CN101575595B - Method for preparing calcium-alginate-immobilized marine bacterium MP-2 esterase - Google Patents
Method for preparing calcium-alginate-immobilized marine bacterium MP-2 esterase Download PDFInfo
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Abstract
The invention relates to a method for preparing calcium-alginate-immobilized marine bacterium MP-2 esterase. Sodium alginate and marine bacterium MP-2 esterase are dissolved in a buffer solution of glycin (NaOH) and then mixed evenly; and the mixture is dripped into CaCl2 solution for balling, standing, immobilization, washing and drying so as to obtain the graininess immobilized marine bacteriumMP-2 esterase. The method has the advantages of stable system, good compatibility, mild reaction and high activity. The calcium-alginate-immobilized marine bacterium MP-2 esterase can be used as an ethyl caproate catalyst, has high synthesis conversion rate, can be used repeatedly and has wide application prospect.
Description
Technical field
The present invention relates to the marine microorganism esterase field, particularly relate to the preparation method of calcium-alginate-immobilized marine bacterium MP-2 esterase.
Background technology
(Esterase EC3.1.1.1) belongs to hydrolase (Hydrolases) to microorganism esterase, derives from the microbe, refers to the general name of all enzymes that can the catalytic hydrolysis carboxylicesters.As biological catalyst, have very high catalysis, substrate specificity and atopic, utilize its hydrolysis reaction, ester conversion and ester synthesis reaction to be widely used in fields such as medicine, chemical industry, food, the energy and environmental protection.Especially in recent years along with the deepening continuously of nonaqueous phase enzymology, esterase demonstrates the potential application prospect having become the focus of attention both domestic and external aspect the fractionation of the synthetic and chiral drug of ester compound.And marine microorganism is for adapting to this limit of life environment of the high salt high pressure of marine low temperature, and the marine microorganism enzyme has showed zymologic property and the physiological function more unique than the Lu Yuan microbial enzyme.Yet because the existence of the problems such as cost, high efficiency and stability of free esterase, it is used and still faces many difficulties.Therefore for modern industry, free esterase is not a kind of ideal catalyzer.In order to overcome its shortcoming, adopt immobilized marine microorganism esterase technology, the characteristic that can not only keep protoenzyme, and will improve the stability of enzyme greatly, life-time service and be difficult for inactivation continuously, also easily with the separating of product, be beneficial to enzyme repeat repeatedly use and the purifying of product, thereby significantly reduce the use cost of enzyme, therefore have more wide application prospect.
In recent years, immobilized enzyme has been carried out the research report both at home and abroad and thought that sodium alginate is the sodium salt polymkeric substance of uronic acid, less and be difficult for being degraded to the toxicity of biology, the gel that forms with it and calcium salt comes the embedding enzyme, the gel physical strength is better, inner one-tenth vesicular structure, reaction conditions gentleness, simple to operate, the loss of enzyme activity is little.The catalysis journal, 2005,26 (11), the P877-981 report is reported with the preparation of sodium alginate immobilization head mold fatty ester, foodstuffs industry science and technology 2006,27 (9), 69-71 report sodium alginate immobilization glutamine transaminage Study on Stability.
Summary of the invention
The objective of the invention is to overcome above-mentioned prior art and have many shortcomings, can provide a kind of system stable through the long-term development research marine microorganism of contriver and secretory product thereof and production practice, consistency is good, reaction temperature and, the enzyme motility rate keeps the preparation method of high calcium-alginate-immobilized marine bacterium MP-2 esterase.
The preparation method of calcium-alginate-immobilized marine bacterium MP-2 esterase provided by the invention comprises the following steps:
1. sodium alginate and marine bacterium MP-2 esterase are dissolved in respectively in the damping fluid of glycine NaOH of 0.1mol/L pH10, after being dissolved into solution, two solution under agitation thorough mixing are even, wherein Na-alginate concentration is 2-4 quality %, be preferably 3 quality %, sodium alginate and marine bacterium MP-2 esterase in mixing solutions 1: 6 to 1: 10 are preferably 1: 8;
2. in immobilization reactor, step mixing solutions is 1. under agitation dropwise injected CaCl
2Balling-up in the solution, CaCl
2The concentration of solution is 1-3 quality %, is preferably 2 quality %; At room temperature for example leave standstill under 25 ℃ after the balling-up and carry out immobilization, the immobilization time is 30-80 minute, be preferably 60 minutes, then for several times with distilled water wash, to remove unnecessary ion, after cold wind dries up, get particulate state immobilized marine bacterium MP-2 esterase (being called for short the immobilization esterase), its granular size is about 0.5-1.5mm, 4 ℃ of preservations.
Among the preparation method of calcium-alginate-immobilized marine bacterium MP-2 esterase provided by the invention, described marine bacterium MP-2 esterase is to obtain high yield esterase bacterial strain MP-2 by Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science from ocean bed mud culture of isolated, claims that the high yield esterase is a marine bacterium MP-2 esterase; Institute provides by Huanghai Sea aquatic products, and promptly commodity are commercially available.
Esterase is in immobilization process, and along with the increase of esterase amount, the vigor of immobilization esterase is the trend of falling after rising and activity recovery descends gradually.The ratio of esterase and sodium alginate carrier is when being 8: 1 (mass ratio) in 6: 1 to 10: 1, and this moment, enzyme activity was the highest relatively.This is perhaps because along with the esterase amount increases progressively, the esterase that adsorbs on the carrier increases, the immobilized ester enzyme activity is raise gradually, but because carrier can only adsorb limited esterase amount, when the carrier amount reduced relatively, the esterase amount of the carrier of Board Lot institute embedding was more relatively, and it is agglomerating to cause the esterase molecule to assemble mutually, the active centre of enzyme molecule might be covered, and catalysis activity still can descend.Therefore, when having only the suitable proportioning of esterase and carrier, just can make the immobilization esterase show higher activity.
Described sodium alginate exists as carrier in the esterase immobilization process, and sodium alginate concentration not only influences the physical strength of gel, mass transfer, and then can have influence on the vigor of immobilization esterase.When sodium alginate concentration was low, the gel aperture was bigger, and the esterase molecule in the gel runs off easily, so enzyme activity is lower; When sodium alginate concentration was too high, the gel aperture was too little, and limited the diffusion of substrate and product this moment, caused enzyme activity to descend equally.The while excessive concentration, though the intensity of gel increases, embedding medium viscosity also increases, and is not easy to immobilized operation.Therefore, carrier concn should be controlled in the suitable scope, and as being 2-4 quality % when sodium alginate concentration, preferred 3.0% o'clock, enzyme activity was the highest relatively.
CaCl in the described fixing agent
2Ca
2+In immobilization process, form water-fast calcium alginate gel with the Lalgine radical ion, thereby the esterase embedding is fixed.CaCl as fixing agent
2The physical strength that forms gel also there is material impact.Therefore, CaCl
2Concentration should be controlled in the suitable scope.Along with CaCl
2The increase of concentration, the immobilized ester enzyme activity is the trend of falling after rising, and works as CaCl
2Concentration is 2-4 quality %, is at preferred 2% o'clock, and the relative vigor of immobilization esterase is the highest.
The described immobilization time is meant that freshly prepd immobilization esterase gel particle is at CaCl
2The time of leaving standstill in the solution.Solidify the long or too short immobilized ester enzyme activity that all makes of fixing time and descend, for example 30-80 minute, the relative enzyme activity that is preferably 60 minutes immobilization esterases was the highest.This is because along with the immobilization time lengthening, and gel-strength improves, and the polymer link is fine and close, is unfavorable for the matrix transmission, and the substrate diffusional resistance increases, and causes the immobilized ester enzyme activity to reduce thus.
Immobilized marine bacterium MP-2 esterase provided by the invention is under above-mentioned optimum condition, and the vigor of immobilized marine bacterium MP-2 esterase (being called for short the immobilization esterase) reaches 616.2U/g, and this moment, enzymatic activity recovery was 52.8%.
With the zymologic property of immobilized marine bacterium MP-2 esterase not (or claiming free esterase) relatively:
The determination of activity of esterase
A: the mensuration of free esterase activity: adopt the p-NPP method, 20 μ l enzyme liquid are added to 2ml contain 4mmol/LMg
2+In glycine-NaOH (pH10.0) buffer system of the 0.1mol/L of 17mmol/L substrate p-NPP, behind effect 10min under 60 ℃, pH10.0 condition, add 2mol/L NaOH solution and stop enzyme reaction, enzyme liquid replacement original enzyme liquid with inactivation in the boiling water water-bath is a blank in addition, at the light absorption value of 405nm place assaying reaction product p-NP, survey two parallel samples, triplicate at every turn.Discharge the required enzyme amount of 1 μ mol p-NP (p-Nitrophenol) with every min decomposition p-nitrophenyl cetylate (p-Nitrophenyl palmitate) and be defined as 1 enzyme activity unit, represent with U.
B: the mensuration of immobilized ester enzyme activity: replace resolvase with the 0.1g immobilized enzyme, in 60 ℃ of shaking baths, be incubated, press the resolvase method and measure enzyme activity (U/g).
Immobilization esterase and free esterase optimum temperature are relatively
In 4～100 ℃ of temperature ranges, carry out the active mensuration of esterase hydrolyzed.
As shown in Figure 1, the optimum temperuture of immobilization esterase is 80 ℃, the raising of specific ionization esterase 20 ℃, it still keeps greater activity in 50～80 ℃ of scopes, illustrate that esterase is immobilized its thermotolerance of back and increases, this may be owing to enzyme molecule after the immobilization is connected with the carrier multiple spot, can prevent the distortion of enzyme molecular stretching, thereby stablized the conformation of enzyme molecule, and then the critical denaturation temperature of enzyme is improved.
The comparison of immobilization esterase and free esterase thermostability
Immobilization esterase and free esterase are dissolved in respectively in the damping fluid of 0.1mol/L NaOH--glycine of pH10, place under the differing temps (60～80 ℃), 2h is handled in water bath with thermostatic control, take a sample every 20min, cooling rapidly, measuring its remnant enzyme activity, is 100% with not treated enzyme activity, and all the other amount to into the percentage ratio of residual enzyme vigor.
As shown in Figure 2, free esterase insulation 1h enzyme activity only deposits 10% in the time of 70 ℃, and immobilization esterase insulation 2h enzyme activity when still keeping 80%, 80 ℃ free esterase insulation 45min enzyme activity then completely lose, and immobilization esterase insulation 2h enzyme activity still keeps 70%.As seen, the thermostability of immobilization esterase is far above free esterase, and this is that protein molecule is fixed among the gel owing to dissociate esterase after immobilization, and the molecule mass motion is limited, has suppressed self degrading of enzyme, and the stability in active centre also increases thereupon.
Immobilization esterase and free esterase be the comparison of suitable action pH
Get a certain amount of immobilization esterase and join respectively in the damping fluid of different pH, add substrate again and survey its activity with free esterase.
As shown in Figure 3, the suitableeest action pH of immobilization esterase brings up to 11.This may be because sodium alginate is that electronegative carrier can attract the positively charged ion in the solution, comprises H
+, make it attached to carrier surface, the result makes immobilization esterase diffusion layer H
+External solution height around the concentration ratio, the pH in the surrounding environment must alkalitropism be offset like this, could offset the microenvironment effect, makes esterase show maximum vigor.
Immobilization esterase and free esterase pH stability are relatively
A certain amount of immobilization esterase and free esterase are dissolved in respectively in the damping fluid of different pH (2～12), place under 4 ℃, behind the insulation 48h, again enzyme solution is recalled to optimal pH 10, enzyme activity with insulation gained under the optimum condition is 100%, and all the other amount to into the percentage ratio of residual enzyme vigor.
As shown in Figure 4, the immobilization esterase all keeps higher enzyme activity in pH9～11 scopes, and except pH10, its enzyme activity is all lower in this scope for free by comparison esterase.This explanation esterase is immobilized its resistance to acids and bases of back obviously to be strengthened.This may be since behind the enzyme immobilization residing microenvironment and active centre be subjected to the influence of carrier, the dissociating property of active group changes.
Metal ion is to the influence of immobilization esterase and free esterase activity
A certain amount of immobilization esterase and free esterase are added each metal ion species respectively, and concentration of metal ions is 0.01mol/L in the maintenance survey reaction system alive, compares to survey with damping fluid and live.
As shown in Figure 5, Co
2+, Li
+Immobilization esterase and free esterase all there is activation and more remarkable to the activation of immobilization esterase; Ca
2+Free esterase is had no significant effect Cu
2+, Pb
2+, Ag
+, Mn
2+, Zn
2+, Fe
3+, Ba
2+, Mg
2+Free esterase activity there is restraining effect, and Ca
2+, Cu
2+, Mn
2+Influence to immobilized enzyme is not remarkable, simultaneously Pb
2+, Zn
2+, Fe
3+, Ba
2+, Mg
2+Restraining effect to the immobilization esterase has patience; Ag
+Almost completely suppressed the activity of free esterase and still can keep 34.2% the activity of immobilization esterase.The good stability of immobilization esterase specific ionization esterase to metal ion is described thus, and this is because behind the enzyme immobilization, its space structure is relatively stable.
The Michaelis-Menton constant Km of immobilization esterase and free esterase
Get the substrate p-NPP of different concns, add immobilization esterase and free esterase respectively, measure the first speed of reaction of two kinds of enzymic hydrolysis substrates, be Michaelis-Menton equation curve (Fig. 6 by Curve Expert software regression fit, 7) try to achieve Km (Gu)=2.08mmol/L, Km (trip)=3.34mmol/L, this illustrates that this esterase Michaelis-Menton constant Km after immobilization reduces, this may change owing to the higher structure of enzyme and the influence of carrier causes that the avidity of enzyme-to-substrate increases, thereby Km is reduced.
The package stability of immobilization esterase and free esterase
Place 4 ℃ to store down immobilization esterase and free esterase, live every 10d sampling and measuring enzyme, the activity of immobilization esterase and free esterase all reduces along with the prolongation of time, as shown in Figure 8, can only maintain 25.8% after free esterase is placed 60d, and the vigor of immobilization esterase can remain on still more than 45%, perhaps this is because more stable through structure after the immobilization, suppress self degraded, improved stability.
The storage transformation period of immobilization esterase
Transformation period calculates by this formula: t
1/2=0.693t/2.303lg (E
0/ E)
Wherein: t
1/2The immobilized ester half life of enzyme; T is the working hour; E
0/ E is enzyme activity residue fraction immobilization esterase enzyme reservation 82.9% alive behind 4 ℃ of storage 15d behind the working hour t, and free esterase keeps 64.8%; The storage transformation period of immobilization esterase is 55d.This shows that marine microorganism esterase obviously strengthens through the immobilization rear stability.
The preparation method of calcium-alginate-immobilized marine bacterium MP-2 esterase provided by the invention, characteristics are:
1. the calcium alginate gel with low cost and no side effects is a carrier, adopt entrapping method successfully immobilization marine bacterium MP-2 esterase.Sodium alginate and Ca
2+Form hydrophilic porousness calcium alginate gel, its system is stable, consistency is good, reaction temperature and.2. under the condition of optimal fixationization, the vigor of immobilization esterase reaches 616.2U/g, and this moment, enzymatic activity recovery was 52.8%.3. marine bacterium MP-2 esterase is through after the immobilization, optimum temperuture raises to some extent, skew and its thermostability, pH stability, operational stability, package stability have taken place and the equal specific ionization esterase of the stability of metal ion have been strengthened to some extent in optimum pH, Michaelis-Menton constant Km reduces, and also shows that the avidity of this immobilized ester enzyme-to-substrate increases to some extent.Cause existing between the enzyme-to-substrate calcium alginate gel, substrate could be in conjunction with enzymatic reaction takes place by absorption and diffusion process, under similarity condition because the existence of this effect, slowed down apparent reaction rates, thereby the suitableeest enzymatic reaction temperature is raise, the optimal pH scope broadens, and stability is improved.4. be catalyzer with the immobilization esterase, synthesizing ethyl hexanoate in the normal hexane non-aqueous solvent, the synthetic transformation efficiency reaches 65.8%.After reusing 6 times, the immobilization esterase still keeps 85% enzyme activity, and having good stability of this immobilization esterase is described.The immobilization of marine microorganism esterase makes it have good industrialization and application prospect widely, thus the development and utilization of further having opened up marine microorganism enzyme resource.
Description of drawings
Fig. 1 is the influence of temperature to immobilization esterase and free esterase activity
Fig. 2 is the thermostability of immobilization esterase and free esterase
Fig. 3 is the influence of pH to immobilization esterase and free esterase activity
Fig. 4 is immobilization esterase and free esterase pH stability
Fig. 5 is the influence of metal ion to immobilization esterase and free esterase activity
Fig. 6 is an immobilization esterase Michaelis-Menton equation curve
Fig. 7 is free esterase Michaelis-Menton equation curve
Fig. 8 immobilization esterase and free esterase package stability
Embodiment
The present invention further specifies the present invention with the following example, but protection scope of the present invention is not limited to the following example.
Embodiment 1
Get the damping fluid that 1.125 gram sodium alginates and 9 gram marine bacterium MP-2 esterases are dissolved in the glycine NaOH of 0.1mol/L pH10 respectively, under agitation thorough mixing is even after the dissolving, wherein Na-alginate concentration is 3 quality %, and sodium alginate and marine bacterium MP-2 esterase are in mixing solutions 1: 8..In immobilization reactor (commercially available) with above-mentioned mixing solutions under agitation dropwise implantation concentration be the CaCl of 2 quality %
2In the solution, at room temperature leave standstill after the balling-up and carried out immobilization in 60 minutes, then with distilled water wash until removing unnecessary ion, after cold wind dries up then, particle dia is about the particulate state immobilized marine bacterium MP-2 esterase of 1mm.
The present embodiment step is same as embodiment 1, and not being both sodium alginate is 2 grams, and concentration is 2 quality %; Marine bacterium MP-2 esterase 2 grams, CaCl
2Concentration is 1.8 quality %; The immobilization time is 70 minutes.
The application of immobilization esterase
With the immobilization esterase is catalyzer, is solvent with the normal hexane, prepares 20% (volume fraction) ethanolic soln of 1% caproic acid, accurately draws the 1mL caproic acid in the 100mL volumetric flask, with 20% alcohol dilution to scale.Get 20ml caproic acid ethanolic soln in distilling flask, add 1g immobilization esterase, at 50 ℃ of insulation esterification 2h.With gas chromatographic analysis ethyl hexanoate content, and calculate the ester Synthesis conversion, the result shows that this immobilized enzyme catalysis ethyl hexanoate synthetic transformation efficiency reaches 65.8%.And its catalytic activity is not seen obviously and is weakened after reusing for 6 times.Be converted to enzyme activity by the ester Synthesis conversion, be equivalent to after 6 repeated uses, this immobilized enzyme still keeps the catalytic activity more than 85%.This further illustrates this immobilization esterase and has good operational stability.
Claims (2)
1. the preparation method of calcium-alginate-immobilized marine bacterium MP-2 esterase is characterized in that comprising the following steps:
1. sodium alginate and marine bacterium MP-2 esterase are dissolved in respectively in the damping fluid of glycine NaOH of 0.1mol/L pH10, after being dissolved into solution, two solution under agitation thorough mixing are even, wherein sodium alginate and marine bacterium MP-2 esterase mass ratio in mixing solutions is 1: 6 to 1: 10, and Na-alginate concentration is 2-4 quality %;
2. in immobilization reactor, step mixing solutions is 1. under agitation dropwise injected CaCl
2Balling-up in the solution, CaCl
2The concentration of solution is 1-3 quality %, at room temperature leave standstill after the balling-up and carry out immobilization, the immobilization time is 30-80 minute, then with distilled water wash for several times to remove unnecessary ion, after cold wind dried up, getting particle dia was the particulate state immobilized marine bacterium MP-2 esterase of 0.5-1.5mm.
2. according to the preparation method of the calcium-alginate-immobilized marine bacterium MP-2 esterase of claim 1, it is characterized in that described sodium alginate soln concentration is 3.0 quality %; The mass ratio of described sodium alginate and marine bacterium MP-2 esterase is 1: 8; Described CaCl
2Strength of solution is 2.0 quality %; The immobilization time is 60 minutes.
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| 平芮巾等.产海洋细菌MP-2酯酶菌株的鉴定及酯酶理化性质的研究.《渔业科学进展》.2009,第30卷(第2期),全文. * |
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Assignee: Shandong Huachen biochemistry Co., Ltd. Assignor: Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science Contract record no.: 2011990000790 Denomination of invention: Method for preparing calcium-alginate-immobilized marine bacterium MP-2 esterase Granted publication date: 20110622 License type: Exclusive License Open date: 20091111 Record date: 20110816 |
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