CN111334487A - Preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder - Google Patents
Preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder Download PDFInfo
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- CN111334487A CN111334487A CN202010236866.8A CN202010236866A CN111334487A CN 111334487 A CN111334487 A CN 111334487A CN 202010236866 A CN202010236866 A CN 202010236866A CN 111334487 A CN111334487 A CN 111334487A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
- C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
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- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01019—Cyclomaltodextrin glucanotransferase (2.4.1.19)
Abstract
The invention discloses a preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder, which is characterized in that pET22b (+) -gamma-CGTase/E.coli BL21(DE3) strain fermentation liquor which is successfully constructed in the early stage is directly subjected to high-pressure homogenizing crushing to obtain crude enzyme liquid, 5% -30% of maltodextrin is added into the crude enzyme liquid and then spray drying is carried out to prepare powdery enzyme.
Description
Technical Field
The invention belongs to the technical field of enzymes, and particularly relates to a preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder.
Background
Cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) is an important member of α -amylase family, and can catalyze intramolecular and intermolecular glycosyl transfer reactions, such as coupling, cyclization and disproportionation, the main function of the cyclodextrin is to catalyze cheap and easily available glucose polymers such as starch, glycogen, maltooligosaccharide and the like to generate Cyclodextrin (CD), the cyclodextrin is oligosaccharide with a cyclic structure formed by connecting a plurality of D-glucopyranose units end to end through α -1, 4-glycosidic bonds, common natural cyclodextrins include α -CD, β -CD and gamma-CD, and are respectively composed of six, seven and eight glucose units, and the cyclodextrin has a hollow cylindrical structure with internal hydrophobicity and external hydrophilicity, and can be included with hydrophobic molecules through host-object recognition, so that the cyclodextrin can achieve the functions of protecting, stabilizing and solubilizing object molecules, and is widely applied to food, medicine, environmental protection, textile, chemical analysis and the like.
Compared with α -CD and β -CD, the gamma-CD has the largest inside hydrophobic cavity diameter and the highest water solubility, can contain larger hydrophobic molecules and has the most obvious solubilizing effect, has the smallest toxicity and the high biodegradability, is suitable for being used as a food additive, can effectively prevent erythrocyte hemolysis and is more suitable for parenteral administration carriers, so the gamma-CD has larger application potential in the fields of food, medicine, chemical industry and the like.
The industrial production of the gamma-CD is synthesized by adopting a gamma-CGTase enzymatic method. The gamma-CGTases conversion products which are found at home and abroad and come from different strains are a mixture of three cyclodextrins, and the conversion specificity is not high, so that the production cost of gamma-CD is very high. One of the most specific of the transformation is derived fromBacillus clarkii7364 the gamma-CGTase can be converted into three cyclodextrins with gamma-CD content up to 79%. The optimal reaction temperature of the enzyme is 60 ℃, but the enzyme is almost completely inactivated after being kept for 15min at 60 ℃, the enzyme activity is lost by about 20 percent after being kept for 15min at 50 ℃, and the thermal stability of other known gamma-CGTase is not high; in addition, it is reported that the CGTase fermentation liquor is almost completely inactivated after being stored for 7 days at 37 ℃, which seriously influences the industrial application of the gamma-CGTase and increases the production cost of the gamma-CD.
Chinese patent document CNIO1717765A (application number: 200910260985.0) discloses a cyclodextrin glucosyltransferase complex enzyme preparation which comprises cyclodextrin glucosyltransferase, glycerol, PEG400 and gelatin, wherein the enzyme preparation is stored at 40 ℃ for 30 days, and the enzyme activity retention rate reaches 95%. However, the liquid enzyme preparation is not as widely used in industry as the powder enzyme preparation due to its disadvantages of large storage volume, difficult transportation, and easy growth of bacteria in long-term storage. At present, no relevant report on the preparation method and storage stability of the gamma-CGTase powdery enzyme preparation exists in China, so that the preparation method of the gamma-CGTase enzyme powder needs to be developed to improve the storage stability of the gamma-CGTase enzyme powder.
Disclosure of Invention
The technical problem to be solved by the application is to provide a preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder, and solve the problems of large storage volume, difficult transportation and poor storage stability in the prior art.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder comprises the following steps:
step (1), fermenting escherichia coli to produce gamma-cyclodextrin glucosyltransferase;
step (2), carrying out high-pressure homogenizing and crushing on the fermentation liquor obtained in the step (1) to obtain a gamma-cyclodextrin glucosyltransferase crude enzyme liquid, wherein the high-pressure homogenizing and crushing pressure is 5000-;
step (3), adding maltodextrin with the final concentration of 5-30% (w/v) and the DE value of 5-20 to the gamma-cyclodextrin glucosyltransferase crude enzyme liquid prepared in the step (2), and mechanically stirring to fully dissolve the maltodextrin to prepare uniform dextrin solution;
and (4) feeding the dextrin solution prepared in the step (3) into a powder spraying tower, and preparing the powder enzyme by using a spray dryer, wherein the spray drying conditions are as follows: inlet air temperature 100-: 40-80 deg.C, flow rate of 5-15m L min-1After spraying, collecting the enzyme powder in the collection bottle;
and (5) preserving the enzyme powder collected in the step (4) at normal temperature or cold storage, wherein the preservation temperature is 3-37 ℃.
Further, the escherichia coli in the step (1) is an engineering bacterium constructed by using a gamma-cyclodextrin glucosyltransferase gene derived from Bacillus alcalophilus (Bacillus clarkii 7364).
Further, the strain fermentation medium in the step (1) is a TB medium, the components of the TB medium are peptone 18 g/L, yeast powder 18 g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31g/L and dipotassium hydrogen phosphate 16.43 g/L, the culture temperature is 37 ℃, the induction temperature is 28 ℃, and the stirring speed is 200-700 rpm.
Further, the stirring speed is 50-200rpm, and the stirring power is 5-20 kW.
The invention has the beneficial effects that:
(1) the preparation method is simple, and aims at the intracellular expressed gamma-cyclodextrin glucosyltransferase
Carrying out high-pressure homogenization and crushing to obtain a crude enzyme solution;
(2) the maltodextrin not only serves as a protective agent to obviously improve the thermal stability of the enzyme powder, but also can be directly converted into cyclodextrin as a substrate of the enzyme in the subsequent process, thereby reducing the industrial production cost;
(3) the enzyme powder obtained by spray drying has high enzyme activity yield, and the enzyme activity yield of the cyclase can reach more than 80%;
(4) impurities are not introduced into the reaction system, so that the difficulty of subsequent separation and impurity removal is reduced.
(5) The gamma-cyclodextrin glucosyltransferase crude enzyme powder provided by the invention has extremely excellent storage stability, and the enzyme activity retention rate is still up to more than 90% after the gamma-cyclodextrin glucosyltransferase crude enzyme powder is stored for 6 months at 4 ℃; the enzyme activity retention rate of the product can reach more than 85% after being stored for 3 months at normal temperature.
(6) The method provided by the invention has a huge application prospect in the aspect of industrial production of cyclodextrin, and the prepared product has a small storage volume and is convenient to transport and suitable for large-scale industrial production.
Drawings
FIG. 1 is a flow chart of preparation of gamma-CGTase enzyme powder.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples, it being understood that the described examples are only a part of the examples of the present invention, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The method is simple, the crude enzyme liquid is obtained by directly carrying out high-pressure homogenizing crushing on gamma-cyclodextrin glucosyltransferase expressed in cells, the conversion specificity of the enzyme is high, the proportion of gamma-cyclodextrin in a conversion product can reach 90.9%, α -cyclodextrin is not contained, the enzyme powder obtained by spray drying is high in enzyme activity yield, the activity yield of the cyclase can reach more than 80%, the heat stability of the enzyme powder is remarkably improved by taking the maltodextrin as a protective agent, the maltodextrin can be directly converted into the cyclodextrin in the subsequent process, the industrial production cost is reduced, impurities are not introduced into a reaction system, and the difficulty of subsequent separation and impurity removal is reduced.
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention include, but are not limited to, the scope shown in the following examples.
Example 1:
a method for preparing gamma-cyclodextrin glucosyltransferase crude enzyme powder is disclosed, and referring to figure 1, the method comprises the following steps:
the gamma-CGTase is produced by pET22b (+) -gamma-CGTase/E.coli BL21(DE3) strain fermentation, and the fermentation medium is: peptone 18 g/L, yeast powder 18 g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31g/L, dipotassium hydrogen phosphate 16.43 g/L; fermentation culture conditions: a 10L fermentation tank, the liquid loading of the culture medium is 6L, ampicillin and thalli are inoculated, the culture is carried out for 5h under the condition of 37 ℃, IPTG inducer is added for culture for 10h, and then the culture is taken out of the tank.
And (2) carrying out high-pressure homogenizing crushing on the fermentation liquor obtained in the step (1) to obtain a gamma-CGTase crude enzyme solution, wherein the high-pressure homogenizing crushing pressure is 15000 psi.
And (3) adding 25% (w/v) maltodextrin with the DE value of 17.3 into the gamma-CGTase crude enzyme solution prepared in the step (2), and mechanically stirring to fully dissolve the maltodextrin to prepare a uniform dextrin solution. Wherein the mechanical stirring speed is 100rpm, and the power is 11 kw.
And (4) feeding the dextrin solution prepared in the step (3) into a powder spraying tower, and preparing the dextrin solution into powdery enzyme by a spray dryer, wherein the spray drying conditions are as follows: inlet air temperature 125 ℃, outlet temperature: 60 ℃ and a flow rate of 10m L & min-1. And after spraying, collecting enzyme powder in a collection bottle, respectively detecting the activity of the gamma-CGTase hydrolase and the activity of the cyclase before and after powder spraying, and calculating the enzyme activity yield.
And (5) respectively storing the enzyme powder collected in the step (4) at 4 ℃, 15 ℃ and 37 ℃ for 3 months, detecting the hydrolase activity and the cyclase activity, and calculating the enzyme activity yield.
This embodiment is the most preferred embodiment.
Example 2:
a preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder comprises the following steps:
step (1) Gamma-CGTase was produced by fermentation of pET22b (+) -Gamma-CGTase/E.coli BL21(DE3) strain, and the fermentation medium and culture conditions were the same as those of example 1.
And (2) carrying out high-pressure homogenizing crushing on the fermentation liquor obtained in the step (1) to obtain a gamma-CGTase crude enzyme solution, wherein the high-pressure homogenizing crushing pressure is 25000 psi.
And (3) adding 15% (w/v) maltodextrin with the DE value of 13.4 into the gamma-CGTase crude enzyme solution prepared in the step (2), and mechanically stirring to fully dissolve the maltodextrin to prepare a uniform dextrin solution. Wherein the mechanical stirring speed is 50rpm, and the power is 5 kw.
And (4) feeding the dextrin solution prepared in the step (3) into a powder spraying tower, and preparing the dextrin solution into powdery enzyme by a spray dryer, wherein the spray drying conditions are as follows: inlet air temperature 100 ℃, outlet temperature: flow rate 15m L. min at 40 DEG C-1. After spraying, collecting the enzyme powder in the collection bottle, andand (3) respectively detecting the activity of the gamma-CGTase hydrolase and the activity of the cyclase before and after powder spraying, and calculating the enzyme activity yield.
And (5) respectively storing the enzyme powder collected in the step (4) at 4 ℃, 15 ℃ and 37 ℃ for 3 months, detecting the hydrolase activity and the cyclase activity, and calculating the enzyme activity yield.
Example 3:
a preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder comprises the following steps:
step (1) Gamma-CGTase was produced by fermentation of pET22b (+) -Gamma-CGTase/E.coli BL21(DE3) strain, and the fermentation medium and culture conditions were the same as those of example 1.
And (2) carrying out high-pressure homogenizing crushing on the fermentation liquor obtained in the step (1) to obtain a gamma-CGTase crude enzyme solution, wherein the high-pressure homogenizing crushing pressure is 5000 psi.
And (3) adding 5% (w/v) maltodextrin with the DE value of 5.7 into the gamma-CGTase crude enzyme solution prepared in the step (2), and mechanically stirring to fully dissolve the maltodextrin to prepare a uniform dextrin solution. Wherein the mechanical stirring speed is 200rpm, and the power is 20 kw.
And (4) feeding the dextrin solution prepared in the step (3) into a powder spraying tower, and preparing the dextrin solution into powdery enzyme by a spray dryer, wherein the spray drying conditions are as follows: inlet air temperature 150 ℃, outlet temperature: 80 ℃ and a flow rate of 5m L & min-1. And after spraying, collecting enzyme powder in a collection bottle, respectively detecting the activity of the gamma-CGTase hydrolase and the activity of the cyclase before and after powder spraying, and calculating the enzyme activity yield.
And (5) respectively storing the enzyme powder collected in the step (4) at 4 ℃, 15 ℃ and 37 ℃ for 3 months, detecting the hydrolase activity and the cyclase activity, and calculating the enzyme activity yield.
TABLE 1 enzyme activity yield of gamma-CGTase enzyme powder
The significant progress of the invention is further demonstrated by the two comparative examples presented below.
Comparative example 1:
the enzyme powder is prepared by a modified starch adsorption method, the yield of the hydrolase activity after spray drying is only 33%, and the yield of the cyclase activity is only 51%;
a preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder is characterized by comprising the following steps:
step (1) Gamma-CGTase was produced by fermentation of pET22b (+) -Gamma-CGTase/E.coli BL21(DE3) strain, and the fermentation medium and culture conditions were the same as those of example 1.
And (2) carrying out high-pressure homogenizing crushing on the fermentation liquor obtained in the step (1) to obtain a gamma-CGTase crude enzyme solution, wherein the high-pressure homogenizing crushing pressure is 15000 psi.
And (3) adding 5% (w/v) of modified starch into the gamma-CGTase crude enzyme solution prepared in the step (2), mechanically stirring at the rotating speed of 100rpm, filtering after 6 hours, collecting a filter cake, drying at 60 ℃, and detecting that the yield of the hydrolase activity is 33.09% and the yield of the cyclase activity is 51.40% after the modified starch is adsorbed.
The preparation method of the modified starch comprises the following steps: preparing saturated ammonium sulfate solution, adding corn starch according to the amount of 15% (w/v), stirring for 3h at 50 ℃, filtering, collecting filter cake, drying at 60 ℃, and storing at room temperature for later use.
And (4) respectively storing the enzyme powder collected in the step (3) at 4 ℃, 15 ℃ and 37 ℃ for 3 months, detecting the hydrolase activity and the cyclase activity, and calculating the enzyme activity yield. The hydrolase yields are 90.87%, 85.18% and 70.60% respectively, and the enzyme yields are 93.34%, 88.50% and 72.95% respectively.
Comparative example 2:
a preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder is characterized by comprising the following steps:
step (1) Gamma-CGTase was produced by fermentation of pET22b (+) -Gamma-CGTase/E.coli BL21(DE3) strain, and the fermentation medium and culture conditions were the same as those of example 1.
And (2) carrying out high-pressure homogenizing crushing on the fermentation liquor obtained in the step (1) to obtain a gamma-CGTase crude enzyme solution, wherein the high-pressure homogenizing crushing pressure is 15000 psi.
And (3) adding 5% (w/v) of cassava starch into the gamma-cyclodextrin glucosyltransferase crude enzyme solution prepared in the step (2), and mixing the slurry.
Step (ii) of(4) And (4) feeding the starch slurry prepared in the step (3) into a powder spraying tower, and preparing the starch slurry into powdery enzyme by using a spray drying machine, wherein the spray drying conditions are as follows: inlet air temperature 120 ℃, outlet temperature: 60 ℃ and a flow rate of 10m L & min-1. And after spraying, collecting enzyme powder in a collection bottle, respectively detecting the activity of the gamma-CGTase hydrolase and the activity of the cyclase before and after powder spraying, and calculating the enzyme activity yield. The yield of the hydrolase activity is 36.87%, and the yield of the generated enzyme activity is 60.24%.
And (5) respectively storing the enzyme powder collected in the step (4) at 4 ℃, 15 ℃ and 37 ℃ for 3 months, detecting the hydrolase activity and the cyclase activity, and calculating the enzyme activity yield. The hydrolytic enzyme activity yields are respectively 95.44%, 92.18% and 78.63%, and the generated enzyme activity yields are respectively 94.79%, 90.20% and 78.91%.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. Any partial modification or replacement within the technical scope of the present disclosure by a person skilled in the art should be included in the scope of the present disclosure.
Claims (4)
1. A preparation method of gamma-cyclodextrin glucosyltransferase crude enzyme powder is characterized by comprising the following steps:
step (1), fermenting escherichia coli to produce gamma-cyclodextrin glucosyltransferase;
step (2), carrying out high-pressure homogenizing and crushing on the fermentation liquor obtained in the step (1) to obtain a gamma-cyclodextrin glucosyltransferase crude enzyme liquid, wherein the high-pressure homogenizing and crushing pressure is 5000-;
step (3), adding maltodextrin with the final concentration of 5-30% (w/v) and the DE value of 5-20 to the gamma-cyclodextrin glucosyltransferase crude enzyme liquid prepared in the step (2), and mechanically stirring to fully dissolve the maltodextrin to prepare uniform dextrin solution;
and (4) feeding the dextrin solution prepared in the step (3) into a powder spraying tower, and preparing the powder enzyme by using a spray dryer, wherein the spray drying conditions are as follows: inlet air temperature 100-: 40-80 deg.C, flow rate of 5-15m L min-1After spraying, collecting the enzyme powder in the collection bottle;
and (5) preserving the enzyme powder collected in the step (4) at normal temperature or cold storage, wherein the preservation temperature is 3-37 ℃.
2. The method according to claim 1, wherein the Escherichia coli in the step (1) is an engineered bacterium constructed using a γ -cyclodextrin glucosyltransferase gene derived from Bacillus alcalophilus (Bacillus clarkii 7364).
3. The production method as claimed in claim 1 or 2, wherein the fermentation medium of the strain in the step (1) is TB medium, and the components of the TB medium are peptone 18 g/L, yeast powder 18 g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31g/L, dipotassium hydrogen phosphate 16.43 g/L, culture temperature 37 ℃, induction temperature 28 ℃, and stirring speed 200-700 rpm.
4. The production method as claimed in claim 4, wherein the mechanical stirring conditions in the step (3) are a stirring rotation speed of 50 to 200rpm and a stirring power of 5 to 20 kW.
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