CN101201356A - Method for rapidly measuring activity of penicillin acidated enzyme - Google Patents

Abstract

The invention relates to a method to detect the activity of enzyme, and discloses a method to quickly determine the activity of penicillin acylase. The invention adopts the dynamic method to detect the activity of enzyme by using a reader without breaking cells; gets a dynamic reaction curve; and calculates the activity of enzyme by using the gradient and the intercept acquired after linear regression of the dynamic curve. In addition, the influence of temperature on measurement of the activity of enzyme is simply studied; and compensation factors are established to facilitate rapid determination of the room temperature. The steps of the method of the invention are simple; the accuracy is high; the cost is saved. In addition, the invention is applicable for detecting the activity of penicillin acylase which is fermented and produced to be high density, and has high promotion value.

Description

A kind of method of fast measuring activity of penicillin acidated enzyme

Technical field

The present invention relates to the detection method that enzyme is lived.

Background technology

In order to solve the raising of germ to antibiotic resistant such as penicillin, people have invented artificial microbiotic such as artificial semisynthetic penicillin.Since the hydrolysis of PA ase method is produced 6-APA and 7-ADCA and is replaced traditional chemical cracking technology; as the main production method of cephalosporin and amoxycillin, from then on PA ase has also climbed up important industrial scene in semi-synthetic at present-lactam antibiotics industry that the PA ase method becomes.So far it is import that the immobilized penicillin acylated enzyme that domestic factory uses in cracking is produced also has half, so domesticly research and produce that PA ase is provided is crucial.Be used at present industrial immobilized penicillin G acylase in the world mainly from the fermenting and producing of Escherichia coli and bacillus megaterium.To its method of enzyme activity determination also in constantly groping to improve.Just introduce the assay method of general PA ase below.

At first, as substrate, enzymolysis obtains product and takes off phenylacetyl generation 6-amino-penicillanic acid (6-APA) people with raw material penicillin, and the growing amount of the volume 6-APA of analytical unit chronomere obtains enzyme and lives.In order to detect 6-APA, people have been developed diverse ways such as PDAB method etc.But found all that substrate penicillin all has the interference of can not ignore to the method for various detection product 6-APA, must extracting fall penicillin before measuring at once.This kind of enzyme not only complex steps of method for measuring of living like this, and sensitivity is very low.

The nineties, the someone has been developed fluorescence detection, uses fluorescamine in conjunction with 6-APA.The method can greatly reduce substrate penicillin to be disturbed, and simultaneously because make sensitivity improve 10 with fluoroscopic examination ⁴Doubly.Polypeptides matter and fluorescamine are serious in conjunction with influence, need to regulate pH to 4 and just can eliminate the effects of the act.The enzyme activity determination environment is changed greatly, make its accuracy and reliability reduce.

In fact use people in the near future just to find a kind of substrate of alternative penicillin at penicillin method: 6-nitro-3-phenylacetylamino benzoic acid (NIPAB), product are 6-nitro-3-aminobenzoic acid.The problem that substrate that this method not only solves disturbs, and product itself is detectable faint yellow material, can use the spectrophotometer detectable concentration under wavelength 405nm.So, simplified measuring process, and made sensitivity rise three more than the order of magnitude (about 5000 times) than the PDAB method.This method is exactly still to be the main NIPAB method of using so far.The NIPAB substrate is expensive, and its enzyme live reaction be not with penicillin this as substrate, consider that saving cost etc. still uses penicillin method so a lot of research is particularly produced.This patent of applying for just is based on the development of NIPAB.Below be traditional NIPAB method, step is more loaded down with trivial details:

The centrifugal supernatant that goes of fermentation liquor keeps precipitation, and sedimentation cell adopts clasmatosis method smudge cellses such as ultrasound wave, high pressure or freeze thawing, centrifuging and taking supernatant with the resuspended back of phosphate buffer.

Get above-mentioned supernatant 20 μ l and add in the phosphate buffer of 980 μ l 50mmol/L pH7.5 37 ℃ of insulations.Add 1ml more at the 0.9mg/ml of 37 ℃ of following preheatings NIPAB solution, accurately react 4min, add 1ml ethanol cessation reaction.

Get the above-mentioned reactant liquor that has stopped and under spectrophotometer wavelength 405nm, detect light absorption value.

The enzyme activity definition: under above-mentioned reaction conditions, the required PA ase of NIPAB of 1min hydrolysis 1 μ mol is 1 enzyme activity unit U.The thalline of enzyme is defined as every liter of every OD than vigor ₆₀₀The enzyme activity unit that thalline had.

The enzyme activity computing formula:

Wherein:

T: reaction time (min)

P: the slope of typical curve

V ₀: reflection system cumulative volume (ml)

A ₄₀₅: the sample optical density readings is poor in the blank sample optical density readings

V1: enzyme liquid amasss (ml)

Cuvette thickness is defaulted as 1, and unit is cm

The said method complex steps is brought very big inconvenience to operation.Usually in the laboratory for the fast measuring small amount of sample not broken cell directly survey enzyme and live, still be that ethanol all is difficult to thorough cessation reaction but test figure shows virose trichloroacetic acid.

Summary of the invention

Fundamental purpose of the present invention is exactly that PA ase for microbial fermentation production provides simple and efficient enzyme activity determination method.

The present invention is directed to the original loaded down with trivial details problem of NIPAB method step,, do not influence the angle that detects effect again and set out, a kind of method of new fast measuring activity of penicillin acidated enzyme is provided, comprise the following steps: from simplifying step

A) getting PA ase genetic engineering fermented liquid uses the phosphate buffer of pH7.4-7.8 or Tris-HCl damping fluid to dilute 10-50 doubly;

B) prepare NIPAB solution with phosphate buffer or the Tris-HCl damping fluid of pH7.4-7.8;

C) get the dilution of step a acquisition and the NIPAB solution of step b acquisition and successively add ELISA Plate, the amount of NIPAB is excessive in the control reaction system;

D) at 37 ℃, under microplate reader vibration mixing,, be horizontal ordinate with time 405nm place performance graph mode continuous detecting absorbance 4-5 minute, light absorption value is an ordinate acquisition dynamic response curve;

E) linear regression got slope k and intercept b after performance graph deducted blank plate and blank sample.

F) calculating enzyme lives:

Wherein enzyme activity unit is U/L.

Wherein:

P: the slope of typical curve

V ₀: reflection system cumulative volume, unit is ml

V ₁: enzyme liquid is long-pending, and unit is ml

Above-mentioned enzyme activity computing formula is by the enzyme activity basic calculating formula:

Conversion, wherein, A ₄₀₅/ t is exactly the variation of light absorption value in the unit interval, i.e. the k that obtains of microplate reader data processing.

Among the above-mentioned steps a, when getting PA ase genetic engineering fermented liquid with phosphate buffer or the dilution of Tris-HCl damping fluid, 50 times of the general dilutions of high density jar fermentation, shake flask fermentation is generally 10-50 doubly by stand density;

Preferably, damping fluid described in above-mentioned steps a and the b is the phosphate buffer of pH7.5.

Preferably, above-mentioned steps d is with in the microplate reader continuous detecting, and the time interval of adjacent twice detection is 0.5 minute.

Needing the amount of NIPAB in the control reaction system among the above-mentioned steps c is that excessive reason is: one timing of enzyme amount, and enzyme reaction will show as 0 order reaction along with concentration of substrate constantly is increased to a certain degree, i.e. and enzyme reaction speed no longer is subjected to the influence of concentration of substrate.Therefore under the excessive far away situation of substrate, enzyme reaction speed is linear to enzyme concentration.Generally speaking, cumulative volume based on reactant liquor, when diluted about 50 times of fermenting enzyme liquid, the concentration of NIPAB can guarantee to reach more than 0.65mg/ml in the entire reaction course to excessive, and 0.9mg/ml is approximately the saturation concentration that deionized water under the room temperature is made solvent.According to content disclosed by the invention and in conjunction with prior art, the personnel of affiliated technical field can control the extension rate of enzyme liquid, the concentration of NIPAB solution and the ratio that both mix neatly, thereby reach the purpose of guaranteeing that NIPAB is excessive.Certainly, control for convenience can be as preferred embodiment, and among the step b, the concentration of preparation NIPAB solution is 0.9mg/ml; Among the step c, the dilution of step a acquisition and the NIPAB solution of step b acquisition are successively added ELISA Plate with isopyknic amount.

In addition, the present invention has also carried out the research that temperature is surveyed value effect to the enzyme biopsy, has provided the correction coefficient when room temperature detects, so that detect more convenient.Wherein, when above-mentioned steps d at room temperature, when detecting with microplate reader, step f calculates the enzyme formula that enzyme lives of living and just is adjusted into:

Wherein:

Enzyme activity unit is U/L

P: the slope of typical curve

V ₀: reflection system cumulative volume, unit is ml

V ₁: enzyme liquid is long-pending, and unit is ml.

Because the characteristics of maximum of the present invention are not stop enzyme reaction, but measure a dynamic response curve, so the called after dynamic method is surveyed activity of penicillin acidated enzyme.

The present invention guarantees again that from the step that promptly simplifies the operation the angle of accuracy goes out to send to consider new activity of penicillin acidated enzyme detection method, and concrete thinking and principle are as follows:

Save the most loaded down with trivial details smudge cells step, this is the periplasmic space at the enzyme place because NIPAB can come in and go out, the not broken reaction that also can not influence NIPAB and enzyme of cell.The technology of producing semisynthetic antibiotics for the cell that contains PA ase in the direct immobilization periplasmic space that occurs not long ago has important meaning especially.

In addition, because sample often needs refrigerator to preserve in the actual detected process, freeze thawing pair cell badly broken, simple centrifugal extraction is very big to the enzyme that records loss influence alive, therefore also considers to have saved unnecessary operation stepss such as centrifugal.

Among the present invention, dynamic method is the key that guarantees (even raising) measurement accuracy after step is simplified in solution.Adopting dynamic method to measure enzyme work is owing to find after deliberation, as smudge cells not and still adopt cessation method, because ethanol is very undesirable to the effect of turnover cell cessation reaction, the fermentation liquor under the high-density large-scale condition of culture particularly, the reaction time of the accurate timing of 4min just can't guarantee at all.And test finds that medium component, cell tissue fragment etc. are very big to the absorbance value effect, and the omission of any sample liquid pre-treatment step all will be lived to enzyme under the end-point method is worth measuring the deviation of bringing the rate of can not neglecting.And after adopting " dynamic method ", just can omit most of unnecessary purifying preprocessing process in the end-point method, and impurity effect is disappeared, also improved the reliability of enzyme measured value alive.

Adopt dynamic method to survey enzyme work and omitted the sample preparation process, more complicated to measuring process.And the function of microplate reader is very suitable to solving the problem that runs in the PA ase measurement alive.Thereby therefore further adopt dynamic method to realize simplifying among the present invention and detect step, improve the purpose of accuracy in detection in conjunction with microplate reader.

Discover that use microplate reader to test in 4-5 minute of script end-point method, enzyme is lived the curve linear relation well, the useable linear regression Calculation gets the rate of curve value, lives thereby calculate the acquisition enzyme.

Improve the back scheme and more be applicable to the use microplate reader performance graph method survey activity of penicillin acidated enzyme that high density fermentation is produced than traditional NIPAB method

Following table is the present invention and the summary and the comparison that detected the activity of penicillin acidated enzyme method in the past:

	Penicillin method					Tradition NIPAB method	Dynamic method
								PDAB	Azanol	The iodine amount	Phenol red	The fluorescamine method
	Broken thalline	Need	Need	Need	Need								Need	Can not need	Do not need
The centrifugal removal of impurity						Need	Need	Need	Need	Need	Need	Do not need
	Measure required time	A few hours	A few hours	A few hours	A few hours								A few hours	About 1 hour	Several minutes
Substrate						Benzyl penicillin salt	Cosmocillin	Cosmocillin	Cosmocillin	Cosmocillin	NIPAB	Ditto
	The colour developing approach	PDAB makes developer	Generate colored substance with azanol under the substrate alkalescence	Consume iodine and survey the iodine coupling by product	Phenol red indicator is to the colour developing of alkaline product								Fluorescence indicator combines with product	Decomposition product colour developing itself	Ditto
Sensitivity (determinand can be surveyed Cmin)						6-APA 2.8*10 -3	Unknown	Unknown	Unknown	6-APA 7.7*10 -8	ANBA 5.5*10 -7	Ditto
	Instrument	The spectrophotometer colorimetric	The spectrophotometer colorimetric	The spectrophotometer colorimetric	The spectrophotometer colorimetric								Fluorospectrophotometer	Common spectrophotometer	Microplate reader
Cost						General reagent	General reagent	General reagent	General reagent	Fluorescamine price 5000-10000 unit	NIPAB price 5000-10000 unit	Every sample cost of determination

/g

/g

＜5 yuan

On the whole, method step of the present invention is simple, and the accuracy height is saved cost, and is fit to detect the activity of penicillin acidated enzyme that high density fermentation is produced, and has excellent popularization and is worth.

Embodiment

Determining of the drafting of embodiment 1 typical curve and slope p value.

What the NIPAB method detected under wavelength 405nm is the light absorption value of its enzymolysis product ANBA (5-amino-6-nitrobenzoic acid), therefore draws the typical curve of standard specimen ANBA earlier.Typical curve is that the concentration (mol/L) with ANBA is horizontal ordinate, A ₁₀₅Be the curve of ordinate, be linearity, can draw its slope p.

Experimental procedure:

A) get the ANBA standard items and be diluted to 10mmol/L, 20mmol/L, 40mmol/L, 50mmol/L, 60mmol/L and 80mmol/L respectively with 50mmol/L pH7.5 potassium phosphate damping fluid.

B) above every kind of concentration is put respectively in right amount in a test tube, total 1-6 test tube; Blank pipe is No. 0, is 50mmol/L pH7.5 potassium phosphate damping fluid.

C) above-mentioned 0--6 pipe places 37 ℃ of constant temperature.Under the 405nm wavelength, detect light absorption value, record with 721 spectrophotometers or microplate reader then.

D) be horizontal ordinate with ANBA concentration (mol/L), light absorption value is the ordinate mapping, and related data is tried to achieve in linear regression.

It is 8976 that experimental result obtains the p value.

Embodiment 2

(construction method is by " alcaligenes faecalis penicillin G acylase is constitutive expression and separation and purification in Escherichia coli " to make up Escherichia coli PA ase genetic engineering bacterium DH5 α/pKKFPGA; " bioengineering journal " 20 the 5th phases of volume; 736～740 is open), its host bacterium is DH5 α (supE44, Δ lacU169 (φ 80 lacZ Δ M15); hsdR17; recA1, endA1, gyrA96; thi-1, reA1).Plasmid pKKCA II, the Pkk235 plasmid of deriving does not contain lacI ^qAllele is expressed as composing type.

Adopt the above-mentioned Escherichia coli PA ase of different condition High Density Cultivation genetic engineering bacterium to contrast.The fermentation period of recombination bacillus coli mycin acylase gene engineering bacteria is 48h, 28 ℃ of fermentation temperatures, 500ml shake flask fermentation 220rpm.Different condition is respectively and contains 0.5mMCu in the fermentation medium ²⁺, contain 0.5mMNi ⁺And three kinds of conditions that do not contain these two kinds of ions.Respectively with the live measuring method and survey with microplate reader performance graph mensuration that enzyme is alive to be compared of centrifugally operated final state enzyme down not of smudge cells not.(annotate: data value is only with noting longitudinal comparison, i.e. difference between distinct methods, u is U/L)

Not smudge cells not the measuring method of living of the final state enzyme under the centrifugally operated survey enzyme and live

Test procedure:

A) get 9 samples of PA ase fermentation liquor under the different condition, be numbered 1-9 number;

B) the 1-9 sample is respectively with 50 times of 50mmol/L pH7.5 potassium phosphate damping fluid dilutions;

C) getting 9 each 1ml of mixed liquor that above-mentioned steps obtains and the pH7.5 potassium phosphate damping fluid 1ml that makes blank pipe places 10 10ml test tubes to be placed on 37 ℃ of water-bath constant temperature respectively;

D) capacity (15ml-20ml) is placed 37 ℃ of water-bath preheatings equally with the 0.9mg/ml NIPAB solution that 50mmol/L pH7.5 potassium phosphate damping fluid prepares;

E) pick up counting after being ready to, in 1-9 test tube and blank pipe, respectively add the NIPAB solution that the 1ml step d) is handled fast successively;

F) accurately during timing to 4 minute, in 1-9 test tube and blank pipe, respectively add 1ml90% (mass percent) ethanol fast successively and come cessation reaction;

G) record successively under 405nm 1-9 number with 721 spectrophotometers at once after stopping and the light absorption value of blank pipe, sample hose deducts blank pipe value and is required A in the formula (1) ₄₀₅, calculate enzyme and live.

It is as follows to obtain final enzyme live data

Sample number into spectrum	1	2	3	4	5	6	7	8	9
										Final state method enzyme u alive	1557.5	1270.1	1377.0	137.0	113.6	150.4	1380.3	1370.3	1377.0

2. microplate reader performance graph mensuration survey enzyme is lived:

Experimental facilities: the Multiskan MK3 type microplate reader of Thermo Labsystems company

Experimental procedure:

A) by operation instruction microplate reader is carried out the parameter setting, it is 1 minute that incubation time is set, 37 ℃ of incubation temperature.Be made as the vibration step after hatching, adopt detection of dynamic, be made as continuous detecting 4 minutes detection time at interval 0.5 minute;

B) get the 1-9 fermentation liquor and dilute 50 times with 50mmol/L pH7.5 potassium phosphate damping fluid;

C) get and add 96 hole ELISA Plate successively after each 100 μ l of step a mixed liquor that obtains and the pH7.5 potassium phosphate damping fluid of making blank pipe are preheated to 37 ℃;

D) in 10 holes of application of sample, add fast each 100 μ l of 0.9mg/ml NIPAB with the preparation of 50mmol/L pH7.5 potassium phosphate damping fluid 37 ℃ of preheatings;

E) back is put into instrument to 96 orifice plates, begins reaction and also measures automatically, and finishing the back acquisition is horizontal ordinate with time, and light absorption value is the dynamic response curve of ordinate;

F) linear regression got slope k and intercept b after performance graph deducted the blank plate scan values, calculated enzyme value alive with formula (1).

Table 1 is that microplate reader records raw data table, and performance graph is referring to Fig. 1.

Table 1

Time (min)	1	2	3	4	5	6	7	8	9
										1.000	0.113	0.092	0.110	0.030	0.021	0.020	0.099	0.076	0.096
1.500	0.123	0.098	0.118	0.031	0.022	0.019	0.111	0.085	0.106
										2.000	0.132	0.107	0.128	0.031	0.021	0.019	0.119	0.092	0.114
2.500	0.140	0.112	0.137	0.030	0.023	0.018	0.130	0.098	0.123
										3.000	0.148	0.118	0.144	0.029	0.021	0.016	0.136	0.105	0.129
3.500	0.157	0.124	0.153	0.029	0.022	0.017	0.146	0.111	0.137
										4.000	0.164	0.131	0.162	0.029	0.022	0.016	0.154	0.116	0.144

Fig. 1 is an example with " 1 group ", and linear regression obtains formula and is shown in the top.

Table 2 is to calculate the enzyme live data that finally obtains with formula (1):

Table 2

Numbering	1	2	3	4	5	6	7	8	9
										Enzyme u alive	188.6	143.2	192.6	0	2.4	0	200.5	147.2	175.9
The intercept enzyme is lived	1083.4	886.5	1033.7	350.5	235.9	236.7	923.1	719.0	911.6

3. smudge cells live measuring method and survey enzyme with microplate reader performance graph mensuration and live that the total enzyme of dynamic method relatively is alive always to be lower than the not end-point method under the centrifugally operated of smudge cells not of the final state enzyme under the centrifugally operated not, this is mainly due in the end-point method, and thoroughly cessation reaction and non-enzyme reaction cause the influence of absorbance ethanol in regulation 4min.

It is 0 o'clock the enzyme value of living that the work of intercept enzyme is defined as the reaction time, does not change in time and changes.Be that also can to occur enzyme work at 0 o'clock be because non-enzyme reaction factor affecting light absorption value why, thereby the formula that enzyme lives is calculated in substitution with light absorption value after, can obtain the corresponding enzyme value of living in the reaction time.Can find that from experiment sample is not done pre-service, and particularly the enzyme non-enzyme reaction factor of living when low is huge to the light absorption value influence of product place wavelength, they do not change in time and change.In this method, the existence that the intercept enzyme is lived can't influence the accuracy of enzyme value alive.

The factor that embodiment 3 utilizes dynamic method survey activity of penicillin acidated enzyme to study and in the measurement light absorption value exerted an influence is at first set the enzyme value alive that all understand the factor correspondence that light absorption value is exerted an influence:

In the solubility nutrient culture media:

The enzyme that born of the same parents are outer: it is secreted into the outer enzyme of born of the same parents on a small quantity for certain mechanism, also has reason fragmentations such as cell freeze thawing, the enzyme in the nutrient culture media, and its enzyme work is made as u1;

The solubility nutrient culture media: its enzyme work is made as ua.

Remove the supernatant material:

Endocellular enzyme: its enzyme work is made as u2;

Thalline and soluble nutrient culture media: its enzyme work is made as ub.

Press foregoing description, in dynamic method was measured, the numerical value of enzyme u alive was u1+u2, and the numerical value that the intercept enzyme is lived is ua+ub

With the sample 1-9 among the embodiment 2 number, respectively get 10ml, remove supernatant after hydro-extractor 3000rpm10 minute, careful washing back is resuspended with pH7.5 potassium phosphate damping fluid, three times in this way.Measure result such as table 3 with microplate reader with the operation steps of dynamic method among the embodiment 2 again:

Table 3

Numbering	1	2	3	4	5	6	7	8	9
										Endocellular enzyme is lived	166.3	126.5	142.4	7.2	2.4	7.2	132.1	124.1	135.3
Go supernatant intercept enzyme to live	843.1	659.3	730.9	200.1	223.2	134.9	559.4	725.7	760.0

Endocellular enzyme work is u2 in the last table, goes the work of supernatant intercept enzyme to be ub.Associative list 2 like this, and calculating can get:

Table 4

Numbering	1	2	3	4	5	6	7	8	9
										u1	22.3	16.7	50.1	-7.2	0.0	-7.2	68.4	23.1	40.6
u2	166.3	126.5	142.4	7.2	2.4	7.2	132.1	124.1	135.3
										ua	240.3	227.2	302.8	150.4	12.7	101.9	363.7	-6.8	151.6
ub	843.1	659.3	730.9	200.1	223.2	134.9	559.4	725.7	760.0

Above-mentioned test findings shows, the enzyme major part in thoughtful space, have some owing to reasons such as freeze thawing broken wall outside born of the same parents.Insoluble matters such as thalline have the greatest impact to the lurid light absorption value of wavelength 405nm, and the influence of nutrient culture media etc. is also very big, and (as shaking bottle situation such as cultivation and composing type cultivation in short-term) was bigger to the contribution of extinction than enzyme reaction product when they were lived at low enzyme.

The not centrifugal removal impurity of embodiment 4 and not in the simple end-point method of smudge cells ethanol stop Research on effect

Below experiment be for not centrifugal removal impurity and not in the simple end-point method of smudge cells ethanol stop Research on effect.Same sample stops effect over time under the termination of different ethanol concentration.Experimental procedure is substantially with example 2, and variation place is listed in table 5 with the result:

Table 5

Bacterium liquid	20μl		20μl		20μL		10μL
									PBS	980μl		980μl		980μl		490μl
NIPAB solution	1ml		1ml		1ml		1ml
									Ethanol	1ml90％		2ml90％		1ml is anhydrous		1ml90％
	Light absorption value	Enzyme is lived	Light absorption value	Enzyme is lived	Light absorption value	Enzyme is lived	Light absorption value	Enzyme is lived
									0min	0.30	1253	0.175	975	0.27	1128	0.175	1218
3min	0.42	1755	0.18	1003	0.28	1170	0.185	1288
									7min	0.31	1295	0.245	1365	0.28	1170	0.19	1323
10min	0.31	1295	0.21	1170	0.29	1212	0.19	1323
									13min	0.31	1295	0.205	1141	0.27	1128	0.185	1288

The result shows that the termination effect of Different concentrations of alcohol has difference, and fluctuation is more obvious in time.Owing to can not the cessation reaction enzyme live and rise, live and descend to some extent owing to reason enzymes such as environmental factor marked change such as temperature and product decomposition in long back of termination time during beginning.

Ethanol stops the bad meeting of effect and brings appreciable error as can be seen, and batch samples when measuring manual cessation reaction will be difficult to the accurate control time.But it is bad little many with respect to the impurity effect degree that comparative example 3 can find that ethanol stops effect, and therefore traditional final state method can drop to error very low in the small amount of sample of centrifugal removal impurity is measured.

Embodiment 5 classic methods and the comparison of dynamic method newly

This example is classic method and the comparison of dynamic method newly, and the result shows that new method is still keeping very high degree of accuracy after omitting most of loaded down with trivial details sample preparation process, overcome the impurity interference fully.The fermentation broth sample of certain a collection of cultivation adopts following tradition and dynamic method two kinds of different processing, measuring methods to measure respectively.

Use two kinds of methods in this example of smudge cells in the tradition method: the freeze-thaw method binding lysozyme is handled, ultrasonic disruption.Tradition method step:

A) get the centrifugal 6000rpm 15min of fermentation liquor 10ml, go supernatant to keep precipitation;

B) sedimentation cell adopts clasmatosis method smudge cellses such as ultrasound wave, freeze thawing with the resuspended back of 50mmol/L pH7.5 potassium phosphate damping fluid, and centrifugal 6000rpm 15min gets supernatant;

C) get above-mentioned supernatant 20 μ l and add in the potassium phosphate damping fluid of 980 μ l 50mmol/L pH7.5 37 ℃ of insulations.Add 1ml more at the 0.9mg/ml of 37 ℃ of following preheatings NIPAB solution, accurately react 4min, add 1ml ethanol cessation reaction;

D) blank effective 1ml 50mmol/L pH7.5 potassium phosphate damping fluid replaces the fermentation liquor of dilution, and other operations are identical;

E) get the above-mentioned reactant liquor that has stopped and under spectrophotometer wavelength 405nm, detect light absorption value.

The light absorption value that obtains the sample of ultrasound wave and the processing of freeze-thaw method smudge cells by above-mentioned steps is respectively 0.024,0.030, and by formula the enzyme that calculates is lived and is 100u and 125u.

The dynamic method step is with example 2.In addition, the sample that aforementioned conventional method sample treatment is handled also uses the microplate reader kinetic measurement to calculate, and the result lists in the lump, as following table.Microplate reader gained performance graph data processing is with example 2.

Table 6

	Dynamic method	Ultrasound wave centrifugation	Ultrasound wave	Freeze-thaw method centrifugation	Freeze-thaw method
						Enzyme is lived	138.5	7.2	102.6	11.9	121.8
The intercept enzyme is lived	1501	1039.7	88.7	814.4	65.6

Be different from classic method, this example has all been measured enzyme to the supernatant of broken back centrifugally operated gained with deposited components and has been lived, and lists in table together.The enzyme that this table shows is lived and coincide with the enzyme of preceding traditional method reality is alive: traditional method may be lost because of the product in the processing procedure a little less than dynamic method; Fragmentation may be thorough inadequately, still has a small amount of enzyme to live in the centrifugation; The ultrasonic disruption enzyme is lived low slightly, may be because the high temperature that specific operational losses product such as ultrasound wave produce; The intercept enzyme is lived significantly, and insolubles precipitation particularly conforms to situation in the previous examples.

This case verification new dynamic method conform to the measurement result of classic method, be reliable.

Embodiment 6 temperature are to the influence test of enzyme biopsy survey method

Because temperature control, vibratory equipment etc. are not the essential accessories of microplate reader, belong to annex, so a lot of microplate reader does not have devices such as temperature control.Wherein temperature control is alive very important to enzyme reaction survey enzyme, and enzyme reaction speed is subjected to the temperature appreciable impact.Consider this, under different temperature, survey enzyme work with microplate reader and probe into rule.

With PA ase genetic engineering bacterium (with embodiment 2) fermented and cultured.The fermentation of 5L jar, liquid amount 1L, inoculum concentration 10%.Throughput 0.5VVM, 28 ℃ of cultivation temperature, dissolved oxygen remains on more than 30%.Fermentation period is 72h, and this is the 48h sampling.Get three parallel samples and be numbered a, b, c, survey enzyme down at 21 ℃, 27 ℃, 32 ℃, 37 ℃, 42 ℃ and 47 ℃ respectively and live, the test procedure of the dynamic method among the logical embodiment 2 of step.The gained data with temperature (℃) be that horizontal ordinate, enzyme are lived (U/L) for total coordinate mapping, as Fig. 2

Near linear, amplitude of variation is less before 37 ℃ for exponential type curve as can be seen, from room temperature to 37 ℃ growth less than and near 20% the enzyme value of living.

After cultivate the fermentation broth sample that obtains with a certain batch fermentation again, use the thermostat water bath temperature control with end-point method, respectively 17 ℃, 22 ℃, 27 ℃, 32 ℃, 37 ℃, 42 ℃, 47 ℃ and 52 ℃ down 721 spectrophotometric instrumentation enzymes live.The gained data are calculated to such an extent that enzyme is lived with formula (1), equally with temperature (℃) be that horizontal ordinate, enzyme are lived (U/L) for total coordinate mapping, get Fig. 3.

The conclusion that obtains that temperature is studied with final state one point method and dynamic method the influence of enzyme reaction speed conforms to, and the optimum temperature of enzyme is about 45 ℃.And before 37 ℃, keeping linear approximate relationship, live from room temperature to 37 ℃ enzyme and rise 20%.Obtain in view of the above enzyme that room temperature detects live with 37 ℃ under record the correction coefficient that enzyme is lived: enzyme (the 37 ℃)=1.2 * room temperature enzyme of living is lived.

With reference to above-mentioned experiment, for the microplate reader that does not have attemperating unit, the enzyme that records under room temperature in laboratory value alive can be multiplied by 1.2 these correction coefficient, also can be multiplied by corresponding coefficient to reduce error for the temperature beyond the room temperature.For 15 ℃ and following low temperature and the inapplicable method that multiply by correction coefficient of high temperature correction coefficient more than 37 ℃.

Claims (5)

1. the method for a fast measuring activity of penicillin acidated enzyme comprises the following steps:

A) get phosphate buffer or the Tris-HCl damping fluid dilution suitable multiple of PA ase genetic engineering fermented liquid with pH7.4-7.8;

B) prepare NIPAB solution with phosphate buffer or the Tris-HCl damping fluid of pH7.4-7.8;

C) get the dilution of step a acquisition and the NIPAB solution of step b acquisition and successively add ELISA Plate, the amount of NIPAB is excessive in the control reaction system;

D) at 37 ℃, under microplate reader vibration mixing,, be horizontal ordinate with time 405nm place performance graph mode continuous detecting absorbance 4-5 minute, light absorption value is an ordinate acquisition dynamic response curve;

E) linear regression got slope k and intercept b after performance graph deducted blank plate and blank sample;

F) calculating enzyme lives:

Wherein:

Enzyme activity unit is U/L

P: the slope of typical curve

V ₀: reflection system cumulative volume, unit is ml

V ₁: enzyme liquid is long-pending, and unit is ml.

2. the method for fast measuring activity of penicillin acidated enzyme according to claim 1 is characterized in that damping fluid described in step a and the b is the phosphate buffer of pH7.5.

3. the method for fast measuring activity of penicillin acidated enzyme according to claim 1; it is characterized in that; the concentration of the solution of NIPAB described in the step b is 0.9mg/ml, and the solution that mixed liquor that a of step described in the step c obtains and step b obtain successively adds ELISA Plate with isopyknic amount.

4. the method for fast measuring activity of penicillin acidated enzyme according to claim 1, it is characterized in that, described steps d is at room temperature, under microplate reader vibration mixing, 405nm place performance graph mode continuous detecting absorbance 4-5 minute, with time is horizontal ordinate, and light absorption value is that ordinate obtains the dynamic response curve; Among the described step f, the formula that the calculating enzyme is lived is:

5. as the method for fast measuring activity of penicillin acidated enzyme as described in claim 1 or 2 or 3, it is characterized in that, in the steps d,

During with the microplate reader kinetic measurement, the time interval of adjacent twice detection is 0.5 minute.

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