PLC, Phosphoric acid esterase does not have selectivity to phosphatidyl group (product of phosphatide after Phospholipid hydrolase effect), is thus better than the phosphatase substrate limitation of aforesaid method; Molybdenum blue method is very ripe to the detection technique of free phosphorus, and thus present method may be used for the work of accurate quantification enzyme; Composite phospholipid (including but not limited to soybean phospholipid, peanut phosphatide, sesame phosphatide, castor-oil plant phosphatide) can be used as Enzyme activity assay substrate, and phosphatidyl group amount low thus Phosphoric acid esterase consumption is little, so testing cost is low, is convenient to a large amount of screening, detection; Reaction volume carries out small quantization, has made it to carry out in 96 orifice plate scales, be convenient to high-throughput and carry out screening or detecting; Present method is improved in the unreacted phospholipid substrate method of removal, directly composite phospholipid is dissolved in chloroform, PLC enzyme is reacted in a two-phase system, be better than like this utilizing organic solvent to extract the method for substrate, not only easy and simple to handle, and the impurity component in composite phospholipid can be isolated in organic phase, the subsequent detection of aqueous phase can not be had influence on.
Accompanying drawing explanation
Fig. 1 is the typical curve that P-Mo blue detects.
Fig. 2 shows phosphatase reaction speed and substrate (PO
4 3-) concentration increase relation.
Fig. 3 shows the relation that Phosphoric acid esterase (CIAP) reaction product production rate and its concentration increase.
Fig. 4 shows the relation of P-Mo blue complex compound in 700nm place light absorption value and PLC concentration.
Embodiment
" scope " disclosed herein is with the form of lower limit and the upper limit.One or more lower limit can be respectively, and one or more upper limit.Given range is limited by a selected lower limit and a upper limit.Selected lower limit and the upper limit define the border of special scope.All scopes that can carry out by this way limiting comprise and may be combined with, and namely any lower limit can be combined to form a scope with any upper limit.Such as, list the scope of 60-120 and 80-110 for special parameter, be interpreted as that the scope of 60-110 and 80-120 also expects.In addition, if the minimum extent value listed 1 and 2, and if list maximum range value 3,4 and 5, then the scope below can all expect: 1-3,1-4,1-5,2-3,2-4 and 2-5.
In the present invention, unless otherwise indicated, new technical scheme can be mutually combined to form between the content range of each component of composition and its preferable range.
In the present invention, unless otherwise indicated, " its combination " represents the multicomponent mixture of described each element, such as two kinds, three kinds, four kinds and until the multicomponent mixture of maximum possible.
In the present invention, unless otherwise indicated, all " part " and percentage ratio (%) all refer to weight percentage.
In the present invention, unless otherwise indicated, in all compositions, the percentage ratio sum of each component is 100%.
In the present invention, unless otherwise indicated, the breviary of any real combinings that numerical range " a-b " represents between a to b represents, wherein a and b is real number.Such as numerical range " 0-5 " represents the whole real numbers all listed between " 0-5 " herein, and the breviary of " 0-5 " just these combinations of values represents.
In the present invention, unless otherwise indicated, the breviary of the arbitrary integer combination that integer numerical range " a-b " represents between a to b represents, wherein a and b is integer.Such as integer numerical range " 1-N " represents 1,2 ... N, wherein N is integer.
If do not particularly not pointed out, this specification sheets term " one " used refers to " at least one ".
If do not particularly not pointed out, the benchmark of percentage ratio of the present invention (comprising weight percentage) is all the gross weight of described composition.
In this article, except as otherwise noted, the ratio of each component or weight all refer to dry weight.
In the present invention, if do not illustrated especially, all embodiments mentioned in this article and preferred implementation can be combined to form new technical scheme mutually.
In the present invention, if do not illustrated especially, all technical characteristics mentioned in this article and preferred feature can be combined to form new technical scheme mutually.
In the present invention, if do not illustrated especially, mentioned in this article sequentially can to carry out in steps, also can carry out at random, but preferably order is carried out.Such as, described method comprises step (a) and (b), represents that described method can comprise the step (a) and (b) of sequentially carrying out, also can comprise the step (b) and (a) of sequentially carrying out.Such as, describedly mention described method and also can comprise step (c), represent that step (c) random order can join described method, such as, described method can comprise step (a) and (b) and (c), also step (a), (c) and (b) be can comprise, step (c), (a) and (b) etc. also can be comprised.
In the present invention, if do not illustrated especially, " comprising " mentioned in this article represents open, also can be closed.Such as, described " comprising " can represent other elements that can also comprise and not list, and also can only comprise the element listed.
In the present invention, if do not illustrated especially, the concrete numerical value herein in embodiment and concrete material can with other integrate features describing part herein.Such as, the temperature that description part herein mentions reaction is 10-100 DEG C, and the temperature of reaction that embodiment is mentioned is 20 DEG C, so can think the scope having specifically disclosed 10-20 DEG C herein, or the scope of 20-100 DEG C, and other integrate features that this scope can describe part get up to be formed new technical scheme.Again such as, description part herein mentions a compounds alcohol, and the concrete alcohol that embodiment is mentioned is ethanol, and so ethanol can get up formed new technical scheme with other integrate features of description part.
The present inventor finds, use Phospholipase C (such as phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine specific phospholipase C, phosphatidic acid specific phospholipase C) and/or dilution after Phospholipase C enzymolysis composite phospholipid (include but not limited to soybean phospholipid, peanut phosphatide, sesame phosphatide, castor-oil plant phosphatide), then by enzymolysis solution solid-liquid separation, the solution of acquisition is joined in phosphatase reaction system and reacts, again reaction solution and molybdic acid or molybdate (are such as included but not limited to ammonium molybdate, Sodium orthomolybdate, potassium molybdate) solution contact, detect light absorption value, under certain extent of dilution, the enzyme of PLC is lived and the positive correlation of molybdenum blue light absorption value, therefore, the method can be used to detect PLC enzyme live.Contriver also finds, the method can be directly used in the detection that in fermented liquid, PLC enzyme is lived.Contriver also finds, under suitable extent of dilution, only just can determine therefore, to can be used for the rapid screening of bacterial strain or the quick selection of fermentation condition by the relative height that the enzyme of fermented liquid is lived according to the size of light absorption value.Contriver also finds, with an organic solvent and/or water-soluble composite phospholipid, all can be used for the present invention, but when (including but not limited to trichloromethane) when with an organic solvent and dissolve composite phospholipid, its speed of response is obviously faster than the speed of response using water dissolution composite phospholipid.
In the present invention, term " Phosphoric acid esterase " refers to can the enzyme of catalytic phosphatase list ester hydrolysis, but can not the Phosphoric acid esterase of catalytic phosphatase diester and tricresyl phosphate ester hydrolysis.
Therefore, the invention provides a kind of method detecting PLC enzyme and live, the method comprises the following steps:
1) Phospholipase C and/or its diluent enzymolysis composite phospholipid is used;
2) solid-liquid separation, after after using separation, solution contacts with Phosphoric acid esterase, contacts with molybdic acid or molybdate solution, detects light absorption value;
3) extent of dilution of the work of Phospholipase C enzyme and molybdenum blue light absorption value linear dependence is determined;
4) calculate described Phospholipase C enzyme to live.
Present invention also offers a kind of method of screening the bacterial strain of expressing Phospholipase C, the method comprises the following steps:
1) Phospholipase C and/or its diluent enzymolysis composite phospholipid is used;
2) solid-liquid separation, after after using separation, solution contacts with Phosphoric acid esterase, contacts with molybdic acid or molybdate solution, detects light absorption value.
Present invention also offers a kind of method detecting Phospholipase C enzyme work in fermented liquid, the method comprises the following steps:
1) fermented liquid and/or its diluent enzymolysis composite phospholipid is used;
2) solid-liquid separation, after after using separation, solution contacts with Phosphoric acid esterase, contacts with molybdic acid or molybdate solution, detects light absorption value;
3) extent of dilution of the work of Phospholipase C enzyme and molybdenum blue light absorption value linear dependence is determined;
4) calculate described Phospholipase C enzyme to live.
In one embodiment of the invention, described composite phospholipid is the conventional composite phospholipid used in this area, includes but not limited to: soybean phospholipid, peanut phosphatide, sesame phosphatide, one or more in castor-oil plant phosphatide.
In one embodiment of the invention, described Phospholipase C is phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine specific phospholipase C and/or phosphatidic acid specific phospholipase C.
In one embodiment of the invention, in the mixed solvent that water-soluble, the organic solvent of described composite phospholipid or water and organic solvent form.
In another embodiment of the invention, described composite phospholipid is dissolved in organic solvent.
In another embodiment of the invention, described composite phospholipid is dissolved in trichloromethane.
In one embodiment of the invention, described phosphatase reaction system is for comprising Phosphoric acid esterase, Mg
2+with the phosphatase reaction system of damping fluid.
In one embodiment of the invention, described phosphatase reaction system is for comprising Phosphoric acid esterase, Ca
2+with the phosphatase reaction system of damping fluid.
In one embodiment of the invention, the Phosphoric acid esterase in described phosphatase reaction system is 10-15U/mL.
In one embodiment of the invention, the Mg in described phosphatase reaction system
2+for 2-20mM.
In one embodiment of the invention, the Ca in described phosphatase reaction system
2+for 2-20mM.
In one embodiment of the invention, the damping fluid of described phosphatase reaction system is 40-100mMTris-Cl (pH8.0-9.0).
In one embodiment of the invention, described phosphatase reaction system comprises 10-15U/mL Phosphoric acid esterase, 2-20mM Mg
2+and/or 40-100mM Tris-Cl (pH8.0-9.0).
In one embodiment of the invention, described phosphatase reaction system comprises 10-15U/mL Phosphoric acid esterase, 2-20mM Ca
2+and/or 40-100mM Tris-Cl (pH8.0-9.0).
In one embodiment of the invention, described phosphatase reaction system is: 50mM Tris-Cl(pH9.0), 10mM MgCl
2, alkaline phosphatase 10U/mL.
In one embodiment of the invention, the molybdate of use be ammonium molybdate, one or more in Sodium orthomolybdate, potassium molybdate.
In one embodiment of the invention, the concentration of the molybdate of use is 2-3%, is preferably 2.4-2.6%.
In one embodiment of the invention, the concentration of the molybdate of use is 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0%.
In one embodiment of the invention, after described separation, solution contacts with Phosphoric acid esterase and is: solution after separation and Phosphoric acid esterase are reacted at least 30min in 30-45 DEG C.
In one embodiment of the invention, after being separated, the temperature of reaction of solution and Phosphoric acid esterase is 35-42 DEG C.
In one embodiment of the invention, after being separated, the temperature of reaction of solution and Phosphoric acid esterase is 37-40 DEG C.
In one embodiment of the invention, after being separated, the temperature of reaction of solution and Phosphoric acid esterase is 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, 41 DEG C, 42 DEG C, 43 degrees Celsius, 44 DEG C, or 45 DEG C.
In one embodiment of the invention, after described separation, the reaction times of solution and Phosphoric acid esterase is 0.5-2h.
In one embodiment of the invention, compare with P-Mo blue typical curve, under determining different extent of dilution, Phospholipase C enzyme is lived and light absorption value linear dependence region.
In one embodiment of the invention, detect light absorption value in 600-810nm, be preferable over 650-750nm and detect light absorption value.
In one embodiment of the invention, in 600nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, 770nm, 780nm, 690nm, 800nm, or 810nm detects light absorption value.
In one embodiment of the invention, calculating the work of described Phospholipase C enzyme is: under calculating the extent of dilution of light absorption value linear dependence, Phospholipase C enzyme is lived, and calculates the mean value that Phospholipase C enzyme under above-mentioned extent of dilution is lived.
Present invention also offers a kind of product detecting Phospholipase C enzyme and live.
Product provided by the invention comprises: composite phospholipid or composite phospholipid solution, Phosphoric acid esterase storage system solution, phosphatase reaction system solution, and molybdenum blue color reaction thing solution.
In one embodiment of the invention, product provided by the invention comprises Phospholipid hydrolase reaction system solution.In the present invention, the system of spendable Phospholipid hydrolase reaction system solution known by those skilled in the art, also according to perhaps actual needs in disclosed by the invention, suitably can adjust.In one embodiment of the invention, Phospholipid hydrolase reaction system solution comprises buffer system and provides the active metallic ion needed for PLC reaction.In one embodiment of the invention, the active metallic ion needed for PLC reaction is Ca
2+, Zn
2+in one or more; Buffer system is the buffered soln of pH6.5-8.0, and preferred buffered soln is Tris-Cl.In one embodiment of the invention, the solution for PLC reaction comprises: 25-50mM Tris-Cl(pH6.5-8.0), 2-5mM CaCl
2.
In one embodiment of the invention, described Phosphoric acid esterase storage system solution comprises Phosphoric acid esterase and stablizes the solution of described Phosphoric acid esterase.In one embodiment of the invention, described Phosphoric acid esterase storage system solution comprises 400-10, the Phosphoric acid esterase of 000U/mL.In one embodiment of the invention, the solution stablizing described Phosphoric acid esterase in described Phosphoric acid esterase storage system solution comprises: 50-150mM Tris-Cl(pH8.0-9.0), 5-10mM MgCl
2.In one embodiment of the invention, described Phosphoric acid esterase storage system solution is: 50-150mM Tris-Cl(pH8.0-9.0), 5-10mM MgCl
2, 500U/mL Phosphoric acid esterase.
In the present invention, the system of spendable phosphatase reaction system solution known by those skilled in the art, also according to perhaps actual needs in disclosed by the invention, suitably can adjust.In one embodiment of the invention, comprise buffer system for phosphatase reaction system solution and the active metallic ion needed for phosphatase reaction is provided.In one embodiment of the invention, the active metallic ion needed for phosphatase reaction is Mg
2+, Ca
2+in one or more; Buffer system is the buffered soln of pH8.0-9.0, and preferred buffered soln is Tris-Cl.In one embodiment of the invention, the solution for phosphatase reaction comprises: 200-500mM Tris-Cl(pH8.0-9.0), 10-100mM MgCl
2.
In the present invention, the system of spendable molybdenum blue color reaction thing solution known by those skilled in the art, also according to perhaps actual in of the present invention, suitably can adjust.In one embodiment of the invention, molybdenum blue color reaction thing solution comprises molybdic acid or molybdate solution and sulfuric acid.In one embodiment of the invention, described molybdate is ammonium molybdate, one or more in Sodium orthomolybdate and potassium molybdate.In one embodiment of the invention, molybdenum blue color reaction thing solution comprises 0.1-0.2% (w/v) ammonium molybdate solution, 1-5% (v/v) sulfuric acid.
In one embodiment of the invention, this product is detection kit.
In one embodiment of the invention, the product detecting Phospholipase C enzyme alive comprises:
The reagent 1 of the substrate of PLC enzyme reaction is provided, such as, can uses 1-5%(w/v) with chloroform dissolve composite phospholipid.
Carry out the buffer system of phosphatase reaction and the reagent 2 of the active metallic ion needed for phosphatase reaction be provided, such as, can use 200-500mM Tris-Cl(pH8.0-9.0), 10-100mM MgCl
2.
The reagent 3 of the Phosphoric acid esterase needed for reaction is provided, such as, can uses 400-10,000U/mL alkaline phosphatase, 50-150mM Tris-Cl(pH8.0-9.0), 5-10mM MgCl
2, 20-50% glycerine.
The reagent 4 of the reactant needed for molybdenum blue color reaction is provided, such as, can uses 0.1-0.2% (w/v) ammonium molybdate solution, 1-5% (v/v) sulfuric acid.
In one embodiment of the invention, detect in Phospholipase C enzyme product alive and also comprise:
Carry out the buffer system of PLC reaction and the reagent 5 of the active metallic ion needed for PLC reaction be provided, such as, can use 25-50mM Tris-Cl(pH6.5-8.0), 2-5mM CaCl
2.
When product of the present invention uses, also needing to add the reductive agent provided needed for molybdenum blue color reaction, for using reductive agent better, usually needing Fresh, therefore, in most cases made by oneself by user, also can comprise reductive agent in the product.In the embodiment using product of the present invention, described reductive agent is 8%-10%(w/v) xitix, 1%-2% tin protochloride, or 0.5%-1% phenol.
embodiment
In following embodiment of the present invention, the alkaline phosphatase (AlkalinePhosphatase-Calf intestine, AP) in calf intestinal source is purchased from Takara company, and phosphatidylcholine specific phospholipase is purchased from DSM (China) company limited.
Prepared by embodiment 1, typical curve
Get the dipotassium hydrogen phosphate reagent powder 0.1431g of oven dry, be dissolved in deionized water, be settled to 100mL, be mixed with 1mg/mL PO
4 3-dipotassium hydrogen phosphate solution.
From 1mg/mL PO
4 3-dipotassium hydrogen phosphate solution take out 0 respectively, 2,5,8,10,15,20,50 μ L, add deionized water and complement to 940 μ L, then add the 10%(w/v of 20 μ L) ascorbic acid solution, add the 2.5%(w/v of 40 μ L after shaking up 30s) ammonium molybdate solution (solvent is 30% sulphuric acid soln), finally be settled to 1mL, final PO
4 3-concentration is 0,2,5,8,10,15,20,50 μ g/mL.Each concentration point is parallel does 3 repeat samples.
After 37 DEG C of water-bath 10min, under absorbancy 700nm, carry out light absorption value detection, carry out linear fit to the data obtained, namely obtain the typical curve that P-Mo blue detects, result as shown in Figure 1.
Fig. 1 result shows: PO
4 3-concentration is in the scope of 0-10 μ g/mL, and P-Mo blue detects the method for inorganic phosphorus in linear interval, obtains equation of linear regression as follows: y=0.058*x, R through matching
2=0.9989.
Embodiment 2,
Prepare 10 μ g/ μ L PO
4 3-phosphorylcholine solution, get 0 respectively, 5,10,20,40,50,80 μ gPO
4 3-phosphorylcholine solution in total system 100 μ L, this reaction system is also containing 50mM Tris-Cl(pH9.0), 10mM MgCl
2, add alkaline phosphatase to 10U/mL.
37 DEG C of water-bath 30min, join in 840 μ L deionized waters, then add 10% (w/v) ascorbic acid solution of 20 μ L, add 2.5% (w/v) ammonium molybdate solution of 40 μ L, be finally settled to 1mL after shaking up 30 seconds.
37 DEG C of water-bath 10min, carry out light absorption value detection under absorbancy 700nm, obtain corresponding PO after the light absorption value obtained is carried out linear regression
4 3-quality, by the product P O obtained thus
4 3-total mass number is divided by the substrate PO originally added
4 3-total mass number, then divided by the reaction times, namely obtain the alkaline phosphatase of identical amount at different substrate PO
4 3-speed of response during total mass number.With PO
4 3-substrate total mass number is X-coordinate, and with the speed of response calculated for ordinate zou, the relation of both acquisitions, result as shown in Figure 2.
Fig. 2 result shows: in 30min reaction process, PO
4 3-when amount of substrate is 50 μ g, speed of response no longer obviously increases, and therefore, selects PO
4 3-amount of substrate is 50 μ g, as the optimal conditions of follow-up test.
Embodiment 3,
Alkaline phosphatase is carried out gradient dilution, gets 0,0.05,0.1,0.2,0.4,0.5,1.0,1.5U respectively in 100 μ L systems, this reaction system is also containing 50mM Tris-Cl(pH9.0), 10mM MgCl
2, 50 μ g PO
4 3-phosphorylcholine solution.
37 DEG C of water-bath 30min, carry out molybdenum blue method colour developing and detection by the method for embodiment 2, obtain corresponding PO after the light absorption value obtained is carried out linear regression
4 3-quality, by the product P O obtained thus
4 3-total mass number is divided by the 50 μ g substrate PO originally added
4 3-, then be multiplied by 100%, namely obtain reaction product PO
4 3-production rate.Take alkaline phosphatase concentration as X-coordinate, with reaction product production rate for ordinate zou, the relation of both acquisitions, result as shown in Figure 3.
Fig. 3 result shows: along with the increase of CIAP enzyme amount, the product production rate of reaction also increases, and when reaching 10U/mL, product rate is close to gently, refers at PO
4 3-in substrate (50 μ g) situation more than needed, the alkaline phosphatase enzyme amount of 10U/mL can carry out maximum enzyme shear force.Therefore, using the alkaline phosphatase enzyme amount of 10U/mL as the optimal conditions of follow-up test.
Embodiment 4
In the present embodiment, using soybean phospholipid as phosphatidylcholine specific phospholipase (PC-PLC, hereinafter referred to as PLC) reaction substrate, detect PLC enzyme to live, and it is alive to detect PLC enzyme with the method (Kook-HwaSeo & Jong Il Rhee.High-level expression of recombinant phospho lipase C fromBacillus cereus in Pichia pastoris and its characterization. (2004) BiotechnologyLetters26:1475 – 1479) of p-NPPC synthesis substrate, contrast, detailed process is as follows:
4.1 two phase reaction
Soybean phospholipid is dissolved in chloroform, carries out two phase reaction.
In the reaction system of 200 μ L, the soybean phospholipid containing final concentration 0.5% (w/v), 25mM Tris-Cl(pH7.5), 5mM CaCl
2.Dilute PLC enzyme, Dilution ratio is respectively 1:25 again, 1:50,1:100,1:200,1:400,1:500,1:800,1:1000,1:1600,1:2000,1:3200,1:4000,1:6400,1:12800,1:25600, join each dilution enzyme liquid 20 μ L in reaction system.Control group adopts the PLC of boiling water bath 5min and carries out 1:500 dilution.Sample is placed in the reaction 37 DEG C of shaking tables carrying out 15min, 30min, 60min, shaking speed is 150rpm.Again in the centrifugal 2min of 12,000rpm.
Draw centrifugal after supernatant 80 μ L in the phosphatase reaction system of 100 μ L, this system is also containing 50mM Tris-Cl(pH9.0), 10mM MgCl
2, AP10U/mL.At 37 DEG C, water-bath 30min.
After reaction, add the ionized water of 840 μ L, then add the 10%(w/v of 20 μ L) xitix, and the 2.5%(w/v of 40 μ L) ammonium molybdate solution.37 DEG C of colour developing 10min.Get the solution after colour developing and detect absorbancy in 700nm, wherein, the detected result (PLC enzyme Dilution ratio is 1:500-1:6400) of reaction 30min is as shown in table 1, and to contrast in the PLC enzyme of boiling water bath 5min deactivation as sex change.
Table 1 molybdenum blue method is to the viability examination result of the commercial enzyme PLC after gradient dilution
| PLC enzyme concn * | Absorbance value | Vigor (U/mL) | |
| 1 | Sex change contrasts | 0 | 0 |
| 2 | 1.56E-04 | 0.157 | 759.830611 |
| 3 | 2.50E-04 | 0.333 | 1007.259528 |
| 4 | 3.13E-04 | 0.37 | 895.3418028 |
| 5 | 5.00E-04 | 0.645 | 975.4990926 |
| 6 | 6.25E-04 | 0.662 | 800.9679371 |
| 7 | 1.00E-03 | 1.077 | 814.4283122 |
| 8 | 1.25E-03 | 0.883 | 534.1802783 |
| 9 | 2.00E-03 | 1.038 | 392.4682396 |
*: PLC enzyme concn is defined as with PLC enzyme original liquid concentration for 1, by the value of this value divided by the multiple gained of dilution.
With phosphatidylcholine specific phospholipase Dilution ratio (Dilution ratio is for 1:500-1:6400) for X-coordinate, 700nm light absorption value is ordinate zou, obtain the relation curve of the phosphatidylcholine specific phospholipase Dilution ratio-700nm light absorption value of differential responses time, result as shown in Figure 4.
4.2 water react
In order to contrast the efficiency difference of two phase reaction and single water react, devise the PLC water react experiment of 30min, detailed process is as follows simultaneously:
Soybean phospholipid is soluble in water, be mixed with the substrate solution of 1% (w/v).In the reaction system of 200 μ L, containing soybean phospholipid 0.5% (w/v), 25mM Tris-Cl(pH7.5), 5mM CaCl
2, by the method for embodiment 4.1, gradient dilution is carried out to PLC enzyme.At 37 DEG C, water-bath 30min.
Add 200 μ L methyl alcohol after reaction, mixing 10s, in the centrifugal 2min of 12,000rpm.
Draw centrifugal after supernatant 160 μ L in the phosphatase reaction system of 200 μ L, this system is also containing 50mM Tris-Cl(pH9.0), 10mM MgCl
2, AP10U/mL.At 37 DEG C, water-bath 30min.
Respectively add the ionized water of 740 μ L after reaction, then add the 10%(w/v of 20 μ L) xitix, and the 2.5%(w/v of 40 μ L) ammonium molybdate solution.37 DEG C of colour developing 10min.Get the solution after colour developing and carry out absorbancy 700nm detection.With phosphatidylcholine specific phospholipase Dilution ratio (PLC enzyme Dilution ratio is for 1:500-1:6400) for X-coordinate, 700nm light absorption value is ordinate zou, when obtaining water react, the relation curve of phosphatidylcholine specific phospholipase Dilution ratio and 700nm light absorption value, result as shown in Figure 4.
Fig. 4 result shows: along with PLC concentration increases, the light absorption value at the 700nm place recorded increases gradually.Wherein, the light absorption value that each PLC extent of dilution of the two phase reaction of 30min presents all is greater than the light absorption value of the water react of 30min, and be about its twice, namely compared with the speed of response of water react, the speed of response of two phase reaction significantly improves, and is about its 2 times.
In conjunction with the typical curve that P-Mo blue detects, data analysis is returned: light absorption value data are substituted in typical curve equation, convert and obtain the mass concentration of phosphate radical corresponding to this light absorption value, be multiplied by the cumulative volume that light absorption value detects again, obtain the total mass number of phosphate radical corresponding to each light absorption value, through carrying out volume conversion, divided by the molar mass number of phosphate radical, obtain the total mole number of phosphate radical corresponding to each light absorption value, divided by the reaction times, amass divided by the PLC enzyme liquid joined in total reaction again, can calculate in the dilution reaction of each PLC, the unit of activity number that PLC presents, thus find PLC to increase (extent of dilution minimizing) with concentration, between the linear section that the positive correlation of molybdenum blue light absorption value increases.
Result shows, and in extent of dilution 1:1000-1:4000 interval, the concentration of light absorption value and PLC has linear relationship.After being multiplied by extension rate with the data in this interval, as the energy value of commercial enzyme PLC.PLC vigor size is recorded a level after data linear regression analysis, average 898U/mL, thinks that this value is the energy value being recorded commercial enzyme PLC by molybdenum blue method, utilize contrast method to detect, result shows this commercial enzyme PLC enzyme and lives as 834U/ml, and difference is 7.12%.
Embodiment 5, wavelength chooses
For determining the wavelength that suitable light absorption value detects, respectively 500,550,600,650,700,750,800,850,900nm detects molybdenum blue light absorption value, result shows, the molybdenum blue light absorption value that 600-800nm detects and the dilution good relationship of PLC, further, 560,570,580,590,810,820,830,840nm detects molybdenum blue light absorption value, result shows, the molybdenum blue light absorption value detected at 810nm and the dilution good relationship of PLC.
According to this result, molybdenum blue light absorption value can be detected at 600-810nm.
Embodiment 6, fermented liquid detect
6 bacillus shown in use table 2 belong to bacterial strain (purchased from Guangdong institute of microbiology Culture Collection), carry out fermenting (with reference to J.SHILOACH et.al.Phospholipase-C from Bacilluscereus:Production, Purification, and Properties.Biotechnology and bioengineering.1973, Vol9:551-560), the fermented liquid obtained is carried out 1:10 separately, 1:20 dilutes, detect according to the method for embodiment 4.1, the reaction times of PLC is 30min, and method (the Kook-HwaSeo & Jong Il Rhee.High-level expression of recombinant phospho lipase C fromBacillus cereus in Pichia pastoris and its characterization.Biotechnology Letters of substrate is synthesized with p-NPPC, 2004, 26:1475 – 1479) detect the work of PLC enzyme, contrast, wherein, the method of data processing, comprise the method for calculation of PLC energy value, reference example 4.Detected result is in table 2.
The PLC viability examination of table 2 bacterial strain fermentation liquor
According to table 2 result, when belonging to the PLC Enzyme activity assay of the fermented liquid of bacterial strain to 6 bacillus for testing, use the result of the detection of method of the present invention, the result of synthesizing the detection of the method for substrate with p-NPPC is as a comparison more or less the same, therefore, the impact of fermented liquid on method of the present invention is less, method of the present invention can be used directly to live to the PLC enzyme of fermented liquid and detect.
According to above-mentioned result, method of the present invention not only may be used for the Enzyme activity assay of PLC enzyme liquid, and can be directly used in the detection of the PLC enzyme work in fermented liquid.Under suitable extent of dilution, molybdenum blue light absorption value and the positive correlation of PLC concentration, therefore, method of the present invention can also be used, directly judge that according to molybdenum blue light absorption value the relative enzyme of the PLC of fermented liquid is lived, thus high flux screening is carried out to the bacterial strain of expressing PLC, PLC enzyme high bacterial strain alive in rapid screening fermented liquid.
The foregoing is only preferred embodiment of the present invention, and be not used to limit substantial technological context of the present invention, substantial technological content of the present invention is broadly defined in the right of application, any technology entities that other people complete or method, if with application right define identical, also or a kind of change of equivalence, be all covered by being regarded as among this right.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. detect the method that Phospholipase C enzyme is lived, it is characterized in that, said method comprising the steps of:
1) Phospholipase C and/or its diluent enzymolysis composite phospholipid is used;
2) solid-liquid separation, after after using separation, solution contacts with Phosphoric acid esterase, contacts with molybdic acid or molybdate solution, detects light absorption value;
3) extent of dilution of the work of Phospholipase C enzyme and molybdenum blue light absorption value linear dependence is determined,
4) calculate described Phospholipase C enzyme to live.
2. screen a method for the bacterial strain of expressing Phospholipase C, it is characterized in that, said method comprising the steps of:
1) Phospholipase C and/or its diluent enzymolysis composite phospholipid is used;
2) solid-liquid separation, after after using separation, solution contacts with Phosphoric acid esterase, contacts with molybdic acid or molybdate solution, detects light absorption value.
3. detect the method that in fermented liquid, Phospholipase C enzyme is lived, it is characterized in that, said method comprising the steps of:
1) fermented liquid and/or its diluent enzymolysis composite phospholipid is used;
2) solid-liquid separation, after after using separation, solution contacts with Phosphoric acid esterase, contacts with molybdic acid or molybdate solution, detects light absorption value;
3) extent of dilution of the work of Phospholipase C enzyme and molybdenum blue light absorption value linear dependence is determined;
4) calculate described Phospholipase C enzyme to live.
4. the method according to any one of claim 1-3, is characterized in that,
Described composite phospholipid is: soybean phospholipid, peanut phosphatide, sesame phosphatide, one or more in castor-oil plant phosphatide.
5. the method according to any one of claim 1-3, is characterized in that, in the mixed solvent that described composite phospholipid is water-soluble, organic solvent or water and organic solvent form, preferred organic solvent is: trichloromethane.
6. the method according to any one of claim 1-3, it is characterized in that, after described separation, solution contacts with Phosphoric acid esterase and is: after being separated, solution and Phosphoric acid esterase react more than 30min in 30-45 DEG C, after being separated, the preferable reaction temperature of solution and Phosphoric acid esterase is 35-42 DEG C, be more preferably 37-40 DEG C, the preferred reaction times is 0.5h-2h.
7. the method according to any one of claim 1-3, is characterized in that, described detection light absorption value is the light absorption value detecting 600-810nm, is preferably the light absorption value of 650-750nm.
8. detect the product that Phospholipase C enzyme is lived, it is characterized in that, described product comprises: composite phospholipid or composite phospholipid solution, Phosphoric acid esterase storage system solution, phosphatase reaction system solution, and molybdenum blue color reaction thing solution; Preferred product is detection kit.
9. product as claimed in claim 8, it is characterized in that, described product also comprises Phospholipase C reaction system solution.
10. the method as described in claim 1-7 or the product as described in claim 8-9, it is characterized in that, described Phospholipase C is phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine specific phospholipase C and/or phosphatidic acid specific phospholipase C.
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| CN106701712A (en) * | 2015-11-13 | 2017-05-24 | 丰益(上海)生物技术研发中心有限公司 | New phospholipase |
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| CN112522363A (en) * | 2019-09-18 | 2021-03-19 | 丰益(上海)生物技术研发中心有限公司 | Improved method and product for detecting enzymatic activity of phospholipase C |
| CN113215205A (en) * | 2021-04-29 | 2021-08-06 | 华南理工大学 | Method for preparing hydroxy fatty acid |
| CN113933362A (en) * | 2021-10-19 | 2022-01-14 | 辽宁师范大学 | Preparation method of phospholipase C sensor based on atom transfer radical polymerization |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106701712A (en) * | 2015-11-13 | 2017-05-24 | 丰益(上海)生物技术研发中心有限公司 | New phospholipase |
| CN106701712B (en) * | 2015-11-13 | 2021-05-28 | 丰益(上海)生物技术研发中心有限公司 | Novel phospholipase |
| CN108239667A (en) * | 2016-12-27 | 2018-07-03 | 丰益(上海)生物技术研发中心有限公司 | A kind of detection method of phospholipase C vigor |
| CN108239667B (en) * | 2016-12-27 | 2022-08-12 | 丰益(上海)生物技术研发中心有限公司 | Detection method for activity of phospholipase C |
| CN112522363A (en) * | 2019-09-18 | 2021-03-19 | 丰益(上海)生物技术研发中心有限公司 | Improved method and product for detecting enzymatic activity of phospholipase C |
| CN112522363B (en) * | 2019-09-18 | 2024-09-10 | 丰益(上海)生物技术研发中心有限公司 | Improved methods and products for detecting phospholipase C enzyme activity |
| CN113215205A (en) * | 2021-04-29 | 2021-08-06 | 华南理工大学 | Method for preparing hydroxy fatty acid |
| CN113215205B (en) * | 2021-04-29 | 2023-09-01 | 华南理工大学 | A method for preparing hydroxy fatty acid |
| CN113933362A (en) * | 2021-10-19 | 2022-01-14 | 辽宁师范大学 | Preparation method of phospholipase C sensor based on atom transfer radical polymerization |
| CN113933362B (en) * | 2021-10-19 | 2023-09-05 | 辽宁师范大学 | A preparation method of phospholipase C sensor based on atom transfer radical polymerization |
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