CN105754968B - Modified lipase and preparation method thereof - Google Patents
Modified lipase and preparation method thereof Download PDFInfo
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C12Y301/00—Hydrolases acting on ester bonds (3.1)
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Abstract
The invention relates to a modified lipase, which is prepared by carrying out a crosslinking reaction on a substrate containing lipase under the action of glutamine transaminase. Compared with the thermal stability of the existing modified lipase, the thermal stability of the modified lipase is improved to a greater extent, and the modified lipase is suitable for industrial popularization and use. The invention relates to a preparation method of modified lipase, which applies glutamine transaminase as a catalyst to the crosslinking reaction of active protein (lipase) to prepare the modified lipase (namely improve the stability of the lipase), and can catalyze the self crosslinking of the lipase without introducing other macromolecular or micromolecular substances and influencing the performance of the lipase; but also can catalyze the co-crosslinking of lipase and other proteins to form mixed network macromolecules, thereby improving the stability of the lipase. The method is simple, safe, obvious in effect and beneficial to industrial production.
Description
Technical Field
The invention belongs to the field of lipase, and particularly relates to modified lipase and a preparation method thereof.
Background
Lipases are one of important industrial enzyme preparations, can catalyze reactions such as lipolysis, ester exchange and ester synthesis, and are widely applied to industries such as oil processing, food, medicine and daily chemical industry. However, the temperature of general industrial production is high, and the application of lipase in industrial production is limited due to poor thermal stability of lipase. The lipase has good thermal stability, is more beneficial to the processes of industrial production, transportation and storage, and simultaneously means that the service life of the lipase is longer. Therefore, it is necessary to provide a safe and convenient method for improving the stability of lipase.
At present, there are many methods for improving the thermostability of lipase, such as molecular modification, chemical modification, and the like. The molecular modification mainly adopts means such as rational design directed evolution and the like, but specific mutation sites are often difficult to determine. Chemical modification includes modification (amination, alkylation, etc.), immobilization, chemical crosslinking, etc. of groups on the surface of the enzyme molecule. The structure of enzyme molecules can be improved through intermolecular crosslinking, the rigidity of the enzyme is increased, and the thermal stability of the enzyme is improved. The cross-linking agent used for chemical cross-linking is glutaraldehyde, but glutaraldehyde has certain toxicity and can damage the initial enzyme activity. Akbulut N et al (Akbulut N,
M T,Pijning T,et al.Improved activity and thermostability ofBacillus pumilus lipase by directed evolution[J]journal of Biotechnology,2013,164(1):123- & 129) by shuffling the lipase gene to extend its half-life to 38.5min at 50 ℃. Khanahmadi S et al (Khanahmadi S, Yusof F, Amid A, et al. optimized prediction and characterization of CLEA-lipase from cocoa pod husk [ J]Journal of Biotechnology 2015,185:153-161) preparation of Cross-linked enzyme aggregates using glutaraldehyde increased the tolerance temperature of the lipase by 10 ℃. Chinese patent CN201410288942.4 provides a method for improving lipase stability, which comprises using sodium periodate as an oxidant, oxidizing polysaccharide to prepare oxidized polysaccharide, and using the oxidized polysaccharide as a modifier to perform chemical modification on rhizopus oryzae lipase. After the modified lipase is treated at 50 ℃ for 30min, the stability of the modified lipase is improved by 78 percent compared with that of the unmodified lipase.
At present, the methods for improving the stability of the lipase, whether molecular modification or chemical modification, cannot be applied to the improvement of the stability of the industrial lipase.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a modified lipase aiming at the defects of the prior art, the thermal stability of the modified lipase is improved to a greater extent than that of the lipase prepared by modification by the prior art, and the modified lipase is suitable for industrial popularization and use.
The invention also provides a preparation method of the modified lipase, which is simple, safe and nontoxic, has obvious effect, and the thermal stability of the prepared modified lipase is improved to a greater extent than that of the lipase prepared by modification in the prior art, and can be applied to the improvement of the stability of the industrial lipase.
To this end, the present invention provides, in a first aspect, a modified lipase obtained by subjecting a substrate containing a lipase to a crosslinking reaction under the action of transglutaminase.
According to the invention, the solution of the substrate is a lipase solution or a mixture of a lipase solution and a protective protein solution.
In some embodiments of the invention, the lipase comprises one or more of lipase powder, extracellular lipase, and fermentation broth after cell wall breaking.
In other embodiments of the invention, the protective protein comprises gelatin and/or bovine serum albumin.
According to the invention, the stability time of the modified lipase at 50 ℃ is more than or equal to 60 minutes.
The second aspect of the invention provides a preparation method of modified lipase, which comprises the step of carrying out crosslinking reaction on a substrate containing lipase under the action of glutamine transaminase to prepare the modified lipase.
According to the invention, the solution of the substrate is a lipase solution or a mixture of a lipase solution and a protective protein solution.
In some embodiments of the present invention, the lipase solution is prepared by using phosphate buffer as a solvent and lipase.
In other embodiments of the present invention, the protective protein solution is prepared by using phosphate buffer as a solvent and protective protein.
According to the invention, the mass concentration ratio of the lipase solution to the protective protein solution in the substrate solution is 1:2-2: 1.
In the present invention, the amount of transglutaminase to be used is 40 to 200U/g based on the total weight of the substrate, and preferably 120-200U/g based on the total weight of the substrate.
In some embodiments of the invention, the concentration of the lipase solution and the protective protein solution are each in the range of 1-30 mg/mL.
In other embodiments of the present invention, the concentration of the phosphate buffer is 50-100mM, preferably the concentration of the phosphate buffer is 80-100 mM.
In the present invention, the pH of the phosphate buffer is 6 to 8, and preferably the pH of the phosphate buffer is 7 to 8.
In some embodiments of the invention, the lipase comprises one or more of lipase powder, extracellular lipase, and fermentation broth after cell wall breaking.
In other embodiments of the invention, the protective protein comprises gelatin and/or bovine serum albumin.
According to the invention, the temperature of the crosslinking reaction is 20-30 ℃, preferably the temperature of the crosslinking reaction is 25-30 ℃; the time of the crosslinking reaction is 22-26h, and preferably 24-26 h.
In some embodiments of the invention, the crosslinking reaction is performed in a shaker; the rotation speed of the shaking table is 100-200rpm, preferably 180-200 rpm.
Drawings
The invention will be described below with reference to the accompanying drawings.
FIG. 1 is a schematic diagram showing intermolecular or intramolecular cross-linking reaction between a TG enzyme-catalyzed lipase or a lipase and a protective protein; the reference numerals in the figures have the following meanings: 1 a lipase; 2 lipase or protected protein; 3 modified lipase.
FIG. 2 shows the results of the effect of crosslinking reaction time on the enzymatic activity of modified lipases.
FIG. 3 shows the effect of the crosslinking reaction temperature on the enzymatic activity of the modified lipase.
FIG. 4 shows the results of the effect of pH of the reaction solution on the enzymatic activity of the modified lipase.
FIG. 5 is a graph showing the comparison of thermal stability before and after crosslinking in the preparation of modified lipase by the method of the present invention.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below.
As described above, neither molecular modification nor chemical modification methods for improving lipase stability can be applied to industrial lipase stability improvement. The inventor researches and discovers that the molecular modification for improving the stability of the lipase has complex operation and long time consumption, so that the molecular modification can not be applied to the improvement of the stability of the industrial lipase; the chemical modification for improving the stability of lipase usually involves toxic modifying agents, has complex operation process and low economic feasibility, and thus cannot be used for improving the stability of industrial lipase.
The inventor further researches and discovers that the use of glutamine transaminase (TG enzyme) can catalyze lipase to generate intermolecular or intramolecular cross-linking, and can also catalyze the lipase to carry out co-cross-linking with other protein molecules so as to form a network macromolecular polymer. Specifically, TG enzymes catalyze the epsilon-NH of lysine (Lys) residues of proteins2And a glutamic acid (Glu) residue2Formation of isopeptide bond [ epsilon- (gamma-glutamine) -lysine]Resulting in intermolecular or intramolecular cross-linking of the protein, as shown in FIG. 1 and reaction scheme (I). The crosslinking has obvious influence on the property, the gelling capacity, the thermal stability and the like of the protein, thereby improving the structure and the functional property of the protein, increasing the rigidity of enzyme molecules and obviously improving the thermal stability of the crosslinked lipase. TG enzyme is generally used in food processing, and the present inventors applied TG enzyme for the first time to active protein (lipase)And (4) modifying. The present invention has been made based on the above-mentioned findings.
Therefore, the modified lipase according to the first aspect of the present invention can be understood as a novel modified lipase with improved thermostability, which is obtained by subjecting a substrate containing the lipase to a crosslinking reaction under the action of glutamine transaminase. In some embodiments, for example, the modified lipase has a stabilization time of 60 minutes or more at 50 ℃.
The "solution of the substrate" referred to herein as "substrate solution" is a lipase solution or a mixture of a lipase solution and a protective protein solution, wherein the substrate is a lipase or a mixture of a lipase and a protective protein, and thus, the substrate solution may also be understood as a mixture solution prepared from a lipase or a mixture of a lipase and a protective protein and a solvent, such as phosphate buffer.
In some embodiments of the invention, the lipase comprises one or more of lipase powder, extracellular lipase and fermentation broth after cell wall breaking; preferably, the lipase is Yarrowia lipolytica (Yarrowia lipolytica) lipase.
The source of the lipase is not particularly limited in the present invention, for example, the lipase can be a commercially available product, such as Novozym 435, or can be prepared by fermentation of Yarrowia lipolytica (see Chinese patent CN200510112638.5) bacterium deposited in China general microbiological culture Collection center (address: Ministry of China academy of sciences, North China, Ministry of China, Haitan, Beijing, and China at 30.9.2005) with the deposit number of CGMCC No. 1470; preferably, the lipase is prepared by fermenting yarrowia lipolytica with the preservation number of CGMCC No. 1470.
In other embodiments of the invention, the protective protein is preferably a protein susceptible to cross-linking by TG enzyme, including gelatin and/or bovine serum albumin.
The TG enzyme is not particularly limited in the present invention, and for example, the TG enzyme may be a commercially available product such as one available from east sage food science and technology Co.
In some embodiments of the invention, the ratio of the mass concentration of the lipase solution to the mass concentration of the protective protein solution in the substrate solution is 1:2-2: 1; preferably, the mass concentration ratio of the lipase solution to the protective protein solution in the substrate solution is 1: 1.
In some embodiments of the invention, the transglutaminase is used in an amount of 40-200U/g based on the total weight of the substrate. The preferred amount of transglutaminase to be used is 120-200U/g based on the total weight of the substrate. It is further preferred that the amount of transglutaminase to be used is 120U/g based on the total weight of the substrate.
According to the method, the lipase solution is prepared by taking a phosphate buffer solution as a solvent and lipase. In some embodiments of the invention, the concentration of the lipase solution is in the range of 1-30 mg/mL.
According to the method, the protective protein solution is prepared by taking phosphate buffer as a solvent and protective protein. In some embodiments of the invention, the concentration of the protective protein solution ranges from 1 to 30 mg/mL.
In some embodiments of the invention, the phosphate buffer has a concentration of 50-100 mM; preferably the concentration of the phosphate buffer is 80-100 mM; it is further preferred that the concentration of the phosphate buffer is 80 mM.
In some embodiments of the invention, the phosphate buffer has a pH of 6 to 8; preferably, the pH value of the phosphate buffer solution is 7-8; further preferably, the phosphate buffer has a pH of 7.
The preparation method of the modified lipase according to the second aspect of the present invention can be understood as a method for improving the stability of lipase, and specifically comprises the following steps:
(1) preparing a phosphoric acid buffer solution.
The concentration of the phosphate buffer solution is 50-100 mM; further preferably, the concentration of the phosphate buffer is 80-100 mM; more preferably, the phosphate buffer has a concentration of 80 mM.
The structure of lipase can be protected in a buffer solution with certain ionic strength. If the pH of the buffer solution is too high or too low, enzyme molecules are inactivated; if the ionic strength is too low, the buffering effect is lost, and if the ionic strength is too high, the enzyme molecules are inactivated.
(2) Preparing a lipase solution.
And (3) adding lipase into the phosphate buffer solution serving as a solvent, and uniformly stirring and dissolving to obtain a lipase solution.
The lipase comprises one or more of lipase powder, extracellular lipase and fermentation liquor after cell wall breaking.
(3) Preparing a protective protein solution.
And (3) adding protective protein by taking the phosphate buffer as a solvent, and heating and dissolving to prepare a protective protein solution.
The addition of protective proteins can provide more groups for crosslinking on the one hand and can protect the lipase from excessive crosslinking on the other hand. The protective protein is preferably a protein susceptible to cross-linking by TG enzymes, including gelatin and/or bovine serum albumin.
(4) A solution of the substrate is prepared.
The lipase solution may be used directly as a solution of the substrate, or the lipase solution may be mixed with the protective protein solution to obtain a solution of the substrate.
The mass concentration ratio of the lipase solution to the protective protein solution in the substrate solution is 1:2-2: 1; preferably, the mass concentration ratio of the lipase solution to the protective protein solution in the substrate solution is 1: 1. The mass concentration ratio of the lipase solution to the protective protein solution in the substrate solution is too low, the crosslinking effect is not obvious, the mass concentration ratio of the lipase solution to the protective protein solution in the substrate solution is too high, the crosslinking reaction is excessive, and the improvement effect on the lipase structure is not great.
(5) A reaction solution was prepared.
Adding TG enzyme into the solution of the substrate, and stirring uniformly to prepare reaction liquid.
The amount of transglutaminase to be used is 40-200U/g based on the total weight of the substrate. The preferred amount of transglutaminase to be used is 120-200U/g based on the total weight of the substrate. It is further preferred that the amount of transglutaminase to be used is 120U/g based on the total weight of the substrate. When the amount of TG enzyme is too small, the catalytic efficiency is low, and when the amount of TG enzyme is too large, the TG enzyme is easy to crosslink excessively to inactivate the TG enzyme.
(6) A crosslinking reaction is carried out.
The reaction solution is put into a shaking table for reaction. The rotating speed of the shaking table is 100-200 rpm; preferably, the rotating speed of the shaking table is 180-200 rpm; it is further preferred that the rotational speed of the shaker is 180 rpm.
The temperature of the crosslinking reaction is 20-30 ℃; preferably, the temperature of the crosslinking reaction is 25-30 ℃; it is further preferred that the temperature of the crosslinking reaction is 25 ℃. The reaction speed is slow when the temperature is too low, and the lipase is easy to inactivate when the temperature is too high.
The time of the crosslinking reaction is 22-26 h; preferably, the time of the crosslinking reaction is 24-26 h; further preferably, the time for the crosslinking reaction is 24 hours. The inventor researches and discovers that the time of the crosslinking reaction is too short, the crosslinking is insufficient, and the performance of the lipase is not obviously improved; if the time for the crosslinking reaction is too long, the crosslinking tends to be excessive, and the enzyme molecules are inactivated.
The term "enzyme activity", also called "enzyme activity", as described in the present invention is defined as: under the measuring conditions, the lipase quantity required for generating 1 mu mol of fatty acid per minute is 1 lipase enzyme activity unit and is expressed by U.
The phrase "stabilization time of the modified lipase or lipase" as used herein refers to a time during which the modified lipase or lipase is stable under certain temperature conditions, for example, a time during which the modified lipase or lipase is stable at 50 ℃; the stable state refers to that the enzyme activity is reduced by less than 5%.
The term "Yarrowia lipolytica" as used herein is also known as Yarrowia lipolytica.
As described above, glutamine transaminase has been widely used in the field of food, but the present inventors applied it as a catalyst to the crosslinking reaction of a protein (lipase) having activity, thereby avoiding the use of a chemical crosslinking agent, and making the entire process for improving the stability of lipase (i.e., preparing a modified lipase) efficient, safe and non-toxic.
The modified lipase prepared by the method (namely the stability of the lipase is improved) can catalyze the self-crosslinking of the lipase, does not introduce other macromolecular or micromolecular substances, and does not influence the performance of the lipase; but also can catalyze the co-crosslinking of lipase and other proteins to form mixed network macromolecules, thereby improving the stability of the lipase. The method is simple and safe, and is more beneficial to industrial production.
Examples
In order that the invention may be more readily understood, the invention will now be described in further detail with reference to the accompanying drawings and examples, which are given by way of illustration only and are not limiting to the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
The Yarrowia lipolytica lipase in the following examples was prepared by fermentation of Yarrowia lipolytica (Yarrowia lipolytica) deposited in China general microbiological culture Collection center (address: China academy of sciences 13, northern Guancun, in the Haita district, Beijing, Japan) at 30.9.2005 with the deposit number CGMCC No.1470 by the inventor's laboratory.
The invention adopts an olive oil emulsification method to determine the enzyme activity, namely a method for hydrolyzing olive oil-polyethylene glycol (PVA) emulsion for 10 minutes at 35 ℃ to determine the enzyme activity, and the specific method is as follows:
(1) preparing an olive oil-polyethylene glycol emulsion: mixing PVA solution with the mass concentration of 3% and olive oil according to the volume ratio of the PVA solution to the olive oil of 3:1, and stirring at a high speed to prepare uniform emulsion;
(2) preparing an emulsion reaction system: mixing 5mL of olive oil-polyethylene glycol emulsion with 4mL of 0.1M phosphoric acid buffer solution with the pH value of 8 to prepare an emulsion reaction system;
(3) reaction: preheating an emulsion reaction system for 5min at 35 ℃, adding 1mL of diluted sample liquid to be detected (enzyme liquid to be detected), adding 15mL of absolute ethyl alcohol to terminate the reaction after reacting for a specified time, and obtaining an enzyme activity detection product;
the above reaction can be carried out in a HZS-HA water bath shaker (Tomings medical instruments, Harbin).
(4) And (4) detecting a result: and adding 1-2 drops of phenolphthalein indicator into the enzyme activity detection product, and titrating with 0.05M NaOH to obtain the volume of NaOH titrated by the sample to be detected.
(5) And (3) calculating enzyme activity: calculating enzyme activity by adopting a formula (II);
enzyme activity
Wherein: the volume difference of NaOH titrated by a sample to be measured and a blank sample is delta V, and the unit is mL;
the micromole number of NaOH contained in each milliliter of NaOH solution;
dilution factor of N sample;
t reaction time, unit min.
Example 1: and (3) investigating the influence of the crosslinking reaction time on the enzyme activity of the modified lipase.
(1) Preparing 80mM phosphate buffer solution with pH 7.0;
(2) preparing a substrate solution by using the phosphate buffer solution prepared in the step (1) as a solvent:
the substrate solution 1-1 is lipase solution, and the concentration of the lipase solution is 1 mg/mL;
the substrate solution 1-2 is a mixture of a lipase solution and a gelatin (protective protein) solution, and the concentration of the lipase in the substrate solution II is 1mg/mL, and the concentration of the gelatin (protective protein) in the substrate solution II is 1 mg/mL;
(3) preparing a reaction solution:
TG enzyme is added into the substrate solution 1-1 to prepare a reaction solution 1-1, and the addition amount of the TG enzyme is 120U per gram of the substrate.
TG enzyme is added into the substrate solution 1-2 to prepare a reaction solution 1-2, and the addition amount of the TG enzyme is 120U per gram of the substrate.
Substrate solution 1-1 was used as a control reaction solution.
(4) The reaction solutions 1-1 and 1-2 and the control reaction solution were reacted at 25 ℃ and 180rpm for 12, 24, 36, and 48 hours, respectively.
After the reaction, the enzyme activity of the modified lipase product was measured by a method of hydrolyzing olive oil-polyethylene glycol (PVA) emulsion at 35 ℃ for 10 minutes, and compared with that of the unmodified lipase, the results are shown in fig. 2.
As can be seen from FIG. 2, the result of co-crosslinking with gelatin is superior to that of lipase alone; compared with the free lipase of a control group, the result after crosslinking is better than that without crosslinking; at the same time, it can also be seen that the optimum reaction time is 24 h.
Example 2: and (3) investigating the influence of the crosslinking reaction temperature on the enzyme activity of the modified lipase.
(1) Preparing 80mM phosphate buffer solution with pH 7.0;
(2) preparing a substrate solution by using the phosphate buffer solution prepared in the step (1) as a solvent:
the substrate solution 2-1 is lipase solution, and the concentration of the lipase solution is 1 mg/mL;
the substrate solution 2-2 is a mixture of a lipase solution and a gelatin (protective protein) solution, and the concentration of the lipase in the substrate solution 2-2 is 1mg/mL, and the concentration of the gelatin (protective protein) is 1 mg/mL;
(3) preparing a reaction solution:
TG enzyme is added into the substrate solution 2-1 to prepare a reaction solution 2-1, and the addition amount of the TG enzyme is 120U per gram of the substrate.
TG enzyme is added into the substrate solution 2-2 to prepare a reaction solution 2-2, and the addition amount of the TG enzyme is 120U per gram of the substrate.
Substrate solution 2-1 was used as a control reaction solution.
(4) The reaction solutions 2-1 and 2-2 and the control reaction solution were reacted at 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C and 180rpm for 24 h.
After the reaction was completed, the enzyme activity of the modified lipase product was measured in the same manner as in example 1 and compared with that of the unmodified lipase, and the results are shown in FIG. 3.
As can be seen from FIG. 3, the enzyme is easily inactivated when the crosslinking temperature is too high, and the crosslinking result is good at 20-25 ℃. Example 3: and (3) investigating the influence of the pH value of the reaction liquid on the enzyme activity of the modified lipase.
(1) Preparing 80mM phosphate buffer solution with pH values of 4, 5, 5.5, 6, 6.5, 7, 7.5 and 8 respectively;
(2) preparing substrate solutions by respectively using the phosphate buffer solution as a solvent in the step (1):
substrate solutions 3-1 to 3-8 are lipase solutions with pH values of 4, 5, 5.5, 6, 6.5, 7, 7.5 and 8 respectively, and the concentration of the lipase solutions is 1 mg/mL;
substrate solutions 3-21 to 3-28 are lipase solutions with pH values of 4, 5, 5.5, 6, 6.5, 7, 7.5 and 8 respectively, and the concentration of the lipase solutions is 1 mg/mL;
the substrate solutions 3-31 to 3-38 were mixture solutions containing lipase and gelatin (protective protein) at pH values of 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, respectively, and the lipase concentration was 1mg/mL and the gelatin (protective protein) concentration was 1mg/mL in the substrate solutions 3-31 to 3-38.
(3) Preparing a reaction solution:
TG enzyme was added to the substrate solutions 3-1 to 3-8 to prepare reaction solutions 3-1 to 3-8, and the amount of added TG enzyme was 120U per gram of substrate.
TG enzyme is added into the substrate solutions 3-31 to 3-38 to prepare reaction solutions 3-31 to 3-38, and the addition amount of the TG enzyme is 120U per gram of the substrate.
Substrate solutions 3-21 to 3-28 were used as control reaction solutions 3-21 to 3-28.
(4) The above reaction solutions 3-1 to 3-8, 3-31 to 3-38 and the control reaction solutions 3-21 to 3-28 were reacted at 25 ℃ and 180rpm, respectively, for 24 hours.
After the reaction was completed, the enzyme activity of the modified lipase product was measured in the same manner as in example 1 and compared with that of the unmodified lipase, and the results are shown in FIG. 4.
As can be seen from fig. 4, the cross-linking results are best at pH7.
Example 4: the method for catalyzing lipase crosslinking by using glutamine transaminase comprises the following specific steps:
(1) preparing 80mM phosphate buffer solution with pH 7.0;
(2) preparing a substrate solution by taking the buffer solution prepared in the step (1) as a solvent and taking lipase as a substrate, wherein the concentration of the substrate solution is 30 mg/mL;
(3) TG enzyme is added into the substrate solution to prepare reaction solution, and the addition amount of the TG enzyme is 120U per gram of the substrate.
(4) Reacting for 24 hours at the temperature of 25 ℃ and the rotating speed of 180rpm to obtain the modified lipase.
(5) Detection of thermal stability of enzymes
The thermal stability of the enzyme was compared with that of the above modified lipase under the same reaction conditions using the non-modified lipase as a control: the modified lipase and the control group lipase were placed at 50 ℃, and sampled at 0, 2, 3, 5, 10, 15, 20, 30, 45, and 60min, respectively, and the enzyme activities thereof were measured by the same method as in example 1, and the change in enzyme activities of the non-modified lipase and the modified lipase after different times at 50 ℃ was compared, and the results are shown in fig. 5.
As can be seen from FIG. 5, the enzyme activity retention rate of the non-modified lipase is only about 30% after 1h at 50 ℃; and the modified lipase obtained after TG enzyme crosslinking has almost no loss of enzyme activity after 1h at 50 ℃. The thermal stability of the obtained modified enzyme is obviously improved after the crosslinking by the method of the invention. Example 5: the enzyme activities of modified lipases obtained by carrying out lipase crosslinking reactions in the presence of glutaraldehyde, genipin, and glutamine transaminases (TG enzymes), respectively, were compared.
(1) Preparing 80mM phosphate buffer solution with pH 7.0;
(2) preparing a substrate solution by using the phosphate buffer solution prepared in the step (1) as a solvent, wherein the concentration of lipase is 0.5mg/mL, and the concentration of gelatin is 0.5 mg/mL;
(3) preparing a reaction solution:
adding 0.5% (v/v) glutaraldehyde into 10mL substrate solution to prepare reaction solution 5-1;
0.1 of genipin is added into 10mL of substrate solution to prepare a reaction solution 5-2;
adding TG enzyme into 10mL of substrate solution to prepare a reaction solution 5-3, wherein the addition amount of TG is 120U per gram of substrate;
(4) reacting the reaction solution 5-1 for 6h at 25 ℃ and the rotation speed of 180rpm, and reacting the reaction solutions 5-2 and 5-3 for 24h to obtain the modified lipase.
The enzyme activity of the modified lipase product was measured by a method of hydrolyzing an olive oil-polyethylene glycol (PVA) emulsion at 35 ℃ for 10 minutes, and the results are shown in table 1.
TABLE 1 comparison of enzyme activities of modified lipases Using Cross-linker and TG enzyme
| Crosslinking agents or catalysts | Glutaraldehyde | Genipin | TG enzyme |
| Enzyme activity retention of modified fats | 27.78％ | 53.33％ | 100％ |
From table 1, it can be seen that the lipase cross-linking using glutaraldehyde and genipin as cross-linking agents all cause the decrease of enzyme activity, especially the loss of enzyme activity is large when the lipase cross-linking using glutaraldehyde as cross-linking agents is performed, while the loss of enzyme activity is basically not caused when the lipase cross-linking is catalyzed by TG enzyme. Compared with a chemical cross-linking agent, the TG enzyme catalytic cross-linking process is milder, and the damage to the lipase activity structure is less.
Example 6: and (3) preparing the modified lipase by catalyzing the lipase with TG enzyme and co-crosslinking with protective protein.
(1) Preparing 80mM phosphate buffer solution with pH 7.0;
(2) preparing a substrate solution by taking the phosphate buffer solution prepared in the step (1) as a solvent and taking a mixture of lipase, lipase and protective protein (gelatin or bovine serum albumin) as a substrate respectively:
in the substrate solution, the concentration of lipase was 0.5mg/mL, and the concentration of other proteins (gelatin or bovine serum albumin) was 0.5 mg/mL.
(3) Preparing a reaction solution:
TG enzyme is added into the substrate solution to prepare reaction solution, and the addition amount of the TG enzyme is 120U per gram of the substrate.
(4) The above reaction solution 6-1 containing lipase, gelatin and TG enzyme, the reaction solution 6-2 containing lipase, bovine serum albumin and TG enzyme and the control reaction solution 6-3 containing lipase and TG enzyme were reacted for 24 hours at 25 ℃ and 180rpm, respectively, to obtain modified lipase.
The enzyme activity of the modified lipase product was measured in the same manner as in example 1, and the results are shown in Table 2.
Table 2 comparison of crosslinking results with addition of different protective proteins
| Protein species | Lipase enzyme | Lipase + gelatin | Lipase + bovine serum albumin |
| Retention rate of |
100％ | 120％ | 100％ |
Table 2 shows the comparison of the enzyme activities after different protective proteins (gelatin, bovine serum albumin) and lipase were added and cross-linked in the method of the present invention. Surprisingly, when the added protective protein is gelatin or BSA, the enzyme activity is not only not lost but also improved to some extent.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (22)
1. A modified lipase is prepared by carrying out a crosslinking reaction on a substrate containing lipase under the action of glutamine transaminase; the substrate solution is a mixture of a lipase solution and a protective protein solution;
the protective protein is gelatin.
2. The modified lipase according to claim 1, wherein the lipase comprises one or more of lipase powder, extracellular lipase and fermentation broth after cell wall breaking.
3. The modified lipase according to claim 1 or 2, which has a stability time of 60 minutes or more at 50 ℃.
4. A preparation method of modified lipase comprises the steps of carrying out crosslinking reaction on a substrate containing lipase under the action of glutamine transaminase to prepare the modified lipase;
the substrate solution is a mixture of a lipase solution and a protective protein solution;
the protective protein is gelatin.
5. The method according to claim 4, wherein the lipase solution is prepared by using phosphate buffer as a solvent and lipase.
6. The method according to claim 5, wherein the protective protein solution is prepared by using phosphate buffer as a solvent and protective protein.
7. The method according to claim 5, wherein the mass concentration ratio of the lipase solution to the protective protein solution in the substrate solution is 1:2-2: 1.
8. The process according to claim 7, wherein the transglutaminase is used in an amount of 40-200U/g based on the total weight of the substrate.
9. The process as claimed in claim 7, wherein the transglutaminase is used in an amount of 120-200U/g based on the total weight of the substrate.
10. The method of claim 5, wherein the concentration of the lipase solution and the protective protein solution is in the range of 1-30 mg/mL.
11. The method according to claim 10, wherein the phosphate buffer is at a concentration of 50-100 mM.
12. The method according to claim 10, wherein the phosphate buffer is present in a concentration of 80-100 mM.
13. The method of claim 10, wherein the phosphate buffer has a pH of 6 to 8.
14. The method of claim 10, wherein the phosphate buffer has a pH of 7 to 8.
15. The method according to any one of claims 5 to 14, wherein the lipase comprises one or more of lipase powder, extracellular lipase and a fermentation broth after cell wall breaking.
16. The method according to any one of claims 5 to 14, wherein the temperature of the crosslinking reaction is 20 to 30 ℃.
17. The method of claim 16, wherein the temperature of the crosslinking reaction is 25-30 ℃.
18. The method according to claim 16, wherein the cross-linking reaction time is 22-26 h.
19. The method according to claim 16, wherein the cross-linking reaction time is 24-26 h.
20. The process according to any one of claims 5 to 14, wherein the crosslinking reaction is carried out in a shaker.
21. The method as claimed in claim 20, wherein the rotational speed of the shaker is 100-200 rpm.
22. The method as claimed in claim 20, wherein the rotational speed of the shaker is 180-200 rpm.
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| CN102260662A (en) * | 2010-05-24 | 2011-11-30 | 中国科学院过程工程研究所 | Carrier used for immobilized enzymes, its purpose and carrier fixed with enzymes |
| CN102711853A (en) * | 2009-12-22 | 2012-10-03 | 生命连结有限公司 | Modification of enzymatic crosslinkers for controlling properties of crosslinked matrices |
| CN103421762A (en) * | 2012-05-22 | 2013-12-04 | 北京化工大学 | Immobilized enzyme and preparation method thereof |
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| CN102260662A (en) * | 2010-05-24 | 2011-11-30 | 中国科学院过程工程研究所 | Carrier used for immobilized enzymes, its purpose and carrier fixed with enzymes |
| CN103421762A (en) * | 2012-05-22 | 2013-12-04 | 北京化工大学 | Immobilized enzyme and preparation method thereof |
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| Lipase Immobilization on Woollen Cloth in the Presence of Transglutaminase;Jing Dong An等;《The 12th Asian Pacific Confederation of Chemical Engineering Congress》;20080831;第1页 * |
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