CN105296595B - A kind of bioenzyme activity detection method based on nanogold growth - Google Patents
A kind of bioenzyme activity detection method based on nanogold growth Download PDFInfo
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- CN105296595B CN105296595B CN201510711739.8A CN201510711739A CN105296595B CN 105296595 B CN105296595 B CN 105296595B CN 201510711739 A CN201510711739 A CN 201510711739A CN 105296595 B CN105296595 B CN 105296595B
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Abstract
The present invention relates to a kind of bioenzyme activity detection methods based on nanogold growth.Generated saccharide compound has reproducibility after biological enzyme substrate hydrolysis, under certain condition, can react with gold chloride and generate nanogold, so that solution is developed the color, the size of enzyme activity can be speculated according to the size of solution absorbance.Method includes the following steps: the first step, the drafting of working curve；Second step, enzyme digestion reaction；Third step, the measurement of bioenzyme activity.Detection method of the present invention is accurate and reliable, practical, instrument requirements are simple, is to measure a kind of reliable method in biological enzyme activity development process suitable for the operation under normal condition.
Description
Technical field
The present invention relates to nanosecond science and technology field and method for detecting enzymatic activity fields more particularly to nano material in biological enzyme activity
Property detection in application.
Background technique
The links such as enzyme preparation production, application and research all be unable to do without the measurement of enzymatic activity, such as lead to during the fermentation
The activity of measurement enzyme is crossed to reach maximum yield of enzyme；The whereabouts of enzyme is grasped, by measurement enzymatic activity in extractive process to reach
Best yield；By measurement enzymatic activity in the application process of enzyme, to grasp handy enzyme amount.Therefore, the production of enzyme preparation,
Using and research process in Accurate Determining enzymatic activity be all it is highly important, exploitation is easy, enzyme assay accurately and fast is ground
The method of studying carefully has great importance.
Currently, spectrophotometry is the detection most common method of bioenzyme activity, 3,5- dinitrosalicylic acid is generally selected
(DNS) it is used as color developing agent.The principle of the method is: degradation of substrates is reduced sugar by biological enzyme, and reduced sugar is under the conditions of boiling water bath
Chromogenic reaction occurs with DNS reagent, the depth of reaction solution color is directly proportional to the reduction sugar amount that enzymatic hydrolysis generates, the generation of reduced sugar
Amount is again directly proportional to the vigor of biological enzyme in reaction solution, by the absorbance of spectrophotometric determination reaction solution, to calculate
The vigor of biological enzyme in reaction solution.Although this method has operability well, but still in place of Shortcomings, such as color developing agent DNS
Solution preparation is cumbersome and waiting for a long time, and the professional that need to have certain technical level prepares, if preparation of reagents is unsuccessful, directly
Connect influence experimental result.Therefore, suitable reagent is found instead of DNS color developing agent, establishes the new spectrophotometry of one kind to detect
The activity of biological enzyme is still problem to be solved.
Nano material is one of research hotspot in recent years, is widely used in physics, chemistry, electricity with its unique performance
The every field such as son, material.Wherein, it since nanogold has excellent optical property, is answered extensively in biochemical analysis field
With.Studies have shown that under certain condition, some determinands with reproducibility can be reacted with gold chloride generates nanogold, make molten
Liquid by it is colourless or it is faint yellow be changed into claret or purple, and the absorbance of nano-Au solution and the concentration of determinand exist it is quantitative
Relationship.Neurotransmitter, hydrogen peroxide, antioxidant, beta-stimulants and tetracycline antibiotics are realized in this way
The detection of equal many kinds of substance.The present invention proposes the reaction detection biological enzyme using nanogold growth under the inspiration of above-mentioned work
Activity.Its principle is: degradation of substrates is reduced sugar by biological enzyme, and reduced sugar reacts life under certain reaction condition with gold chloride
At nanogold, in ultraviolet-visible spectrum, the absorbance of nano-Au solution is directly proportional to the reduction sugar amount that enzymatic hydrolysis generates, reduction
The amount of sugar is again directly proportional to the vigor of biological enzyme in reaction solution, to calculate the vigor of biological enzyme in reaction solution.Institute of the present invention
The detection method of foundation can be used as the supplement of existing bioenzyme activity detection method, enrich the detection method of enzymatic activity, be
The Activity determination of enzyme provides more selections.
Summary of the invention
The purpose of the present invention is to provide a kind of bioenzyme activity detection methods based on nanogold growth.
The purpose of the present invention is achieved by the following technical measures:
A, the drafting of working curve: being diluted to a series of titer for saccharide compound standard items, by a series of mark
Quasi- liquid reacts the nano-Au solution to form various concentration with gold chloride respectively, by measuring the absorbance of each reaction solution, obtains sugar
Class compound concentration and absorbance relationship working curve.
B, the substrate solution V prepared with acetic acid-sodium acetate buffer solution enzyme digestion reaction: is added in test tube1ML is placed in water
5min is preheated in bath, and enzyme dilution V2mL is added and obtains mixed liquor, after mixed liquor is reacted 30min in a water bath, takes V3μ L is mixed
It closes liquid to be added in 1 new test tube, then be reacted with gold chloride, and make the total volume V of solution4μL；Above-mentioned enzyme digestion reaction does 3
Secondary parallel laboratory test, measurement result are averaged.
C, measurement of ultraviolet-visible spectrophotometer appropriate wavelength condition the measurement of bioenzyme activity: is used after the completion of enzyme digestion reaction
The absorbance value of lower mixed solution determines that substrate for enzymatic activity hydrolyzes generated reduced sugar according to solution absorbance and working curve
Concentration；It defines 1g solid enzyme powder (or 1mL liquid enzymes) under optimum conditions, hydrolyzes substrate solution per minute and discharge 1 μm of ol
Enzyme amount needed for reduced sugar is a unit of activity (IU), calculates the vigor of biological enzyme according to the following formula:
Wherein, X is the vigor (IU) of biological enzyme；P is the concentration (g/L) of corresponding reduced sugar on standard curve；C is enzyme solution
Original concentration (g/L)；T is enzyme reaction time (min)；M is the molal weight of reduced sugar；V1、V2、V3、V4As described in b above；
106For transforming factor；DfFor enzyme solution extension rate.
Preferably, the condition that saccharide compound of the present invention is reacted with gold chloride are as follows: containing chlorine gold in reaction mixture
Sour 1-2mM, hexadecyltrimethylammonium chloride (CTAC) or cetyl trimethylammonium bromide (CTAB) 3.6-12mM, Yi Jiqing
Sodium oxide molybdena 3-5M；Reaction mixture is in 70 DEG C of reaction 10-15min.Wherein, the amount of gold chloride is big excessive compared with reduced sugar, with
Guarantee that reducing sugar reaction is complete；CTAC or CTAB can prevent the nanogold coagulation generated, play the role of sensitizer；Sodium hydroxide
It plays a key effect for reduced sugar with reacting for gold chloride, gold chloride can will be in reduced sugar under the conditions of existing for the sodium hydroxide
Aldehyde radical be oxidized to carboxyl, and itself is reduced to nanogold.
Preferably, method for detecting enzymatic activity of the present invention is suitable for meeting all bioenzyme activities of following condition
Detection, i.e., under certain reaction condition, substrate is not reacted with gold chloride, and enzymolysis product reacts with gold chloride and generates nanogold.
For example, substrate is sodium carboxymethylcellulose using the activity of method for detecting enzymatic activity of the present invention measurement cellulase, with
Glucose represents reduced sugar and works curve；The activity of zytase is measured, substrate is xylan, represents reduced sugar with xylose and does
Working curve；The activity of amylase is measured, substrate is starch, represents reduced sugar with maltose and works curve；It is poly- to measure sweet dew
The activity of carbohydrase, substrate are mannosan, represent reduced sugar with mannose and work curve.
Preferably, saccharide compound standard items of the present invention, substrate, biological enzyme sample are with pH=4.5-5.5's
Acetic acid-sodium acetate buffer solution is prepared, to meet the Optimal pH condition of substrate for enzymatic activity reaction.
Detailed description of the invention
Fig. 1 is inventive principle schematic diagram.
Fig. 2 is solution colour variation diagram (the from left to right concentration of glucose after different glucose is reacted with gold chloride
It is respectively as follows: 0.025,0.042,0.058,0.075,0.092g/L)
Fig. 3 is after different glucose is reacted with gold chloride, solution absorbance variation diagram (from a to the e concentration of glucose
It is respectively as follows: 0.025,0.042,0.058,0.075,0.092g/L)
Fig. 4 is concentration of glucose and solution absorbance linear relationship chart.
Specific embodiment
Embodiment 1
Embodiment 1 is used to illustrate to be used to detect the effect of cellulase activity when method provided by the present invention.
A, the drafting of working curve: being diluted to a series of titer for dextrose standard sample, under 70 DEG C of water bath conditions,
With contain gold chloride 1mM, hexadecyltrimethylammonium chloride 3.6mM and sodium hydroxide 4M solution hybrid reaction 15min,
Obtain the different purple solution of the depth (Fig. 2).The absorbance that wavelength is each reaction solution at 550nm is measured after being cooled to room temperature
Amax, obtain concentration of glucose and absorbance relationship working curve (Fig. 4).
B, enzyme digestion reaction: the carboxymethyl cellulose for being 1% with the acetic acid of pH value 4.6-sodium acetate buffer solution compound concentration
Sodium solution.Cellulase sample 0.1000g is weighed, is dissolved with the acetic acid of pH value 4.6-sodium acetate buffer solution, at normal temperature magnetic
Power stirs 30min or so, is settled to 100mL, spare.3 test tubes are taken, 1% carboxymethylcellulose sodium solution is separately added into
1.5mL is placed in 40 DEG C of water-baths and preheats 5min, and enzyme dilution 0.5mL is added, mixed liquor is reacted 30min in 40 DEG C of water-baths
Afterwards, it respectively takes 200 μ L mixed liquors to be separately added into 3 new test tubes, is reacted under the reaction condition of step a with gold chloride, and make
The total volume of solution is 2400 μ L.
C, the measurement of bioenzyme activity: enzyme digestion reaction is after the completion 550nm with measurement of ultraviolet-visible spectrophotometer wavelength
The absorbance for locating reaction solution is 0.328 ± 0.0114.It defines 1g solid enzyme powder under optimum conditions, it is fine to hydrolyze carboxymethyl per minute
Tieing up enzyme amount needed for plain sodium solution discharges 1 μm of ol reduced sugar is a unit of activity (IU), then according to formula can calculate by
The activity for surveying cellulase is 695 ± 19IU.
D, control experiment: the reliability in order to verify the above cellulase activity detection method selects everybody generally to receive
DNS method carry out control experiment.Appropriate DNS reagent is added into the glucose standard of various concentration, boiling water bath heats 5min,
Constant volume after room temperature is naturally cooled to, with the absorbance A of each reaction solution of measurement of ultraviolet-visible spectrophotometermax, it is dense to obtain glucose
Degree and absorbance relationship working curve.Then, 3 test tubes are taken, 1% prepared with acetic acid-sodium acetate buffer solution is separately added into
Carboxymethylcellulose sodium solution 1.5mL and enzyme dilution 0.5mL, after mixed liquor is reacted 30min in 40 DEG C of water-baths, respectively
It takes 200 μ L mixed liquors to be separately added into 3 new test tubes, appropriate DNS reagent, constant volume after reaction is added.It is done with standard blank sample
Blank control measures its maximum absorbance.It is 700 ± 15IU using the activity that DNS method obtains cellulase, is based on receiving with above-mentioned
The result that the cellulase activity detection method of rice gold growth is measured is consistent.It can be seen that life established by the present invention
Detection of the object method for detecting enzymatic activity for cellulase activity is accurately and reliably.
Embodiment 2
Embodiment 2 is used to illustrate to be used to detect the effect of xylanase activity when method provided by the present invention.
A, the drafting of working curve: being diluted to a series of titer for xylose standard product, under 70 DEG C of water bath conditions, with
Solution hybrid reaction 12min containing gold chloride 1mM, hexadecyltrimethylammonium chloride 3.6mM and sodium hydroxide 3M, obtains
The purple solution different to the depth.The absorbance A that wavelength is each reaction solution at 546nm is measured after being cooled to room temperaturemax, obtain
Xylose concentration and absorbance relationship working curve.
B, enzyme digestion reaction: weighing xylan 0.25g, and 0.08g sodium hydroxide is added, and is added 22mL water, magnetic agitation, slowly
Heating stops heating until xylan is completely dissolved, and continues to stir 30min, 0.125mL glacial acetic acid is added, continue magnetic agitation,
PH value is measured, makes its pH value 5.5, is settled to 25mL with acetic acid-sodium acetate buffer solution, is made into the wood that concentration is 100mg/mL
Glycan solution.Xylanase samples (vigor is within the scope of 2500-3500IU) 0.1000g is weighed, it is 5.5 that 40mL pH value, which is added,
Acetic acid-sodium acetate buffer solution, magnetic agitation 30min adds buffer solution and is settled to 100mL, be protected from light under the conditions of 4 DEG C
Save 1h~2h.It is centrifuged 3-5min, takes supernatant, it is spare with 25 times of buffer solution dilution.3 test tubes are taken, it is poly- to be separately added into wood
Sugar juice 1mL is placed in 37 DEG C of water-baths and preheats 5min, and xylan enzyme solution 1mL is added, mixed liquor is reacted in 37 DEG C of water-baths
After 30min, respectively takes 600 μ L mixed liquors to be separately added into 3 new test tubes, is reacted under the reaction condition of step a with gold chloride,
And make the 2400 μ L of total volume of solution.
C, the measurement of bioenzyme activity: enzyme digestion reaction is after the completion 546nm with measurement of ultraviolet-visible spectrophotometer wavelength
The absorbance for locating reaction solution is 0.561 ± 0.0238.Define 1g solid enzyme powder under optimum conditions, hydrolyzed xylan per minute is molten
Enzyme amount needed for liquid discharges 1 μm of ol reduced sugar is a unit of activity (IU), then can calculate tested xylan according to formula
The activity of enzyme is 2876 ± 38IU.
D, control experiment: the reliability in order to verify the above xylanase activity detection method selects everybody generally to receive
DNS method carry out control experiment.Appropriate DNS reagent is added into the Xylose Standard of various concentration, boiling water bath heats 5min, from
It so is cooled to constant volume after room temperature, with the absorbance A of each reaction solution of measurement of ultraviolet-visible spectrophotometermax, obtain xylose concentration with
Absorbance relationship working curve.Then, 3 test tubes are taken, xylan solution 1mL and enzyme dilution 1mL is separately added into, will mix
After liquid reacts 30min in 37 DEG C of water-baths, 600 μ L mixed liquors is respectively taken to be separately added into 3 new test tubes, appropriate DNS examination is added
Agent, constant volume after reaction.Blank control is done with standard blank sample, measures its maximum absorbance.Zytase is obtained using DNS method
Activity is 2609 ± 42IU, and the result measured with the above-mentioned xylanase activity detection method based on nanogold growth keeps one
It causes.It can be seen that detection of the bioenzyme activity detection method established by the present invention for xylanase activity is accurate and reliable
's.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (2)
1. a kind of bioenzyme activity detection method based on nanogold growth, this method are suitable for meeting all lifes of following condition
The detection of object enzymatic activity, i.e., biological zymolyte are not reacted with gold chloride, contain carbohydrate in the product that substrate for enzymatic activity generates
Compound, and these saccharide compounds can react with gold chloride generate nanogold under given conditions.It is characterized by:
A, the drafting of working curve: being diluted to a series of titer for saccharide compound standard items, by a series of titer
The nano-Au solution to form various concentration is reacted with gold chloride respectively, by measuring the absorbance of each reaction solution, obtains carbohydrate
Close object concentration and absorbance relationship working curve.The condition that saccharide compound is reacted with gold chloride are as follows: contain chlorine in reaction mixture
Auric acid 1-2mM, hexadecyltrimethylammonium chloride (CTAC) or cetyl trimethylammonium bromide (CTAB) 3.6-12mM, with
And sodium hydroxide 3-5M；Reaction mixture is in 70 DEG C of reaction 10-15min.
B, the substrate solution V prepared with acetic acid-sodium acetate buffer solution enzyme digestion reaction: is added in test tube1ML is placed in water-bath
5min is preheated, enzyme dilution V is added2ML obtains mixed liquor, after mixed liquor is reacted 30min in a water bath, takes V3μ L mixed liquor adds
Enter in 1 new test tube, then reacted with gold chloride, and makes the total volume V of solution4μL；Above-mentioned enzyme digestion reaction does 3 times in parallel
Experiment, measurement result are averaged.
C, the measurement of bioenzyme activity: enzyme digestion reaction mixes under the conditions of using measurement of ultraviolet-visible spectrophotometer appropriate wavelength after the completion
The absorbance value for closing solution determines that substrate for enzymatic activity hydrolyzes the dense of generated reduced sugar with working curve according to solution absorbance
Degree；Define 1g biological enzyme under optimum conditions, hydrolyzing enzyme amount needed for substrate solution discharges 1 μm of ol reduced sugar per minute is one
Unit of activity is indicated with IU.The vigor of biological enzyme is calculated according to the following formula:
Wherein, X is the vigor of biological enzyme, is indicated with IU；P is the concentration of corresponding reduced sugar on standard curve, unit g/L；C
For the original concentration of enzyme solution, unit g/L；T is enzyme reaction time, unit min；M is the molal weight of reduced sugar；V1、V2、
V3、V4As described in b above；106For transforming factor；DfFor enzyme solution extension rate.
2. the bioenzyme activity detection method according to claim 1 based on nanogold growth, which is characterized in that carbohydrate
Object standard items, substrate, biological enzyme sample is closed to prepare with the acetic acid of pH=4.5-5.5-sodium acetate buffer solution.
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| CN108982462A (en) * | 2018-09-12 | 2018-12-11 | 福建医科大学 | Sulfatase measuring method based on gold nano cluster Ratiometric fluorescent probe |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102618622A (en) * | 2012-04-10 | 2012-08-01 | 湖南大学 | Method for detecting enzyme activity of cellobiose dehydrogenase |
| CN104990913A (en) * | 2015-05-29 | 2015-10-21 | 南开大学 | Gold nanoparticle growth based method for detection of glucose |
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| CN102618622A (en) * | 2012-04-10 | 2012-08-01 | 湖南大学 | Method for detecting enzyme activity of cellobiose dehydrogenase |
| CN104990913A (en) * | 2015-05-29 | 2015-10-21 | 南开大学 | Gold nanoparticle growth based method for detection of glucose |
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| Title |
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| 纳米金在比色传感分析中的应用;沈丽;《材料导报A：综述篇》;20140531;第28卷(第5期);35-41 |
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