CN102747064A - Method for improving stability of bacillus subtilis Zj016 aminopeptidase by chemical modification of succinic anhydride - Google Patents
Method for improving stability of bacillus subtilis Zj016 aminopeptidase by chemical modification of succinic anhydride Download PDFInfo
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- CN102747064A CN102747064A CN2012102605508A CN201210260550A CN102747064A CN 102747064 A CN102747064 A CN 102747064A CN 2012102605508 A CN2012102605508 A CN 2012102605508A CN 201210260550 A CN201210260550 A CN 201210260550A CN 102747064 A CN102747064 A CN 102747064A
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Abstract
The invention discloses a method for improving stability of bacillus subtilis Zj016 aminopeptidase by chemical modification of succinic anhydride, belonging to the technical field of enzyme preparation and food additive. The method is based on residual enzymatic activity and relative enzyme activity; the residual enzymatic activity of modification enzyme liquid is 105.81%; the average amino modification rate of succinic anhydride (SA) for aminopeptidase is 55.86%. Compared with the proenzyme liquid, the optimal reaction temperature of the modification enzyme liquid is improved to 65 DEG C from 60 DEG C; the optimal pH is improved to 9.5-10.0 from 8.5; more than 70% of activity can be maintained after maintaining the temperature for 5 hours at 70 DEG C, so that it is shown that the thermal stability is improved drastically; the kinetic parameter Km is reduced to 0.75mmol.L<-1>, and the affinity of the modified aminopeptidase for primer is improved notably. SA chemical modification is an effective method for notably improving affinity of the bacillus subtilis aminopeptidase for the primer and thermal stability of the bacillus subtilis aminopeptidase, improves application characteristic of the bacillus subtilis aminopeptidase and plays an active role in promoting industrial production and further application of the aminopeptidase.
Description
Technical field
A kind of Succinic anhydried chemically modified improves the method for subtilis Zj016 aminopeptidase stability, belongs to zymin, technical field of food additives.
Background technology
Aminopeptidase is one type of general name of cutting the exopeptidase of amino-acid residue from the N end enzyme of protein or peptide chain.It can be used as the composite enzyme of using of combination in the preparation of proteolysis product, play debitterize, depth hydrolysis, and effects such as preparation multifunction activity peptide also can be used as N end order-checking toolenzyme and medical diagnosis and use enzyme.
Enzyme is having a strong impact on the result of use of enzyme as a kind of biological catalyst because of shortcomings such as short, easy inactivations of its transformation period.The stability of enzyme is meant that the enzyme molecule resists various different factor affecting, keeps the certain space structure, keeps the metastable ability of catalytic activity, comprises aspects such as thermostability, pH stability, oxidation-resistance and organic solvent-resistant.Aminopeptidase is as a kind of industriallization zymin, and its thermostability is a very The key factor.The thermostability raising of enzyme has remarkable advantages, and the storage time, the enzyme of minimizing in transportation that prolong enzyme are lived and lost, and make enzyme under higher reaction temperatures, prolong the reaction times of enzyme, enhance productivity, and effectively reduce production cost.So, how to improve the stability of enzyme, thereby the research that enlarges the range of application of enzyme more and more causes people's attention.
At present, the strategy that improves enzyme stability is mainly improved two aspects from enzyme molecular modification and microenvironment and is started with.The enzyme molecular modification comprises methods such as rite-directed mutagenesis, chemically modified and enzyme immobilization; The microenvironment improvement comprises adds protective material, sanitas, proteinase inhibitor, inhibitor and reductive agent etc.Wherein chemically modified is to improve and improve one of effective ways of enzyme catalysis performance.The chemically modified of enzyme; Utilize some specificity chemical reagent treat enzyme albumen exactly; Make it with the zymoprotein molecule in particular amino acid residue reaction, change the chemical structure and the character of these amino-acid residue side chains, thereby change the original space conformation in active site; Or the composition of the cofactor of change enzyme, and then change the original characteristic of enzyme.The chemically modified of enzyme is eliminated immunogenicity in the transformation period of the specificity that changes enzyme, stability, the medicinal enzyme of raising, even the aspects such as catalyzed reaction type of change enzyme have all obtained major progress.
ε-NH that Lys residue on the aminopeptidase molecule is terminal
2Owing to have stronger nucleophilicity, can react with small molecules chemical modifier Succinic anhydried, thus the electric charge and the structure of change enzyme molecule, and then the character of enzyme is improved.With further using positive effect is arranged to promoting this enzyme suitability for industrialized production from now on.
Summary of the invention
The objective of the invention is under the best modification condition after optimizing, aminopeptidase to be carried out chemically modified with Succinic anhydried; Provide a kind of Succinic anhydried chemically modified to improve the method for subtilis Zj016 aminopeptidase stability; Significantly improved the thermostability of aminopeptidase; Modify of the avidity also to some extent increase of back enzyme, followed the variation of a series of enzymatic property sign aspect simultaneously substrate.
Technical scheme of the present invention:
The fermented liquid frozen centrifugation of subtilis Zj016, clarification, ultrafiltration and concentration, desalination, lyophilize; Get food grade solid ammonia peptase (Chinese patent 201010578579.1 is open, publication number CN102078012A, open day 2011-6-1); Under optimum chemical modification condition; With Succinic anhydried the aminopeptidase of gained being carried out chemically modified, obtain modifying enzyme, is foundation with the enzyme retention rate of living with enzyme activity relatively mainly; Obtain the method that a kind of Succinic anhydried chemically modified improves subtilis Zj016 aminopeptidase stability, its technology is:
A, Succinic anhydried are to the chemical modification method of aminopeptidase
The quality of having examined or check modification reaction pH, modification reaction time, modification reaction temperature, modifier and enzyme through experiment of single factor is than the influence of these four factors to chemical modification reaction, is index with the enzyme retention rate of living, and draws best chemically modified condition:
(1) the aminopeptidase powder that takes by weighing 20mg is dissolved in the Tris-HCl damping fluid of 15mL, 0.05mol/L, pH8.5; The Succinic anhydried (SA) that in mixed solution, slowly adds 10mg then, stirring and dissolving, the pH value of in entire reaction course, using 2mol/L NaOH solution to keep reaction system is 8.0; Under 4 ℃ of conditions, react 1h; After reaction finishes, with the Tris-HCl damping fluid of 0.05mol/L, pH8.5 4 ℃ of dialysed overnight, to remove unnecessary modifier; Obtain the aminopeptidase (modifying enzyme liquid SA-AE) that Succinic anhydried is modified, 4 ℃ of refrigerators are preserved;
(2) the aminopeptidase powder that takes by weighing 20mg is dissolved in the Tris-HCl damping fluid of 15mL, 0.05mol/L, pH8.5,4 ℃ of dialysed overnight, obtains original enzyme liquid (AE) with the Tris-HCl damping fluid of 0.05mol/L, pH8.5, and 4 ℃ of refrigerators are preserved.
The part zymologic property of enzyme before and after B, the modification
(1) adopt Succinic anhydried that aminopeptidase is carried out chemically modified under optimal conditions, SA is 55.86% to the average amido modified rate of AE, and enzyme retention rate alive is 105.81%.
(2) mensuration of kinetic parameter Km
With L-leucine-4-N-methyl-p-nitroaniline is substrate; Under the condition of the best enzyme activity determination of protoenzyme and modifying enzyme, the enzyme of protoenzyme and modifying enzyme was lived when the mensuration concentration of substrate was 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L, 1.4mmol/L.Get two inverses according to the Lineweaver-Burk graphing method and make the 1/v-1/s curve, calculate the Michaelis-Menton constant Km and the maximum response speed Vm of protoenzyme and modifying enzyme.
Its kinetic parameter of aminopeptidase after modification Km is from 1.0 mmolL
-1Be reduced to 0.75 mmolL
-1, explaining that modification back aminopeptidase is significantly improved to the avidity of substrate, maximum reaction velocity Vmax is 2500mmolL
-1Min
-1, result such as Fig. 1.
(3) mensuration of original enzyme liquid and modifying enzyme liquid optimum temperuture
Get original enzyme liquid and each 1mL of modifying enzyme liquid respectively, under the different temperature condition, measure enzyme activity with the Tris-HCl damping fluid of 0.05mol/L, pH8.5, thermograde is 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃.With high enzymatic activity is 100%, calculates relative enzyme activity, and the original enzyme liquid optimum temperuture is 60 ℃, and modifying enzyme liquid optimum temperuture is 65 ℃, result such as Fig. 2.
(4) temperature stability of original enzyme liquid and modifying enzyme liquid
Original enzyme liquid and modifying enzyme liquid are placed in 70 ℃, the Tris-HCl damping fluid of 0.05mol/L, pH8.5, and every ice bath is measured its enzyme activity immediately at a distance from the 1h sampling, and the water bath heat preservation time is 0h, 1h, 2h, 3h, 4h, 5h.With high enzymatic activity is 100%, calculates relative enzyme activity.Behind the 5h, the enzyme of original enzyme liquid retention rate alive only surpluss 35%, and the enzyme of modifying enzyme liquid is lived retention rate still more than 70%, result such as Fig. 3.
(5) mensuration of original enzyme liquid and modifying enzyme liquid ph optimum
Get original enzyme liquid and each 1mL of modifying enzyme liquid respectively, different pH 0.05mol/L, the Tris-HCl damping fluid in, 50 ℃ measure down its enzyme activities, pH value gradient is 6.0,7.0,7.5,8.0,8.5,9.0,9.5,10.0,11.0.With high enzymatic activity is 100%, calculates relative enzyme activity, and the ph optimum of original enzyme liquid is 8.5, and the ph optimum of modifying enzyme liquid is 9.5 ~ 10.0, result such as Fig. 4.
(6) pH of original enzyme liquid and modifying enzyme liquid stability
With original enzyme liquid and modifying enzyme liquid respectively in pH5.0 ~ 7.0 (0.05mol/L Sodium phosphate, dibasic-citrate buffer solution), pH7.0 ~ 10.0 (0.05mol/L glycocoll-sodium hydrate buffer solution) system in 4 ℃ of held, the optimal reaction pH value that sampling transfers to pH them behind the 6h is with the mensuration enzyme activity.With high enzymatic activity is 100%, calculates relative enzyme activity, and what live with the highest enzyme 80% is the pH stable range that the boundary confirms enzyme; Original enzyme liquid is in pH7.0 ~ 9.0 scopes; Enzyme activity is higher and stable, and modifying enzyme liquid is more stable in pH6.0 ~ 9.0 scopes, explains that the pH stability of modifying enzyme liquid is improved.Result such as Fig. 5.
The enzyme of solid ammonia peptase is lived
The LNA method is measured.During mensuration, at first join the Tris-HCl damping fluid, get 6.075g Tris and be dissolved in 900mL zero(ppm) water, transfer to pH8.5, be settled to 1L then with dense HCl., get the 0.1g substrate and be dissolved in 1mL alcohol as substrate with L-leucine p-Nitroaniline L-leu-pNA, then with Tris-HCl damping fluid constant volume to 100mL.Get 0.1g solid ammonia peptase powder with Tris-HCl damping fluid constant volume to 100mL as enzyme liquid to be measured.The Tris-HCl damping fluid and the 1mL substrate (L-leucine p-Nitroaniline L-leu-pNA) that add 2mL, one adds 1mL enzyme liquid, and another adds 1mL Tris-HCl damping fluid as contrast, and behind 50 ℃ of water-bath 10min, the 405nm colorimetric determination of enzyme is lived.
Enzyme formula alive: enzyme work=A * 105.7 * 4 * n/10
Wherein: A is the absorbancy of enzyme liquid to be measured; N is the extension rate of enzyme liquid to be measured.
The enzyme activity definition: 50 ℃, PM decomposition L-leucine-p-Nitroaniline produces the required enzyme amount of 1 micromolar p-Nitroaniline and is enzyme unit alive.
Average amido modified degree is measured
Adopt trinitrobenzenesulphonic acid (TNBS) method modifying enzyme to be averaged the mensuration of amido modified degree.Get 1mL protein enzyme solution (1mg/mL), add the sodium hydrogen carbonate solution of 1mL 4% (pH8.5), the sodium dodecyl sulfate solution of 1mL10%, the TNBS solution of adding 1mL 0.1% behind the 20min, 40 ℃ of insulation 2h are with the hydrochloric acid termination reaction of 0.5mL 1mol/L.325nm photometry absorption value.Same concentration protein enzyme solution after modification with modify before the ratio of absorbance value be residual amino rate.The residual amino rate of modification rate=1-.
Relative enzyme activity determination
On the same group the experiment in enzyme activity the highest be 100, with the ratio of remaining enzyme activity, represent with percentage ratio usually.
Enzyme residual rate alive is measured
Enzyme residual rate alive=initial enzyme of sample enzyme liquid remnant enzyme activity/sample enzyme liquid lives * 100%.
Beneficial effect of the present invention: with the subtilis aminopeptidase in the chemically modified condition after optimizing, obtained the best preparation method of chemically modified enzyme.After the reaction of small molecules chemical modifier Succinic anhydried; Change the structure of enzyme; A series of zymologic properties of enzyme are improved, and especially the thermostability of enzyme after modifying significantly improves, and aspects such as this suitability for industrialized production for enzyme, storage, application have great significance.
Description of drawings
The Lineweaver-Burk curve of Fig. 1 original enzyme liquid and modifying enzyme liquid;
The optimum temperature of Fig. 2 original enzyme liquid and modifying enzyme liquid;
The thermostability of Fig. 3 original enzyme liquid and modifying enzyme liquid;
The righttest action pH of Fig. 4 original enzyme liquid and modifying enzyme liquid;
The pH stability of Fig. 5 original enzyme liquid and modifying enzyme liquid.
Embodiment
The aminopeptidase powder that takes by weighing 20mg is dissolved in the Tris-HCl damping fluid of 15mL, 0.05mol/L, pH8.5; The Succinic anhydried (SA) that in mixed solution, slowly adds 10mg then; Stirring and dissolving, the pH value of in entire reaction course, using 2mol/L NaOH solution to keep reaction system is 8.0, under 4 ℃ of conditions, reacts 1h; After reaction finishes; 4 ℃ of dialysed overnight,, obtain the aminopeptidase (modifying enzyme SA-AE) that Succinic anhydried is modified with the Tris-HCl damping fluid of 0.05mol/L, pH8.5 to remove unnecessary modifier; The aminopeptidase powder that takes by weighing 20mg is dissolved in the Tris-HCl damping fluid of 15mL, 0.05mol/L, pH8.5,4 ℃ of dialysed overnight, obtains original enzyme liquid (AE) with the Tris-HCl damping fluid of 0.05mol/L, pH 8.5, and 4 ℃ of refrigerators are preserved.The enzyme retention rate alive of modifying back SA-AE is 105.81%; Compare with original enzyme liquid, the optimal reactive temperature of modifying enzyme is brought up to 65 ℃ from 60 ℃, and optimal reaction pH brings up to 9.5 ~ 10.0 from 8.5; 70 ℃ of insulations still can keep the activity more than 70% after 5 hours; Compare with enzyme under the equal conditions before the modification alive residual 35%, explain that its thermostability significantly improves, its kinetic parameter of the aminopeptidase after modification Km is from 1.0 mmolL
-1Be reduced to 0.75 mmolL
-1, maximum response constant Vmax is 2500mmolL
-1Min
-1, in the scope of pH6.0 ~ 9.0, modifying enzyme still keeps higher enzyme work and stablizes.
Claims (1)
1. a Succinic anhydried chemically modified improves the stable method of subtilis Zj016 aminopeptidase, it is characterized in that:
A, Succinic anhydried are to the chemical modification method of aminopeptidase
(1) the aminopeptidase powder that takes by weighing 20mg is dissolved in the Tris-HCl damping fluid of 15mL, 0.05mol/L, pH8.5, in mixed solution, slowly adds the Succinic anhydried SA of 10mg then, stirring and dissolving; The pH value of in entire reaction course, using 2mol/L NaOH solution to keep reaction system is 8.0; Under 4 ℃ of conditions, react 1h, after reaction finishes, with the Tris-HCl damping fluid of 0.05mol/L, pH8.5 4 ℃ of dialysed overnight; To remove unnecessary modifier; Obtain the aminopeptidase that Succinic anhydried is modified, modifying enzyme liquid is called for short SA-AE, and 4 ℃ of refrigerators are preserved;
(2) the aminopeptidase powder that takes by weighing 20mg is dissolved in the Tris-HCl damping fluid of 15mL, 0.05mol/L, pH8.5,4 ℃ of dialysed overnight, obtains original enzyme liquid AE with the Tris-HCl damping fluid of 0.05mol/L, pH8.5, and 4 ℃ of refrigerators are preserved;
The part zymologic property of enzyme before and after B, the modification
(1) adopt Succinic anhydried that aminopeptidase is carried out chemically modified under optimal conditions, SA is 55.86% to the average amido modified rate of AE, and enzyme retention rate alive is 105.81%;
(2) kinetic parameter Km: original enzyme liquid Km is 1.0 mmolL
-1, modifying enzyme liquid Km is reduced to 0.75mmolL
-1, explaining that modification back aminopeptidase liquid is significantly improved to the avidity of substrate, maximum reaction velocity Vmax is 2500mmolL
-1Min
-1
(3) optimum temperuture of original enzyme liquid and modifying enzyme liquid: the optimum temperuture of original enzyme liquid is 60 ℃, and the optimum temperuture of modifying enzyme liquid is brought up to 65 ℃;
(4) temperature stability of original enzyme liquid and modifying enzyme liquid: still can keep the activity more than 70% behind 70 ℃ of insulations of modifying enzyme liquid 5h, the enzyme residual rate alive under 70 ℃ of insulations of original enzyme liquid 5h equal conditions is merely 35%;
(5) original enzyme liquid and modifying enzyme liquid ph optimum: the ph optimum of original enzyme liquid is 8.5, and the ph optimum of modifying enzyme liquid brings up to 9.5 ~ 10.0;
(6) pH of original enzyme liquid and modifying enzyme liquid stability: with 80% of the highest enzyme work is the pH stable range that the boundary confirms enzyme, and original enzyme liquid is in pH7.0 ~ 9.0 scopes, and enzyme activity is higher and stable, and under the same conditions, modifying enzyme liquid is more stable in pH6.0 ~ 9.0 scopes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250514A (en) * | 2008-04-11 | 2008-08-27 | 东华大学 | Method for modifying pawpaw prolease by chemical reagent |
| CN101717763A (en) * | 2009-11-23 | 2010-06-02 | 江南大学 | Method for improving stability of liquid aminopeptidase and application of aminopeptidase |
| CN102078012A (en) * | 2010-12-08 | 2011-06-01 | 江南大学 | Application of Bacillus subtilis solid aminopeptidase in compound enzymolysis of porphyra |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250514A (en) * | 2008-04-11 | 2008-08-27 | 东华大学 | Method for modifying pawpaw prolease by chemical reagent |
| CN101717763A (en) * | 2009-11-23 | 2010-06-02 | 江南大学 | Method for improving stability of liquid aminopeptidase and application of aminopeptidase |
| CN102078012A (en) * | 2010-12-08 | 2011-06-01 | 江南大学 | Application of Bacillus subtilis solid aminopeptidase in compound enzymolysis of porphyra |
Non-Patent Citations (2)
| Title |
|---|
| 熊亚红 等: "提高漆酶稳定性的化学修饰方法的研究", 《化学研究与应用》 * |
| 田亚平 须瑛敏: "一种枯草芽孢杆菌氨肽酶的纯化及酶学性质", 《食品与发酵工业》 * |
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