CN106290336A - The analysis method of biologically effective phosphorus in algae and water plant - Google Patents
The analysis method of biologically effective phosphorus in algae and water plant Download PDFInfo
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Abstract
The present invention relates to the analysis method of biologically effective phosphorus in algae and water plant, pass through liquid respectively31P NMR technology and enzyme hydrolysis method are analyzed the detailed phosphorus component in lake algae and water plant and are analyzed, and find that the phosphorus component that two kinds of methods detect has similarity, wherein liquid31P NMR technology detect algae and water plant containing having more than more than 15 kinds phosphorus components, and enzyme hydrolysis method find hydrolysis ability stronger be phytate phosphorus and active monoester phosphorus component, account for the 50.2% and 43.5% of total hydrolyzable organophosphor respectively；Add various enzyme again and cultivate the biologically effective phosphorus studying in lake algae and water plant, find the most of monoesters phosphorus in the two organophosphor and pyrophosphate the most in various degree be hydrolyzed to the orthophosphate that biology can directly utilize, the nutrient substance providing abundance is again broken out for algae, when external environmental condition is suitable, algae will be moved in circles and be broken out, and bring huge hidden danger for lake ecosystem and mankind's safe drinking water.
Description
Technical field
The present invention relates to a kind of analysis method of biologically effective phosphorus, be specifically related to algae and water plant biologically effective phosphorus
Liquid31Method is analyzed in P-NMR and enzyme hydrolysis.
Background technology
Organophosphor all exists water body in lake, deposit and water plant, and they can be by a series of oxidoreduction rings
Biologically effective phosphorus (the i.e. biological phosphorus that can directly absorb, such as orthophosphate etc.) it is converted into during border and enzyme hydrolysis.Zhu[1]Organic phosphorous content average out to 454mg/kg in report deposit, the organic phosphorus concentration of water plant and algae deposit respectively
1.8 times and 12.9 times.Although lake endogenous organophosphor plays an important role during eutrophication, but research algae and
In water plant, the composition structure of organophosphor and the cyclic process in lake endogenous phosphorus thereof also rarely have report.
Liquid phosphorus NMR spectroscopic techniques (31P-NMR) it is applied to the water such as deposit as the effective characterization method of one
In environmental sample.Three major types Forms of Organic Phosphorus, i.e. monoesters phosphorus, diester phosphorus and phosphonate is mainly had in aquatic system.31P-NMR
Monoesters phosphorus can also be further characterised as detailed phosphorus component by spectral technique, such as sugar phosphorus (glucose 6 phosphoric acid, glucose 1 phosphoric acid
Deng), inositolophosphate (IHP), phospholipid, DNA and RNA etc..
In addition, enzyme hydrolysis method be another characterize organophosphor method, typically animal fertilizer, soil and its
His land sample all there is application.Organophosphor can be hydrolyzed to Phos after adding various enzymes by the method.In lake ecological system
In system, the method is generally used in deposit.Such as, this method is divided into active monoester phosphorus, diester the phosphorus in deposit
Phosphorus and class phytate phosphorus.In lake ecosystem, enzyme hydrolysis analysis method is applied in lake algae and water plant also to be rarely had
Report.
Also there is research handle31P-NMR spectral technique and enzyme hydrolysis method combine and are applied to animal fertilizer, soil and deposition
Thing[22]In.But these two kinds of methods are combined and is applied in algae and water plant, analyze in eutrophic lake with this
Source phosphorus recycle recovery and bioavailability have not been reported.Therefore, in this study, by algae and water plant sample profit
Carry out in-depth study by both means, and compare both means organophosphor composition in lake algae and water plant
Difference in structure, and utilize31P-NMR spectral technique analyzes lake algae and the life of water plant organophosphor before and after enzymolysis
Thing utilizability, provides scientific basis for preferably exploring Endogenous Phosphorus cyclic process in eutrophic lake.
[1] Zhu, Y.；Zhang, R.；Wu, F., et al.Phosphorus fractions and
bioavailability in relation to particle size characteristics in sediments
From Lake Hongfeng, Southwest China [J] .Environmental Earth Sciences, 2013,68
(4): 1041-1052.
Summary of the invention
The present invention is directed to defect present in prior art, it is provided that a kind of algae and water plant biologically effective phosphorus31P-
Method is analyzed in NMR and enzyme hydrolysis, and the method can analyze the biologically effective in lake algae and water plant accurately and fast
Phosphorus.
The technical scheme is that a kind of algae and the analysis method of water plant biologically effective phosphorus, including following step
Rapid:
(1) enzyme hydrolysis method
(1) preparation of enzymatic solution
Alkali phosphatase (APase) solution is configured by the Tris-HCl buffer solution of 0.1mol/L, pH=9.0, and activity is
1U/mL；
Alkali phosphatase and phosphodiesterase (PDEase) combination solution are delayed by the Tris-HCl of 0.1mol/L, pH=9.0
Rushing solution allocation, activity is 0.02U/mL；
Alkali phosphatase, phosphodiesterase and phytase combination solution are buffered by the Tris-HCl of 0.1mol/L, pH=7.0
Solution allocation, activity is 0.06U/mL；
(2) preparation of culture fluid
Take algae or water plant lixiviating solution in color comparison tube, add hydrochloric acid and regulate the pH value of described lixiviating solution to neutral,
Add the ultra-pure water of 50 times of described lixiviating solution volumes and the Tris-HCl buffer solution of 0.1mol/L, pH=9.0, add
The described alkaline phosphatase enzymatic solution of 2.5 times of described lixiviating solution volumes, obtains culture fluid 1；
Take algae or water plant lixiviating solution in color comparison tube, add 10 times of described lixiviating solution volume 0.1mol/L, pH=
The Tris-HCl buffer solution of 9.0, adds described alkali phosphatase and the phosphodiesterase of 5 times of described lixiviating solution volumes
(PDEase) combination solution, is subsequently adding the ultra-pure water of 50 times of described lixiviating solution volumes, obtains culture fluid 2；
Take algae or water plant lixiviating solution in color comparison tube, add the ultra-pure water of 50 times of described lixiviating solution volumes, then add
Enter 10 times of described lixiviating solution volume 0.1mol/L, the Tris-HCl buffer solution of pH=7.0, add 5 times of described extraction liquid
Long-pending described alkali phosphatase, phosphodiesterase and phytase combination solution, obtains culture fluid 3；
Described culture fluid 1, culture fluid 2 and the culture fluid 3 prepared, cultivates in 37 DEG C of constant incubators, rotating speed respectively
For 220r/min；
(3) after cultivating 16h, in culture fluid, developer is added, after 10min, before using occluded corrosion cell to survey enzyme cultivation
After phosphate；
(4) phosphoric acid measured before using UV-visible spectrometer analysis, the phosphate measured after cultivation and cultivation after colour developing
The difference of salt is organophosphor amount of hydrolysis, i.e. biologically effective phosphorus amount；
(2)31P-NMR analytic process
(1) take 100mg cold dry after algae or water plant NaOH-EDTA extracting solution powder 1mL 1mol/LNaOH-
0.1mol/LEDTA dissolves again；
(2) add heavy water, stand 30min at ambient temperature, then be centrifuged 30min with 10000r/min rotating speed；
(3) it is transferred in nuclear magnetic tube, under the conditions of being stored in 4 DEG C, within 24h, measures liquid31P-NMR；
(4) it is integrated obtaining biologically effective phosphorus amount to the spectrogram obtained in step (3).
Further, described algae or water plant lixiviating solution use 0.5mol/LNaOH+25mmol/LEDTA extractant
Extracting, the quality volume of described algae or water plant powder and described extractant is 1: 60.Use 0.5mol/LNaOH+
25mmol/LEDTA is as extractant, water plant TP and PoExtraction ratio be 92.4% and 88.1% respectively, algae TP and Po's
Extraction ratio is 96.4% and 90.8% respectively, and higher extraction ratio need not the solution such as HCl and carries out second extraction.
Further, described phytase obtains after being purified by crude product phytase (Phytase), and method of purification is:
A. take crude product phytase, dissolve with pH=5.0-5.5,8-12mmol/LNaAc-HAc buffer solution, transfer
To Dialysis tubing, it is suspended in pH=5.0-5.5,8-12mmol/LNaAc-HAc buffer solution dialysis, changes every 2～4h and burn
In Bei, dialysis solution is once, changes 5～7 times continuously；
B. the centrifugal 5-15min under 8000-12000r/min rotating speed of the enzymatic solution after dialysis；
C. the phytase solution after purifying is saved in 4 DEG C of environment, uses in one week.
Further, phytase after described purification being carried out active testing, the method for described active testing includes following
Step:
Take two centrifuge tubes, be divided into check groups and matched group, in two groups of centrifuge tubes, all add the 1mmol/L nitre of 0.5mL
Base benzenephosphonic acid disodium salt (PNPP) solution, then add in check groups 0.2mL 1U/mL purify after phytase solution and
The Tris-HCl buffer solution of 1mL0.01mol/L pH=7.0, only adds 1.2mL0.01mol/L pH=7.0 in matched group
Tris-HCl buffer solution, wait 3-5 minute, if the solution turned yellow color in check groups, and the solution in matched group is still nothing
Color, then the phytase after explanation purifies is the most active, can continue next step experimental implementation.
Further, described culture fluid is possibly together with sodium lauryl sulphate (the Sodium Dodecyl of 0.5-2%
Sulfate, SDS), the sodium lauryl sulphate of preferably 1%, to prevent during measuring probably due to be acidified and produce enzyme
The interference phosphorus yield such as albumen precipitation.
Further, described31P-NMR analyzes and uses BRUKER standard chamber 5mmBBO probe,31The resonant frequency of P is
161.98Hz, measuring temperature is 20 DEG C, and spectral peak width is 5Hz, and obtaining time AQ is D1 time delay in 0.2102s, NMR parameter
Be set to 5s, the analysis testing time is 15h.
The present invention preferably activity is the alkaline phosphatase enzymatic solution of 1U/mL, and activity is alkali phosphatase and the phosphorus of 0.02U/mL
Acid diesters enzyme combination solution, activity is the alkali phosphatase of 0.06U/mL and phosphodiesterase and phytase solution combination solution,
Under this activity, the various components in the organophosphor in algae and water plant farthest hydrolyze out, and being biology has
Effect phosphorus.Biologically effective phosphorus directly can be absorbed by the biology in water body in lake, can be that their normal growth breeding carries
Originate for material.
After algae or water plant add enzymatic solution cultivation, hydrolysis four organophosphorus, it is respectively (1) active monoester phosphorus,
It is to add the organophosphor hydrolyzing out after alkaline phosphatase enzymatic solution；(2) diester phosphorus, is to add alkali phosphatase and phosphodiesterase
Rear hydrolysis organophosphor out；(3) class phytate phosphorus, be add alkali phosphatase, phosphodiesterase and phytase after hydrolyze out
Organophosphor；(4) stable state phosphorus, is to add the organophosphor not being hydrolyzed out yet after three kinds of enzymes.
31The phosphorus component that P-NMR characterizes is summarised as 6 kinds of phosphorus components, and it is orthophosphate, phytate phosphorus, activity list respectively
Ester phosphorus (the monoesters phosphorus in addition to phytate phosphorus), diester phosphorus, pyrophosphate and phosphonate.In water plant, main phosphorus component
It is orthophosphate (account for total phosphorus 27.3～56.6%), active monoester phosphorus (10.8～33.5%), pyrophosphate (0.9～18.8%)
With diester phosphorus (1.6～6.2%), similar, algae orthophosphate (27.1～67.5%), active monoester phosphorus (8.6～
44.3%), phytate phosphorus (5.6～41.9%), pyrophosphate (1.8～2.3%) and diester phosphorus (1.3～14.4%).Phosphonate
Only small part (0～0.7%).Polyphosphate is not the most detected in water plant and algae.
Major part and whole pyrophosphate in lake algae and water plant monoesters phosphorus are all converted into orthophosphate, add
After alkaline phosphate enzyme, 6 kinds of algae and the phytate phosphorus of water plant average 18.9% and the active monoester phosphorus of 20.7% and
The pyrophosphate of 6.6% is converted into orthophosphate；After adding alkali phosphatase and phosphodiesterase, algae and water plant are average
The phytate phosphorus of 12.3% and the active monoester phosphorus of 20.8% and 7.0% pyrophosphate be converted into orthophosphate；Add alkalescence
After phosphatase, phosphodiesterase and phytase, algae and the active monoester phosphorus of water plant average 22.8% and 17.7% and
The pyrophosphate of 7.0% is converted into orthophosphate.These data show big portion in lake algae and water plant decay process
Point monoesters phosphorus and pyrophosphoric acid translate into the Phos such as the orthophosphate that biology can directly absorb.
Utilize enzyme hydrolysis method and liquid31The distribution pattern of P-NMR Technical comparing algae and the main phosphorus component of water plant has
Certain similarity (r=0.712, p ＜ 0.05).Liquid31P-NMR technology provides ratio phosphorus component information in greater detail, but surveys
Trying expensive, the testing time is relatively long.Enzyme hydrolysis can estimate hydrolyzable organophosphor amount, the i.e. phosphorus of bioavailability
Content, but a few class phosphorus component can only be detected.Therefore, the present invention uses both approaches to study eutrophic lake algae
Detailed phosphorus component chemical feature and biological effectiveness thereof with in water plant, the analysis method of the present invention can be accurately and fast
Analyze lake algae and water plant biologically effective phosphorus, with the circulation of Endogenous Phosphorus in research lake algae further and water plant
Mechanism is significant.
Accompanying drawing explanation
Fig. 1；Under various enzyme effects in algae and water plant NaOH-EDTA extracting solution31P-NMR spectrogram
Fig. 2；Utilize liquid31P-NMR technology and enzyme hydrolysis method are to the comparison of phosphorus component in lake algae and water plant
Fig. 3；Add lake algae and water plant phosphorus component degraded feature after various enzyme is cultivated
Fig. 4；Add lake algae and each phosphorus component of water plant after various enzyme is cultivated and convert ratio
Wherein A1, A2, A3 represent the watermifoil in water plant, phragmites communis and hydrilla verticillata respectively, B1, B2, B3 generation respectively
Microcystis aeruginosa, chlorella and spirulina in table algae.
In conjunction with drawings and Examples, the invention will be further described:
Detailed description of the invention
Embodiment 1
A kind of algae and the analysis method of water plant biologically effective phosphorus, comprise the following steps:
(1) enzyme hydrolysis method
(1) preparation of enzymatic solution
Activity for alkali phosphatase (APase) the solution allocation method of 1U/mL is: takes 3.33mg and buys from Sigma company
Alkali phosphatase (numbered EC3.1.3.1, activity is 30U/mg) pressed powder, with the Tris-of 0.1mol/L, pH=9.0
HCl buffer solution is settled to 100mL.
Activity for the alkali phosphatase of 0.02U/mL and phosphodiesterase (PDEase) combination solution collocation method is: take
The phosphodiesterase pressed powder (numbering EC3.1.4.1, activity is 0.02U/mL) that 10mg buys from Sigma company, uses 10mL
Activity goes to dissolve for 1U/mL alkaline phosphatase enzymatic solution.
Activity is the alkali phosphatase of 0.06U/mL, phosphodiesterase and phytase (Phytase) combination solution configuration side
Method is: (numbering EC3.1.3.26, activity is 0.03U/ to take the crude product phytase pressed powder that 600mg buys from Sigma company
ML), be settled to 100mL with 1mol/LHAc-NaAc solution, then the phytase activity purified is 0.18U/mL, takes 5mL and purifies
Good phytase solution, adds the Tris-HCl buffer solution of 5mL0.1mol/L, pH=7.0, and now phytase solution activity is
0.045U/mL, takes 20mg phosphodiesterase pressed powder, adds the phytase solution of 10mL0.045U/mL, adds
The alkaline phosphatase enzymatic solution of 10mL1U/mL, i.e. configuration have got well alkali phosphatase, phosphodiesterase and phytase combination solution.
(2) preparation of culture fluid
Take 0.5g algae or water plant powder, add 30mL0.5mol/LNaOH+25mmol/LEDTA extractant and carry out
Extract, take 0.1mL lixiviating solution after extraction and be placed in 10mL color comparison tube, do 3 parallel and set control treatment, add
0.025mLHCl, regulation solution, to neutral, all adds 5mL ultra-pure water and 1mL 0.1mol/L, pH=to matched group, experimental group
The Tris-HCl buffer solution of 9.0, then the alkali phosphatase that activity is 1U/mL that addition 0.25mL prepares in experimental group is molten
Liquid, adds the Tris-HCl buffer solution of the 0.1mol/L, pH=9.0 of equivalent (i.e. 0.25mL) simultaneously in matched group, obtains training
Nutrient solution 1；
Take 0.5g algae or water plant powder, add 30mL0.5mol/LNaOH+25mmol/LEDTA extractant and carry out
Extract, take 0.1mL lixiviating solution after extraction and be placed in 10mL color comparison tube, do 3 parallel and set control treatment, add in experimental group
The alkaline phosphatase that activity is 0.02U/mL that the Tris-HCl buffer solution of 1mL0.1mol/L, pH=9.0 and 0.5mL configure
Enzyme and phosphodiesterase combination solution, add the Tris-HCl buffering of 1.5mL0.1mol/L, pH=9.0 in matched group simultaneously
Solution, then in experimental group and matched group, add 5mL ultra-pure water respectively, obtain culture fluid 2；
Take 0.5g algae or water plant powder, add 30mL0.5mol/LNaOH+25mmol/LEDTA extractant and carry out
Extract, take 0.1mL lixiviating solution after extraction and be placed in 10mL color comparison tube, do 3 parallel and set control treatment, to experimental group and comparison
Group all adds 5mL ultra-pure water, in matched group, adds the Tris-HCl buffer solution of 1.5mL0.1mol/L, pH=7.0, to
Experimental group adds the Tris-HCl buffer solution of 1.0mL0.1mol/L, pH=7.0 and alkali phosphatase that 0.5mL configures,
Phosphodiesterase and phytase combination solution, obtain culture fluid 3；
Described culture fluid 1, culture fluid 2 and the culture fluid 3 prepared, cultivates in 37 DEG C of constant incubators, rotating speed respectively
For 220r/min；Every time the sample sets of the processing mode that experiment is corresponding all prepares standard curve, and all of standard curve all with
Step is tested, to correct the interference that substrate causes.The method for drafting of its standard curve is: take 7 10mL color comparison tubes, respectively
(collocation method of phosphate standard solution is to draw to add 0,0.3,0.5,1.0,1.5,2.0,2.5mL phosphate standard solution
2mL phosphorus standard reserving solution, is settled to 100mL with ultra-pure water, and now phosphate standard solution is 0.12 μm ol/mL containing phosphorus concentration,
Need now with the current；Wherein the collocation method of phosphorus standard reserving solution is: by potassium dihydrogen phosphate pure for top grade in 110 DEG C of dry 2h,
Exsiccator cools down, weighs 0.816g and be dissolved in ultra-pure water, move in 1L volumetric flask, add 1: 1 sulphuric acid 19mL, dilute with ultra-pure water
To graticule 1L, now phosphorus standard reserving solution is 6 μm ol/mL containing phosphorus concentration, can store 6 months).
(3) in culture fluid and standard curve solution, add 1.6ml developer respectively, after shaking up, add 0.5mL's 20%
Sodium lauryl sulphate (Sodium Dodecyl Sulfate, SDS) solution, is settled to 10mL with ultra-pure water, after 10min, adopts
The phosphate before and after enzyme is cultivated is surveyed with occluded corrosion cell；
(4) phosphoric acid measured before using UV-visible spectrometer analysis, the phosphate measured after cultivation and cultivation after colour developing
The difference of salt is organophosphor amount of hydrolysis, is biologically effective phosphorus amount；
(2)31P-NMR analytic process
(1) take 100mg cold dry after algae or water plant NaOH-EDTA extracting solution powder 1mL1mol/LNaOH-
0.1mol/LEDTA dissolves again；
(2) add heavy water, stand 30min at ambient temperature, then be centrifuged 30min with 10000r/min rotating speed；
(3) it is transferred in nuclear magnetic tube, under the conditions of being stored in 4 DEG C, within 24h, measures liquid31P-NMR；31P-NMR analyzes and adopts
Pop one's head in BRUKER standard chamber 5mm BBO,31The resonant frequency of P is 161.98Hz, and measuring temperature is 20 DEG C, and spectral peak width is
5Hz, obtain time AQ be in 0.2102s, NMR parameter time delay D1 be set to 5s, the analysis testing time is 15h；
(4) it is integrated obtaining biologically effective phosphorus amount to the spectrogram obtained in step (3).
Wherein, phytase obtains after being purified by crude product phytase (Phytase), and method of purification is:
A. take crude product phytase, dissolve with pH=5.0-5.5,8-12mmol/L NaAc-HAc buffer solution, transfer
To Dialysis tubing, it is suspended in pH=5.0-5.5,8-12mmol/L NaAc-HAc buffer solution dialysis, changes every 2～4h
In beaker, dialysis solution is once, changes 5～7 times continuously；
B. the centrifugal 5-15min under 8000-12000r/min rotating speed of the enzymatic solution after dialysis；
C. the phytase solution after purifying is saved in 4 DEG C of environment, uses in one week.
Phytase after purifying is carried out active testing, and the method for described active testing comprises the following steps:
Take two centrifuge tubes, be divided into check groups and matched group, in two groups of centrifuge tubes, all add the 1mmol/L nitre of 0.5mL
Base benzenephosphonic acid disodium salt (PNPP) solution, then add in check groups 0.2mL 1U/mL purify after phytase solution and
The Tris-HCl buffer solution of 1mL0.01 mol/L pH=7.0, only adds 1.2mL0.01 mol/L pH=in matched group
The Tris-HCl buffer solution of 7.0, waits 3-5 minute, if the solution turned yellow color in check groups, and the solution in matched group is still
Colourless, then the phytase after explanation purifies is the most active, can continue next step experimental implementation.
The collocation method of developer is: 50mL5mol/LH2SO4+ 15mL ammonium molybdate solution+30mL0.1mol/L is anti-bad for solution
Hematic acid solution+5mL1 mg Sb/mL antimony tartrate potassium solution, reagent stores under 4 DEG C of environment, at most can keep 24h, on-demand joins
Put, expired, discard and reconfigure, wherein 5mol/LH2SO4Solution allocation method is that 70mL concentrated sulphuric acid ultra-pure water is diluted to
500mL, 4 DEG C of preservations；Ammonium molybdate solution is to take 4g analytical pure ammonium molybdate, is dissolved in 100mL ultra-pure water fully dissolving and shakes up, room
Temperature preserves, and can not put in 4 DEG C of environment with crystallization-preventive；0.1mol/L ascorbic acid solution be take 1.76g ascorbic acid in
100mL ultra-pure water fully dissolves and shakes up, 4 DEG C of preservations；1mg Sb/mL antimony tartrate potassium solution is to take 0.2743g antimony tartrate
Potassium, is dissolved in 100mL ultra-pure water fully dissolving and shakes up, 4 DEG C of preservations.
Interpretation of result
1, utilize31P-NMR analyzes lake algae and water plant P form
As it is shown in figure 1, lake algae and the liquid of water plant31P-NMR collection of illustrative plates, by the liquid to Fig. 131P-NMR schemes
Spectrum carries out the integration of each phosphorus component, and detailed phosphorus component is shown in Table 1, table 2, utilizes liquid31P-NMR technology, has more than more than 15 kinds
Phosphorus component characterize, wherein orthophosphate (6.0ppm) is peak-to-peak signal maximum in all collection of illustrative plates, algae orthophosphate
Concentration is between 2646-5094mg/kg, and the concentration of water plant orthophosphate is between 670-990mg/kg, although algae and
The orthophosphate concentration of water plant has bigger difference, but in the NaOH-EDTA extracting solution of these samples, orthophosphate accounts for
The percentage ratio of total phosphorus is similar, i.e. algae and water plant orthophosphate accounts for the 42.5% and 43.2% of its corresponding total phosphorus respectively
(table 1, table 2).Pyrophosphate all detects at all of sample, and total monoesters phosphorus (i.e. phytate phosphorus and the sum of active monoester phosphorus) is
Big Forms of Organic Phosphorus, in these biological samples, phytate phosphorus accounts for the percentage ratio of total phosphorus is 5.6～41.9%, and monoesters phosphorus comprises fruit
Saccharide, glyceride, nucleotide, adenylic acid (AMP) etc., wherein fructose class comprises fructose 6 phosphoric acid, glucose 1 phosphoric acid and glucose 6
Phosphoric acid, glyceride includes α glyceride and β glyceride, and it is commonly considered as phospholipid catabolite under alkaline hydrolysis conditions, core
Thuja acid includes 2 ' guanyls (guanosine 2 ' monophosphate) and 5 ' cytidylic acid (cytidine 5 '
Monophosphate), it is commonly considered as the catabolite of DNA and RNA.In addition, monoesters phosphorus also has phosphatidic acid (3-
Sn phosphatidic acid), phosphoethanolamine (O-phosphorylethanolamine), under the effect of enzyme, these
Monoesters phosphorus is easier to hydrolyze (table 1, table 2), and the ratio that diester phosphorus accounts for total phosphorus is lower than the ratio that other phosphorus components account for total phosphorus
(less than 6%) (Fig. 1, table 1).
Phosphorus component in order to produce with enzymolysis compares,31It is big that 15 kinds of phosphorus components that P-NMR characterizes are summarised as 6
Class phosphorus component, it is orthophosphate, phytate phosphorus, active monoester phosphorus (the monoesters phosphorus in addition to phytate phosphorus), diester phosphorus, burnt phosphorus respectively
Hydrochlorate and phosphonate.In water plant (table 1), main phosphorus component is orthophosphate (accounts for total phosphorus 27.3～56.6%), lives
Property monoesters phosphorus (10.8～33.5%), pyrophosphate (0.9～18.8%) and diester phosphorus (1.6～6.2%), similar, algae
Orthophosphate (27.1～67.5%), active monoester phosphorus (8.6～44.3%), phytate phosphorus (5.6～41.9%), pyrophosphate
(1.8～2.3%) and diester phosphorus (1.3～14.4%).Phosphonate only has small part (0～0.7%).In water plant and algae
Apoplexy due to endogenous wind does not the most detect polyphosphate.
2, enzyme hydrolysis method is utilized to analyze lake algae and water plant P form
As shown in table 3, utilize enzyme hydrolysis method that algae and water plant are divided into Phos, active monoester phosphorus, diester phosphorus, class
Phytate phosphorus and 5 components of stable state phosphorus.Such as table 3, algae there is 3033mg/kg (account for algae Po54.6%) PoCan be by water
Solve, water plant has 584mg/kg (account for water plant Po58.1%) PoCan be hydrolyzed.Hydrolyzable phosphorus group in algae
Point: active monoester phosphorus ＞ class phytate phosphorus ＞ diester phosphorus, it accounts for algae total hydrolyzable P respectivelyoThe 51.6% of component, 40.7% and
7.7%；Hydrolyzable phosphorus component in water plant: class phytate phosphorus ＞ active monoester phosphorus ＞ diester phosphorus, it accounts for water plant respectively
Total hydrolyzable PoThe 65.1% of component, 30.8% and 4.1%.In addition, the stable state phosphorus in algae and water plant (can not
Hydrolysis Po) account for the 31.2% and 25.7% of NaOH-EDTA extraction state TP respectively.
In water plant and algae, the distribution pattern of phosphorus is Phos ＞ stable state phosphorus ＞ class phytate phosphorus ＞ active monoester phosphorus
＞ diester phosphorus.The percentage ratio that lake algae stable state phosphorus accounts for extraction state total phosphorus is more slightly higher than water plant, shows, not every
Organophosphor all can hydrolyze under the effect of enzyme.
3、31P-NMR analyzes lake algae and the comparison of water plant P form with enzyme hydrolysis method
Utilize liquid31It is similar that P-NMR technology obtains four classes with enzymatic isolation method analysis lake algae with water plant phosphorus composition
Phosphorus component, respectively orthophosphate or Phos, phytate phosphorus or class phytate phosphorus, active monoester phosphorus and diester phosphorus.Both sides
Inorganic phosphorus concentration in method detection phragmites communis (A2), chlorella (B2), these several samples of spirulina (B3) is more or less the same, remaining sample
Inorganic phosphorus concentration in product (A1, A3, B1) has certain difference (Fig. 2).In terms of overall, liquid is used31P-NMR technology for detection
Algae and water plant Phos account for the inorganic of percentage ratio (meansigma methods is 49.8%) the ratio enzymatic isolation method detection of extraction state total phosphorus
Phosphorus percentage ratio (43.3%) is slightly higher.Six kinds of samples of phytate phosphorus with the detection of two kinds of methods have four kinds similar, but for phragmites communis
(A2) and for chlorella (B2) sample, liquid is compared by the phytate phosphorus concentration of enzymatic isolation method detection31P-NMR spectral technique is on the low side.From
Fig. 2 is it can be seen that other monoesters phosphorus concentration ratio liquid of detecting with enzymolysis31P-NMR spectral technique is on the low side.It is similar to,
The diester phosphorus concentration ratio liquid detected with enzymolysis31P-NMR spectral technique is the most on the low side, and this may be with the instability of diester phosphorus
Property is relevant, and it is easy to be degraded to monoesters phosphorus.In a word, not every phosphorus component (such as stable state phosphorus) all can by enzyme hydrolysis,
The stable state phosphorus (table 3) of difference about 25.7% and 31.2% in water plant and algae.
4、31P-NMR desmoenzyme Hydrolyze method analyzes algae and water plant organophosphor biological effectiveness
Fig. 3 can be clearly seen that, various enzyme (APase, APase+PDEase, APase+PDEase+Phytase) is trained
Phosphorus component change before and after Yanging.In Fig. 3, the histogram graph representation enzyme below abscissa contains than the phosphorus component declined before cultivating after cultivating
Amount, after histogram graph representation enzyme more than abscissa is cultivated, ratio cultivates the front phosphorus component content raised.General trend is seen from Fig. 3,
No matter add which kind of enzyme, all have beyond pyrophosphate and monoesters phosphorus region (including phytate phosphorus and other monoesters phosphorus) depolarization are indivedual
The block diagram declined, represents and adds after enzyme, monoesters phosphorus component content declines, and orthophosphate constituent content all abscissa with
On, expression orthophosphate is after adding enzyme cultivation, and its constituent content raises.From the liquid of Fig. 131P-NMR spectrogram can also obtain
Corresponding checking, in Fig. 1, adds the liquid after various enzyme31The spectral peak of monoesters phosphorus region (3～6ppm) and Jiao in P-NMR spectrogram
The spectral peak (-4～-5ppm) of phosphate acid region, compared with matched group, all has decline in various degree or disappearance trend, this
After indicating the various enzymes of interpolation, monoesters phosphorus and pyrophosphate all have hydrolysis in various degree.
As a example by watermifoil (A1) in 6 kinds of algae and water plant sample, before and after carrying out enzyme cultivation, phosphorus component is detailed
Data analysis (table 1).In watermifoil sample, after adding alkali phosphatase, orthophosphate content adds 1014mg/kg
(46.9%), meanwhile, organophosphor and pyrophosphate have dropped 44.5% and 2.4% respectively, the wherein organophosphor of 44.5%
Component has the phytate phosphorus of 35.9% and other monoesters phosphorus of 9.3% to decline.From table 1 and Fig. 3 it can also be seen that add alkaline phosphatase
After enzyme, diester phosphorus slightly raises (0.7%), and chiro-IHP, neo-IHP, 2 ' guanyls, these phosphorus components of AMP and DNA do not have
Significant change.In addition, enzyme occurs in that a new peak-to-peak signal after cultivating at chemical shift 4.6ppm, and this peak is reflected
It is set to phosphoethanolamine.
In watermifoil sample, after adding alkali phosphatase and phosphodiesterase, pyrophosphate and monoesters phosphorus component content
Degradable (Fig. 3), the peak-to-peak signal (chemical shift 3.5～6.0ppm) of pyrophosphate and monoesters phosphorus is wholly absent (table 1, table 2).
After adding alkali phosphatase and phosphodiesterase, Forms of Organic Phosphorus is remarkably decreased, and have dropped 39.8%, including 31.5%
Phytate phosphorus, other monoesters phosphorus of 8.9% and the diester phosphorus of 0.7%, meanwhile, the content of orthophosphate dramatically increases, and increases
861mg/kg (table 1, Fig. 3).After adding alkali phosphatase and phosphodiesterase, diester phosphorus content degraded (Fig. 3), diester phosphorus
Peak-to-peak signal (chemical shift-1～2.5ppm) disappears (table 1), and diester phosphorus content then will not be made when only adding alkali phosphatase to drop
Solving, which show the specificity of enzyme, diester phosphorus is hydrolyzed into orthophosphate by diesterase.
After adding alkali phosphatase, phosphodiesterase and phytase, the orthophosphate content in watermifoil sample significantly increases
Adding, add 1078mg/kg, meanwhile, diester phosphorus is also increased slightly, and adds 44mg/kg (2.1%), meanwhile, also has phosphorus
Significantly reducing of component, reduces 52.1%, wherein comprises the phytate phosphorus of 38.9%, other monoesters phosphorus and 2.4% of 10.8%
Pyrophosphate reduce.In addition, phytate phosphorus is also referred to as inositolophosphate (IHP), and it has a lot of isomers, but at environment
Typically seen in sample have four kinds of isomerss, is chiro-IHP, neo-IHP, myo-IHP and scyllo-IHP respectively.
In this study, liquid is utilized31P-NMR technology can detect in algae and water plant containing chiro-IHP and neo-IHP
Material (table 1, table 2, Fig. 3), but after adding enzyme, this component there occurs degraded.
5, algae breaks out and the biogeochemical cycle process of inland lake Endogenous Phosphorus
As the major part in Fig. 4, lake algae and water plant monoesters phosphorus and whole pyrophosphate are all converted into orthophosphoric acid
Salt, after adding alkaline phosphate enzyme, 6 kinds of algae and the phytate phosphorus of water plant average 18.9% and the active monoester phosphorus of 20.7%
And the pyrophosphate of 6.6% is converted into orthophosphate；After adding alkali phosphatase and phosphodiesterase, algae and water plant
The phytate phosphorus of average 12.3% and other monoesters phosphorus of 20.8% and 7.0% pyrophosphate be converted into orthophosphate；Add
After alkali phosphatase, phosphodiesterase and phytase, algae and the active monoester phosphorus of water plant average 22.8% and 17.7%
And the pyrophosphate of 7.0% is converted into orthophosphate.This shows monoesters phosphorus in lake algae and water plant decay process
The Phos such as orthophosphate that biology can directly absorb are translated into pyrophosphoric acid.
Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, and this embodiment is also not used to
Limiting the scope of the claims of the present invention, all equivalences done without departing from the present invention are implemented or change, are intended to be limited solely by the technology of the present invention
In the range of scheme.
Claims (5)
1. algae and water plant biologically effective phosphorus enzyme hydrolysis and liquid31P-NMR analyzes method, comprises the following steps:
(1) enzyme hydrolysis method
(1) preparation of enzymatic solution
Alkaline phosphatase enzymatic solution is configured by the Tris-HCl buffer solution of 0.1mol/L, pH=9.0, and activity is 1U/mL；
Alkali phosphatase and phosphodiesterase combination solution are configured by the Tris-HCl buffer solution of 0.1mol/L, pH=9.0, live
Property is 0.02U/mL；
Alkali phosphatase, phosphodiesterase and phytase combination solution are by the Tris-HCl buffer solution of 0.1mol/L, pH=7.0
Configuration, activity is 0.06U/mL；
(2) preparation of culture fluid
Take algae or water plant lixiviating solution in color comparison tube, add hydrochloric acid and regulate the pH value of described lixiviating solution to neutral, then add
Enter the ultra-pure water of 50 times of described lixiviating solution volumes and the Tris-HCl buffer solution of 10 times of 0.1mol/L, pH=9.0, add
The described alkaline phosphatase enzymatic solution of 2.5 times of described lixiviating solution volumes, obtains culture fluid 1；
Take algae or water plant lixiviating solution in color comparison tube, add 10 times of described lixiviating solution volume 0.1mol/L, pH=9.0's
Tris-HCl buffer solution, adds described alkali phosphatase and the phosphodiesterase combination solution of 5 times of described lixiviating solution volumes,
It is subsequently adding the ultra-pure water of 50 times of described lixiviating solution volumes, obtains culture fluid 2；
Take algae or water plant lixiviating solution in color comparison tube, add the ultra-pure water of 50 times of described lixiviating solution volumes, add 10
The most described lixiviating solution volume 0.1mol/L, the Tris-HCl buffer solution of pH=7.0, add 5 times of described lixiviating solution volumes
Described alkali phosphatase, phosphodiesterase and phytase combination solution, obtain culture fluid 3；
Described culture fluid 1, culture fluid 2 and the culture fluid 3 prepared, cultivates respectively in 37 DEG C of constant incubators, and rotating speed is
220r/min；
(3) after cultivating 16h, in culture fluid, developer is added, after 10min, before and after using occluded corrosion cell to survey enzyme cultivation
Phosphate；
(4) using UV-visible spectrometer analysis after developing the color, after cultivation, the phosphate of mensuration mensuration front with cultivation is phosphatic
Difference is organophosphor amount of hydrolysis, i.e. biologically effective phosphorus amount；
(2)31P-NMR analytic process
(1) take 100mg cold dry after algae or the NaOH-EDTA extracting solution powder 1mL1mol/L NaOH-of water plant
0.1mol/LEDTA dissolves again；
(2) add heavy water, stand 30min at ambient temperature, then be centrifuged 30min with 10000r/min rotating speed；
(3) it is transferred in nuclear magnetic tube, under the conditions of being stored in 4 DEG C, within 24h, measures liquid31P-NMR；
(4) it is integrated obtaining biologically effective phosphorus amount to the spectrogram obtained in step (3).
Method the most according to claim 1, it is characterised in that described algae and water plant lixiviating solution use 0.5mol/
LNaOH+25mmol/LEDTA extractant extracts, described algae and water plant powder and the quality volume of described extractant
For 1:60.
Method the most according to claim 1, it is characterised in that described phytase obtains after being purified by crude product phytase, carries
Pure method is:
A. take crude product phytase, dissolve with pH=5.0-5.5,8-12mmol/LNaAc-HAc buffer solution, be transferred to
In analysis pipe, it is suspended in pH=5.0-5.5,8-12mmol/L NaAc-HAc buffer solution dialysis, changes beaker every 2～4h
Middle dialysis solution once, is changed 5～7 times continuously；
B. the centrifugal 5-15min under 8000-12000r/min rotating speed of the enzymatic solution after dialysis；
C. the phytase solution after purifying is saved in 4 DEG C of environment, uses in one week.
Method the most according to claim 1, it is characterised in that possibly together with the dodecyl of 0.5-2% in described culture fluid
Sodium sulfate.
Method the most according to claim 1, it is characterised in that described31P-NMR analyzes and uses BRUKER standard chamber 5mm
BBO pops one's head in,31The resonant frequency of P is 161.98Hz, and measuring temperature is 20 DEG C, and spectral peak width is 5Hz, obtains time AQ and is
In 0.2102s, NMR parameter time delay D1 be set to 5s, the analysis testing time is 15h.
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| CN107478670A (en) * | 2017-08-08 | 2017-12-15 | 中国环境科学研究院 | A kind of deposit mysoinositol phosphate and the analysis method of humic acid interaction |
| CN112326848A (en) * | 2020-10-23 | 2021-02-05 | 杭州师范大学 | Methyldiazomethane methyl esterification phytic acid analysis method based on trimethylsilyl |
| CN113834805A (en) * | 2020-06-24 | 2021-12-24 | 中国农业科学院农业资源与农业区划研究所 | Method for visually detecting inorganic phosphorus distribution of plant cell level |
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Cited By (4)
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| CN107478670A (en) * | 2017-08-08 | 2017-12-15 | 中国环境科学研究院 | A kind of deposit mysoinositol phosphate and the analysis method of humic acid interaction |
| CN113834805A (en) * | 2020-06-24 | 2021-12-24 | 中国农业科学院农业资源与农业区划研究所 | Method for visually detecting inorganic phosphorus distribution of plant cell level |
| CN113834805B (en) * | 2020-06-24 | 2024-05-28 | 中国农业科学院农业资源与农业区划研究所 | Method for visually detecting inorganic phosphorus distribution at plant cell level |
| CN112326848A (en) * | 2020-10-23 | 2021-02-05 | 杭州师范大学 | Methyldiazomethane methyl esterification phytic acid analysis method based on trimethylsilyl |
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