CN104046602A - Method for enhancing activity and stability of Rhizopus oryzae lipase - Google Patents
Method for enhancing activity and stability of Rhizopus oryzae lipase Download PDFInfo
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Abstract
The invention discloses a method for enhancing activity and stability of Rhizopus oryzae lipase, belonging to the technical field of zymochemistry. The method comprises the following steps: carrying out oxidizing treatment on polysaccharide by using sodium periodate as an oxidizer to prepare oxidized polysaccharide, carrying out chemical modification on Rhizopus oryzae lipase by using the oxidized polysaccharide as the modifier, and carrying out dialysis and freeze-drying to obtain the polysaccharide-modified Rhizopus oryzae lipase. The activity of the Rhizopus oryzae lipase is enhanced by 2.2 times after modification under the following conditions: the pH value is 7.63, the reaction time is 15.53 hours, the reducer concentration is 0.2 mol/L, and the glucan concentration is 0.3%. After the Rhizopus oryzae lipase is treated at 50 DEG C for 30 minutes, the stability of the modified lipase is enhanced by 78% as compared with the unmodified lipase. The method has the characteristics of low cost, high safety and no toxicity, compromises the activity and stability of the lipase, and is suitable for industrialized popularization and application.
Description
Technical field
The present invention relates to a kind of method that improves Rhizopus oryzae lipase activity and stability, belong to zymochemistry technical field.
Technical background
Rhizopus oryzae lipase (Rhizopus oryzae lipase, ROL) is the lipase that the one that produced by filamentous fungus has strict 1,3 location specific and centering long-chain Substratspezifitaet.The Rhizopus oryzae bacterial classification lipase activity of general wild-type is lower, needs can obtain high efficiency lipase through further gene cloning and expression.
To catalytic activity of lipase, stability, in the research of the aspects such as selectivity, find, the yielding lipase wild strain screening from occurring in nature, its expression output is subject to the strict regulation and control of Host Strains gene, can only reach and meet microorganism self metabolism, and can not reach the requirement that the high efficient expression in market improves output.Existing enzyme performance modification technology mainly comprises the site-directed mutagenesis technique of immobilization, chemically modified, lactam enzyme by directional anagenesis in vitro, the biological marking and protein engineering.In numerous methods, chemically modified can be in the situation that not knowing zymoprotein structure, allows large-scale chemical group to be incorporated into fast in the structure of enzyme, and zymoprotein folded conformation is changed, and then changes enzymatic structure and catalytic performance.Chemical modification method can be divided into again crosslinked enzyme crystal, zymoprotein side chain functional group covalent modification, zymoprotein finishing, in conjunction with the chemically modified of rite-directed mutagenesis, carry out the methods such as chemical mutation by enzyme active sites amino acid atomic substitutions.
But, in existing Chemical Modification of Enzyme, in changing lipase activity, also can make the stability of enzyme be greatly affected, when being difficult to reach active and stability, improve.Select polysaccharide, as modifier, lipase is carried out to chemically modified, can improve lipase activity and stability simultaneously, there is fine application potential.
Summary of the invention
For addressing the above problem, the invention provides a kind of method that improves Rhizopus oryzae lipase activity and stability.Utilize oxidation of polysaccharides to carry out chemically modified to Rhizopus oryzae lipase, improved enzyme simultaneously and lived and stability, there is very high application and popularization value.The technical solution used in the present invention is as follows:
A kind of method that improves Rhizopus oryzae lipase activity and stability, taking sodium periodate as oxygenant, oxide treatment polysaccharide is prepared oxidation of polysaccharides, then taking oxidation of polysaccharides as modifier, Rhizopus oryzae lipase is carried out to chemically modified, after dialysis and freeze-drying, obtains polyose modification Rhizopus oryzae lipase.
The step of described method is as follows:
1) polysaccharide is made into the solution that concentration is 10mg/mL, and in solution, to add sodium periodate to its concentration be 50mmol/L, after dark lower reaction 2h, under 4 DEG C of dark, react again 24h, after having reacted, add ethylene glycol termination reaction, obtain oxidation of polysaccharides solution;
2) in the Rhizopus oryzae lipase phosphate buffer soln of pH7.6, add step 1) the oxidation of polysaccharides solution of gained, mix rear acquisition mixing solutions, react 15.5h at 20 DEG C;
3) to the step 2 of having reacted) in solution in to add solid sodium borohydride to its concentration be 0.2mol/L, obtain polyose modification solution react 1h at 20 DEG C after;
4) utilize dialysis tubing dialysis step 3) gained polyose modification solution, after freeze-drying, obtain polyose modification Rhizopus oryzae lipase.
Described method steps 1) described in polysaccharide be dextran, ficoll or synanthrin.
The molecular weight of dextran described in described method is 20KDa or 200KDa, and the molecular weight of described ficoll is 70KDa or 400KDa, and the molecular weight of described synanthrin is 5KDa.
Step 2 in described method) described in the concentration of mixing solutions oxidation of polysaccharides be 0.3%.
Step 4 in described method) described dialysis tubing, molecular retention amount is 3500Da.
The concrete steps of described method are as follows:
1) dextran of molecular weight 200KDa is mixed with to the solution that concentration is 10mg/mL, and in solution, to add sodium periodate to its concentration be 50mmol/L, react 24h at 20 DEG C, add after completion of the reaction ethylene glycol termination reaction, obtain oxidized dextran solution;
2) in the Rhizopus oryzae lipase phosphate buffer soln of pH7.6, add step 1) the oxidized dextran solution of gained, mix rear acquisition mixing solutions, react 15.5h at 20 DEG C;
3) to the step 2 of having reacted) in solution in to add solid sodium borohydride to its concentration be 0.2mol/L, obtain glucan-modified solution react 1h at 20 DEG C after;
4) utilize the dialysis tubing dialysis step 3 of molecular retention amount 3500Da) the glucan-modified solution of gained, after freeze-drying, obtain glucan-modified Rhizopus oryzae lipase.
Beneficial effect of the present invention:
1. the present invention is directed to the problem of the low and poor stability of the catalytic activity of natural rice rizolipase, utilize macromolecular polysaccharide to modify Rhizopus oryzae lipase, the catalytic activity of modifying rear enzyme is improved, stability also significantly improves.Overcome and in prior art, can not realize the technological deficiency that enzymic activity and enzyme stability improve simultaneously, the performance of Rhizopus oryzae lipase is significantly improved, be more suitable for industrial applications.
2. the present invention has determined the optimum process condition of modifying enzyme reaction: be that 0.20mol/L, dextran concentration are 0.30% at pH 7.63, time 15.53h, reductant concentration, Rhizopus oryzae lipase activity has improved 2.2 times.
3. the modifier safety adopting in the present invention, nontoxic, modifying enzyme is applied to food-processing industry, higher security is provided.
Brief description of the drawings
Fig. 1 is the impact of reaction times on modification enzyme activity.
Fig. 2 is the impact of pH on modification enzyme activity.
Fig. 3 is the impact of reductant concentration on modification enzyme activity.
Fig. 4 is the impact of dextran concentration on modification enzyme activity.
Fig. 5 is different modifying reaction factor response surface trend map;
(a.pH and the time surface chart on acid-decomposed activity impact; B.pH and the reductant concentration surface chart on acid-decomposed activity impact; C. time and the dextran concentration surface chart on acid-decomposed activity impact).
Fig. 6 is the optimum temperature of protoenzyme and modifying enzyme.
Fig. 7 is the impact of temperature on protoenzyme and modification enzyme activity.
Fig. 8 is the impact of pH on protoenzyme and modification enzyme activity.
Embodiment
The present invention is directed to the problem of the low and poor stability of the catalytic activity of Rhizopus oryzae lipase, utilize oxidation of polysaccharides to modify Rhizopus oryzae lipase, after modifying, activity and the stability of enzyme significantly improve simultaneously.Modifier safety non-toxic used, is more suitable for industrial applications.Below by embodiment, the present invention will be further described, but the present invention is not subject to the restriction of embodiment.
Preparation and detection that embodiment 1 oxidation of polysaccharides is modified Rhizopus oryzae lipase
1. the oxidation of polysaccharide
Polysaccharide is dissolved as after 10mg/mL solution, is 50mmol/L to adding sodium periodate to make its concentration in solution.After above-mentioned system is reacted to 2h under room temperature dark, under 4 DEG C of dark, react 24h again, finally add the ethylene glycol of 500 μ L to mix with termination reaction.After 1h, by after product dialysis treatment, obtain polysaccharide oxide compound, be packed as 1mL and store in 4 DEG C of refrigerators.
2. the chemically modified of lipase
Take 20mg ROL at 2mL pH6-8 phosphoric acid salt (0.02mol/L Na
2hPO
4/ NaH
2pO
4) dissolve in damping fluid.The polysaccharide oxide compound of certain volume is joined in enzyme solution, after mixing, at room temperature cultivate after 8～48h, add the sodium borohydride of different concns, constantly stirring and evenly mixing, removes solvent by sample through dialysis treatment after 1h, through freeze-drying, obtain the Rhizopus oryzae lipase of polyose modification again.
3. lipase acid hydrolysis method
Lard and sad ratio with 3:1 are put in 5mL centrifuge tube, after vibration mixes, are put in 40 DEG C of water-baths, then add the Lipase catalyzed acidolysis of substrate quality 5%, after two hours, take out centrifuge tube, centrifugal 5min under 9000r/min.Draw upper oil phase and be stored in 4 DEG C of refrigerators, to be analyzed.
4. thin-layer chromatography separates
Separation condition: silica-gel plate G plate; Developping agent: V (sherwood oil-ether-acetic acid)=80/20/2.Plank immerses the depth distance plank base 0.5～1.0cm of developping agent.After expansion completes, take out plank and dry, evenly spray the colour developing of 0.1%2,7-dichlorofluorescein.After glycerine fatty acid separates, the retention parameter (Rf value) of each component is respectively: sterol Rf=0.81, sweet three ester Rf=0.69,1,3-DAG Rf=0.31,1,2-DAG Rf=0.22, a sweet ester Rf=0.02, lipid acid Rf=0.50.
5. esterification method
Sweet three esters and DAG after Thin-layer separation are placed in to 25mL round-bottomed flask, add the KOH-methanol solution vibration of 2mL0.5mol/L to mix, in 70 DEG C of water-baths, react 10min, take out cooling after, add the BF3-methanol solution 3mL[V (BF3-ether) of instant preparation: V (methyl alcohol)=1:3], again in 70 DEG C of water-bath back flow reaction 5min, take out cooling, add 2mL normal hexane fully to vibrate, sample is extracted completely in normal hexane, add again saturated nacl aqueous solution, leave standstill after 5min, draw upper strata normal hexane and preserve in sample bottle.
6. gas Chromatographic Determination condition
Experimental analysis adopts Agilent 7890 gas chromatographs and hydrogen flame ionization detector (FID).(100m × 0.25mm × 0.20 μ m) for chromatographic column: FFAP; Combustion gas: H2, flow 30mL/min; Combustion-supporting gas: air, flow 300mL/min; Carrier gas: He, flow 1mL/min; Before post, press: 37.5MPa; Splitting ratio 50:1; Sample size 1 μ L; Injector temperature: 240 DEG C; Furnace temperature: 140 DEG C, maintain 5min; Detector temperature: 250 DEG C.Use Agilent chem workstation (HP3398A GChemStation) to carry out data collection and analysis, adopt area normalization method to calculate.
7. enzymic activity definition
Thereby being triglyceride level, acidolysis reaction under the catalysis of lipase and between lipid acid, there is the method that acyl group transfer change triglyceride structure forms.Taking lard and sad as substrate, by measuring sad combination rate on triglyceride level, reflect the acid-decomposed activity of lipase.
The optimization of embodiment 2 dextran (Dextran) to the chemically modified of Rhizopus oryzae lipase
1. experiment of single factor
Adopt dextran 200,000, as modifier, Rhizopus oryzae lipase is carried out to chemically modified, taking reaction times 8～48h, reaction pH6～8, reductant concentration 0.05%～0.4% and dextran concentration 0.1%～1% as major influence factors, respectively it is carried out to experiment of single factor, investigate each factor to enzyme modification influential effect.
Fig. 1 is for being 7.5 as reaction pH, and reductant concentration is 0.2mol/L, when dextran concentration is 0.5%, and the impact of reaction times on modification enzyme activity.As can be known from Fig. 2, in the time that the reaction times is less than 16h, prolongation in time of enzymic activity and raising peaks in the time of 16h, after reduce in time again.So when follow-up optimization experiment, the central value in reaction times is chosen near 16h.
Fig. 2 is for being 20h when the reaction times, and reductant concentration is 0.2mol/L, when dextran concentration is 0.5%, and the impact of reaction pH on modification enzyme activity.As can be known from Fig. 2, after enzyme is modified under acidic conditions, activity is lower, and raise and be significantly increased with pH, be to reach the highest at 7.6 o'clock at pH, fast reducing again subsequently.
Fig. 3 is for being 20h when the reaction times, and reaction pH is 7.5, when dextran concentration is 0.5%, and the impact of reductant concentration on modification enzyme activity.As can be known from Fig. 3, enzymic activity slowly reduces after first raising with reductant concentration, in the time of 0.2mol/L, peaks.So in follow-up optimization experiment, the central value of reductant concentration is chosen near 0.2mol/L.
Fig. 4 is for being 20h when the reaction times, and reaction pH is 7.5, when reductant concentration is 0.2mol/L, and the impact of dextran concentration on modification enzyme activity.As can be known from Fig. 4, enzymic activity first raises and reduces afterwards with dextran concentration, peaks 0.30% time.So in follow-up optimization experiment, the central value of reductant concentration is chosen near 0.30%.
2. response surface optimization
According to response surface experimental design principle, on the basis of above-mentioned experiment of single factor, the response surface experiment of design four factor three levels amounts to 29.Taking reaction times, reaction pH, reductant concentration and dextran concentration as independent variable(s), taking acid-decomposed activity as response value, set up quadratic polynomial regression model, experimental design factor and code levels are in table 1.
Table 1 experimental design factor and code levels
Response surface optimization experiment is according to Box-Behnken principle of design, on the basis of table 1, designs 29 groups of response surface optimizations and analyzes experiment, experimental design and the results are shown in Table 2.
Box-Behnken experimental design and the result of the glucan-modified ROL of table 2
Experimental data in above-mentioned table 2 is utilized to Design Expert7.0.0 software analysis, obtains the secondary multinomial regression model of acidolysis rate to coding independent variable(s) pH, time, reductant concentration and dextran concentration:
Acidolysis rate=36.96+2.16 × A-2.57 × B+0.067 × C-1.42 × D-2.78 × A × B+10.07 × A × C+0.60 × A × D+1.24 × B × C-0.29 × B × D+1.56 × C × D-11.25 × A
2-12.25 × B
2-12.09 × C
2-15.72 × D
2
Model is carried out to variance analysis and regression coefficient test of significance, in table 3.Model equation variance analysis can be found out, quadratic polynomial model difference remarkable (P < 0.01, R2=0.9917); A P=0.0518 > 0.05 is not remarkable for disappearance.As can be seen from Table 3, A-PH, B-TIME, mutual AC and quadratic term are extremely remarkable (P < 0.01); D-dextran concentration, a mutual AB and CD are remarkable (P < 0.05); Other are all not remarkable.Checked from regression equation coefficient, in 4 models, the size of coefficient entry absolute value is followed successively by B > A > D > C, illustrate that the modification time has the greatest impact to acid-decomposed activity, next is pH and dextran concentration, and the concentration of reductive agent is the most weak to the activity influence of modifying enzyme.
The variance analysis of table 3 response surface regression equation
Note: * represents P<0.05, significantly; * represents P<0.01, extremely remarkable.
Fig. 5 is different modifying reaction factor response surface trend map.Be according to different factors, response value to be affected to strong and weak solid intuitively to reflect by response surface three-dimensional space curve figure, in the time that curvilinear plane is precipitous, show on response value impact greatly, in the time that curvilinear plane is mild, to show response value impact little.Be two kinds of interactive results of factor from two-dimentional level line, can judge the interactive size of each factor by its shape, the oval two factor significant interaction that represent, circular illustrate that the interaction of two factors affects not remarkable on response value.
Fig. 5 a is pH and the surface chart of time on acid-decomposed activity impact, finds out level line shape ovalize by figure, shows significant interaction.Curved surface is precipitous, and pH and time are on significantly (P < 0.05) of the impact of acid-decomposed activity.When reductant concentration and dextran concentration are fixed in 0 level, in the time that the reaction times is constant, along with the variation of pH, acid-decomposed activity presents the trend that first raises and reduce afterwards, and in the time that pH is near 7.6, it is maximum that acid-decomposed activity reaches.In the time that pH is constant, along with the prolongation in reaction times, the active maximum value that reaches in the time that the reaction times is 16h left and right.In the condition and range of experimental design, interaction between them shows as the increase of acid-decomposed activity.
Fig. 5 b is pH and the surface chart of reductant concentration on acid-decomposed activity impact, as seen from the figure, the gradient of response surface is comparatively precipitous, shows that two factors of pH and reductant concentration are larger on acid-decomposed activity impact, the level line shape of the two is obvious ellipse, shows that interaction is extremely remarkable.
Fig. 5 c time and the dextran concentration surface chart on acid-decomposed activity impact, as seen from the figure, when reductant concentration and PH are fixed in 0 level, in the time that dextran concentration is constant, with the prolongation in reaction times, reduces after acidolysis takes the lead in raising; When constant in the time, with the variation of dextran concentration, acid-decomposed activity is also first to raise and reduce afterwards.So the reaction times, dextran concentration was between 0.25%～0.35% time between 14～18h, acid-decomposed activity reaches maximum.And interaction between them shows as acid-decomposed activity and increases, but level line shape subcircular shows that two factor interactions are remarkable not on acidolysis impact.
Based on above response surface analysis and further software analysis calculating, each level of factor while obtaining acid-decomposed activity maximum is: pH7.63, time 15.53h, reductant concentration are that 0.20mol/L and dextran concentration are 0.30%, and the predictor of acidolysis rate is 37.28%.
For the accuracy of verification model prediction, be to repeat 3 experiments under 0.20mol/L, the dextran concentration condition that is 0.30% at pH7.63, time 15.53h, reductant concentration.Experimental result acidolysis rate is respectively 37.80%, 36.07% and 38.83%, and mean value is 37.57%, and relative error is 0.77%, and in analyzing with optimization, gained theoretical value is basically identical, proves that this model is rationally effective.
The mensuration of embodiment 3 enzymic activitys and stability
1. enzyme optimum temperature
At 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C and 60 DEG C of temperature, taking lard and sad as substrate, measure the acid-decomposed activity of ROL and Dextran-ROL, measurement result is as shown in Figure 6.As can be seen from Figure 6, ROL and Dextran-ROL variation with temperature trend are basically identical, after taking the lead in raising, ROL and Dextran-ROL acidolysis reduce, all in the time of 40 DEG C, reach maximum value, so the optimum temperature of ROL and Dextran-ROL is 40 DEG C, now the acid-decomposed activity of Dextran-ROL is 2.2 times of ROL, and ROL almost loses activity at 55 DEG C, and Dextran-ROL still has certain activity in the time of 60 DEG C, so Dextran-ROL has certain heat-resisting ability.
2. the thermostability of enzyme
ROL and Dextran-ROL solution, respectively at being incubated after 30min in 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C and 65 DEG C of water-baths, then are measured to the acid-decomposed activity of enzyme, and the relative enzyme work that definition enzymic activity is the highest one group is 100%, and result as shown in Figure 7.As shown in Figure 7, the better heat stability of Dextran-ROL between 40～45 DEG C, after insulation 30min, activity still can remain on more than 98%, and slow decreasing is subsequently incubated after 30min in the time of 65 DEG C, relative activity remains less than 5%, and enzyme almost all loses activity.The less stable of ROL, raises with temperature, and enzyme activity loss is strong.
3. enzyme pH stability
Enzyme is dissolved in the phosphoric acid buffer of different pH4-9, in 4 DEG C of refrigerators, stores after 1h, then the acid-decomposed activity of enzyme is measured.The relative enzyme work that definition enzymic activity is the highest one group is 100%, and result as shown in Figure 8.As shown in Figure 8, Dextran-ROL and ROL are less to its activity influence after storing in neutral solution, still keep the highest vigor.Dextran-ROL obviously improves than the resistance to acids and bases of ROL, after storing in the weakly acidic solution that is 6 at pH, Dextran-ROL still retains more than 95% vigor and ROL is only 90%, under weak basic condition, Dextran-ROL retains vigor higher than protoenzyme 20%, and be 9 o'clock at pH, ROL is inactivation almost, and Dextran-ROL still retains 50% above vigor.
Claims (7)
1. one kind is improved the method for Rhizopus oryzae lipase activity and stability, it is characterized in that, taking sodium periodate as oxygenant, oxide treatment polysaccharide is prepared oxidation of polysaccharides, taking oxidation of polysaccharides as modifier, Rhizopus oryzae lipase is carried out to chemically modified again, after dialysis and freeze-drying, obtain polyose modification Rhizopus oryzae lipase.
2. method described in claim 1, is characterized in that, step is as follows:
1) polysaccharide is made into the solution that concentration is 10mg/mL, and in solution, to add sodium periodate to its concentration be 50mmol/L, after reacting 2h under 20 DEG C of dark, under 4 DEG C of dark, react 24h again, after having reacted, add ethylene glycol termination reaction, obtain oxidation of polysaccharides solution;
2) in the Rhizopus oryzae lipase phosphate buffer soln of pH7.6, add step 1) the oxidation of polysaccharides solution of gained, mix rear acquisition mixing solutions, react 15.5h at 20 DEG C;
3) to the step 2 of having reacted) in solution in to add solid sodium borohydride to its concentration be 0.2mol/L, obtain polyose modification solution react 1h at 20 DEG C after;
4) utilize dialysis tubing dialysis step 3) gained polyose modification solution, after freeze-drying, obtain polyose modification Rhizopus oryzae lipase.
3. method described in claim 2, is characterized in that step 1) described polysaccharide is dextran, ficoll or synanthrin.
4. method described in claim 3, is characterized in that, the molecular weight of described dextran is 20KDa or 200KDa, and the molecular weight of described ficoll is 70KDa or 400KDa, and the molecular weight of described synanthrin is 5KDa.
5. method described in claim 2, is characterized in that step 2) described in the concentration of mixing solutions oxidation of polysaccharides be 0.3%.
6. method described in claim 2, is characterized in that step 4) described dialysis tubing, molecular retention amount is 3500Da.
7. method described in claim 2, is characterized in that, concrete steps are as follows:
1) dextran of molecular weight 200KDa is mixed with to the solution that concentration is 10mg/mL, and in solution, to add sodium periodate to its concentration be 50mmol/L, react 24h at 20 DEG C, add after completion of the reaction ethylene glycol termination reaction, obtain oxidized dextran solution;
2) in the Rhizopus oryzae lipase phosphate buffer soln of pH7.6, add step 1) the oxidized dextran solution of gained, mix rear acquisition mixing solutions, react 15.5h at 20 DEG C;
3) to the complete step 2 of reaction) in solution in to add solid sodium borohydride to its concentration be 0.2mol/L, obtain glucan-modified solution react 1h at 20 DEG C after;
4) utilize the dialysis tubing dialysis step 3 of molecular retention amount 3500Da) the glucan-modified solution of gained, after freeze-drying, obtain glucan-modified Rhizopus oryzae lipase.
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| CN106093270A (en) * | 2016-08-27 | 2016-11-09 | 江南大学 | A kind of sweet three ester α position fatty acid determination methods of animal and plant fat |
| CN111944783A (en) * | 2018-12-13 | 2020-11-17 | 浙江大学 | Lipase mutant with improved thermal stability and application thereof |
| CN114052085A (en) * | 2021-12-03 | 2022-02-18 | 东北农业大学 | 0-6 month infant formula milk powder for hierarchically simulating breast milk lipid and preparation method thereof |
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| CN111944783A (en) * | 2018-12-13 | 2020-11-17 | 浙江大学 | Lipase mutant with improved thermal stability and application thereof |
| CN111944783B (en) * | 2018-12-13 | 2021-09-07 | 浙江大学 | Lipase mutant with improved thermal stability and application thereof |
| CN114052085A (en) * | 2021-12-03 | 2022-02-18 | 东北农业大学 | 0-6 month infant formula milk powder for hierarchically simulating breast milk lipid and preparation method thereof |
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