CN108359664B - Preparation method and application method of immobilized amylosucrase-containing cells - Google Patents
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a preparation method and a use method of immobilized cells containing amylosucrase; the method comprises the following steps: (1) adding a buffer solution and distilled water into the thalli containing the amylosucrase to prepare a bacterial suspension, then adding a carrier, and uniformly stirring; (2) adding sodium alginate and polyvinyl alcohol, and stirring uniformly to obtain a mixed solution; (3) dropping the mixed solution into CaCl2Curing the mixture in the boric acid solution for 5 to 15 hours to obtain spherical immobilized cells containing amylosucrase; the preparation method provided by the invention has the advantages of low cost of immobilized materials, simple operation, good reusability and the like, the prepared cells containing the amylosucrase are used for catalyzing sucrose and hydroquinone to produce alpha-arbutin, the enzyme activity of the immobilized cells is still kept to be more than 50% of that of free cells after 20 batches of continuous conversion, and the preparation method has good industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a preparation method and a use method of immobilized cells containing amylosucrase.
Background
Alpha-arbutin is an epimer of beta-arbutin and has the chemical name of 4-hydroxyphenyl-alpha-D-glucopyranoside. Researches show that compared with beta-arbutin, the alpha-arbutin has more stable property and better anti-inflammatory, repairing and whitening effects, and is often used as a whitening agent to be applied to high-end skin care products and cosmetics.
At present, the main preparation method of alpha-arbutin is a free cell catalysis method, and the main problems are that: the subsequent treatment process for producing the alpha-arbutin product by utilizing the free cells to catalyze is complex, and the equipment investment and the operation cost are high; the catalyst has poor tolerance to the environment and can not be recycled, thus increasing the production cost. For example, patent No. CN201210508199.X, which describes a method for synthesizing alpha-arbutin by converting Xanthomonas campestris, the conversion efficiency is low, and the content of alpha-arbutin in the conversion solution is about 10 g/L. For example, patent CN201610130457.3 describes a purification process of synthesizing α -arbutin by catalytic synthesis of enzymatic protein and repeated recrystallization, which requires preparation of enzymatic protein for reaction, and repeated recrystallization with pure water during purification, resulting in high production cost and low conversion efficiency.
Disclosure of Invention
The primary object of the present invention is to provide a method for preparing immobilized amylosucrase-containing cells with improved cell activity and stability.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for preparing immobilized cells containing amylosucrase, comprising the following steps:
(1) adding a buffer solution and distilled water into the thalli containing the amylosucrase to prepare a bacterial suspension, then adding a carrier, and uniformly stirring;
(2) adding sodium alginate and polyvinyl alcohol, and stirring uniformly to obtain a mixed solution;
(3) dropping the mixed solution into CaCl2And (3) solidifying for 5-15 h in the boric acid solution to obtain the spherical immobilized cells containing the amylosucrase.
The beneficial effect that above-mentioned technique was sent lies in: the preparation method provided by the invention has the advantages of low cost of immobilized materials, simple operation, good reusability and the like, the prepared cells containing the amylosucrase are used for catalyzing sucrose and hydroquinone to produce alpha-arbutin, the enzyme activity of the immobilized cells is still kept to be more than 50% of that of free cells after 20 batches of continuous conversion, and the preparation method has good industrial application prospect.
Another object of the present invention is to provide a method for using the above immobilized amylosucrase-containing cells, which can increase the yield of alpha-arbutin and shorten the production cycle.
The method comprises the following steps: adding spherical immobilized cells containing amylosucrase into sucrose and hydroquinone, and converting the mixed solution at 28-35 ℃ for 3-6 h to obtain alpha-arbutin;
the concentration of each substance in the mixed solution is as follows: 180-300 g/L of sucrose and 5-20 g/L of hydroquinone; 260-300 g/L of spherical immobilized cells containing amylosucrase.
Drawings
FIG. 1 is a graph comparing the yield of the reuse batches of immobilized cells (prepared in example 2) and free cells (prepared in example 1).
Detailed Description
The invention is further disclosed in the following 3 examples in which the diatomaceous earth has the formula SiO2The molecular weight is 60.08, the median particle size is 19.6 mu m, and the specific gravity is 0.32, so that water with the specific gravity more than 4 times that of the water can be absorbed; soluble in concentrated alkali and hydrofluoric acid, insoluble in water, acid or dilute alkali, available from the company alatin. Perlite, chemical composition SiO2：70～75％，Al2O3：12～16％，Na2O：1.0％～4.0％，K2O: 1.0% -4.0%, white particles with appearance, specific gravity of 0.09, melting point: 1280-1350 ℃ and purchased from Hangzhou brocade green industry technologyThe company Technique, Inc. Activated carbon, molecular formula C, molecular weight 12.01, specific gravity 1.8, has high capacity adsorption capacity for organic pigments and nitrogenous bases, and is available from Aladdin.
Example 1: preparation and performance measurement of thalli containing amylosucrase
Inoculating the recombinant gene engineering bacteria containing the amylosucrase genes to a slant culture medium, and culturing at 37 ℃ for 12h to obtain slant thalli; the slant culture medium comprises: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast powder, 20g/L agar and water as a solvent, wherein the pH value is natural;
inoculating the slant thallus to a seed culture medium, and culturing at 37 ℃ for 12h to obtain a seed solution; the seed culture medium comprises the following components: 10g/L peptone, 10g/L sodium chloride, 5g/L yeast powder and a solvent of water, wherein the pH value is natural;
inoculating the seed solution into a fermentation culture medium with the volume concentration of 3.3%, culturing for 2h at 37 ℃ and 150rpm, adding 10g/L lactose inducer, inducing for 12h at 28 ℃ and 150rpm, centrifuging the fermentation liquor, and collecting wet thalli to obtain thalli containing amylosucrase;
the fermentation medium comprises the following components: 1% of monopotassium phosphate, 1% of dipotassium phosphate, 0.5% of ammonium sulfate, 0.1% of magnesium sulfate, 0.1% of citric acid monohydrate, 0.1% of fumaric acid, 0.1% of sodium glutamate, 0.05% of ferric ammonium citrate, 0.1% of sodium dihydrogen phosphate, 0.1% of disodium hydrogen phosphate, 1% of glycerol, 0.005% of ferrous sulfate heptahydrate, 0.005% of anhydrous calcium chloride, 0.001% of copper sulfate pentahydrate, 0.001% of cobalt chloride hexahydrate, 0.001% of zinc sulfate heptahydrate, 0.005% of manganese sulfate monohydrate, 0.005% of sodium molybdate dihydrate, 0.005% of nickel chloride hexahydrate, 0.005% of boric acid and 0.002% of defoaming agent.
It should be noted that the recombinant genetically engineered bacterium containing the amylosucrase gene is obtained according to the construction method of the genetically engineered bacterium producing alpha-arbutin disclosed in Chinese patent application publication No. CN 106148256A.
Measuring the enzyme activity and reusability of the free cells:
collecting wet thallus, adding into 10ml phosphate buffer solution with pH of 7.0 to make thallus final concentration 10OD, adding sucrose 2.4g and hydroquinone 0.132g, reacting at 30 deg.C for 10min, sampling, detecting product alpha-arbutin concentration by HPLC, and calculating free cell enzyme activity according to enzyme activity definition.
The free cells prepared in step (1) were taken out and put in a 250mL three-necked flask containing 100mL of phosphate buffer (pH 7.0, 100mM) at a cell concentration of 10OD, 24g of sucrose as a substrate and 1.32g of hydroquinone were added, the reaction was carried out at 30 ℃ and a stirring speed of 200rpm for 4 hours, solid-liquid separation was carried out by vacuum filtration, and the concentration of the product was measured by HPLC from the transformed liquid, and the yield was calculated. After three washes of the free cells, the next batch of transformation was performed, and the yield of each batch was calculated, and the enzyme activity was calculated.
Definition of enzyme activity: the amount of enzyme required per OD cell to catalyze the production of 1mmol of α -arbutin per hour at 30 ℃ and pH7.0 was defined as 1U.
HPLC conditions: column: WondaSil C18 reverse phase column, 4.6 x 150 mm. Sample introduction amount: 10 uL. Column temperature: 35 ℃ is carried out. Mobile phase: 5% aqueous methanol. Flow rate: 1.0 mL/min. Detection wavelength: 280 nm. The peak emergence time of alpha-Arbutin: 4.8 min; hydroquinone peak time: 6.0 min;
according to the above method, the obtained free cells had the following properties: the enzyme activity of free cells containing sucrose phosphorylase is 29.5U, and the enzyme activity is 11.5% after 3 batches of continuous operation under the concentration of 240g/L sucrose and 13.2g/L hydroquinone, and the result is shown in figure 1.
Example 2: preparation of immobilized cells containing amylosucrase
(1) Taking the amylosucrase-containing thallus in example 1, adding 0.05mol/L Tris-HCl solution (pH 7.0, 100mM) and distilled water to prepare 100ml bacterial suspension with the bacterial concentration of OD 20, adding 40g of diatomite, and stirring uniformly;
(2) adding 100ml of 6 wt% sodium alginate and 20 wt% polyvinyl alcohol, and uniformly stirring to obtain a mixed solution, wherein the mixed solution contains 10OD of amylosucrase thallus, 20g/L of carrier, 3 wt% of sodium alginate and 10 wt% of polyvinyl alcohol;
(3) dropping the mixed solution into 10g/L CaCl at a dropping speed of 100 drops/min2Curing the mixture at 25 ℃ for 10 hours in 10g/L boric acid solution (pH 7.0) to obtain spherical immobilized amylosucrase-containing cells with the diameter of 2-5 mm, and storing the cells at 4 ℃.
Example 3: synthesis and performance measurement of alpha-arbutin
Taking 3.4g of the spherical immobilized cells containing the amylosucrase prepared in the example 2 into 10ml of phosphate buffer solution with pH7.0, adding 2.4g of sucrose and 0.132g of hydroquinone, reacting at 30 ℃ for 10min, sampling, detecting the concentration of the product alpha-arbutin by HPLC, and calculating the enzyme activity of the immobilized cells according to the enzyme activity definition; the enzyme activity of the immobilized cells was measured in the same manner as in example 1. The enzyme activity recovery rate of the immobilized cell is calculated by dividing the enzyme activity of the immobilized cell by the enzyme activity of the free cell.
34g of the spherical immobilized amylosucrase-containing cells obtained in example 2 were put in a 250mL three-necked flask containing 100mL of a phosphate buffer (pH 7.0, 100mM), 24g of sucrose and 1.32g of hydroquinone were added thereto, the mixture was reacted for 4 hours at 30 ℃ under a stirring speed of 200rpm, solid-liquid separation was carried out by vacuum filtration, and the concentration of the product was measured by HPLC to calculate the yield. After three washes of the immobilized cells, the next conversion was performed and the yield of each batch was calculated.
The results show that: the immobilized cells performed as follows: the enzyme activity of the immobilized cells is 27U, the enzyme activity recovery rate is 91.5%, the enzyme activity of 20 batches of continuous operation is kept by 53.0%, and the result is shown in figure 1.
Example 4: preparation of immobilized cells containing amylosucrase
The preparation method was the same as example 2, except that the carrier in step (1) was activated carbon, the sodium alginate concentration in step (2) was 4 wt%, and the polyvinyl alcohol concentration was 16 wt%.
The performance of the obtained immobilized cells is determined as follows: the enzyme activity of the immobilized cell is 26.11U, the enzyme activity recovery rate is 88.5 percent, and the enzyme activity of 20 batches of continuous operation is kept by 52.0 percent.
Example 5: preparation of immobilized cells containing amylosucrase
The preparation method was the same as example 2, except that the carrier in step (1) was perlite, the sodium alginate concentration in step (2) was 8 wt%, and the polyvinyl alcohol concentration was 12.4 wt%.
The performance of the obtained immobilized cells is determined as follows: the enzyme activity of the immobilized cell is 24.6U, the recovery rate of the enzyme activity is 83.5 percent, and the enzyme activity of 20 batches of continuous operation is kept by 50.5 percent.
Example 6: preparation of immobilized cells containing amylosucrase
The preparation method is the same as example 2, except that the carrier in the step (1) is perlite, and CaCl in the step (3)2The concentration was 8g/L and the boric acid concentration was 15 g/L.
The performance of the obtained immobilized cells is determined as follows: the enzyme activity of the immobilized cell is 23.6U, the recovery rate of the enzyme activity is 80 percent, and the enzyme activity is kept by 50 percent after 20 batches of continuous operation.
Claims (1)
1. A method for synthesizing alpha-arbutin by using immobilized cells containing amylosucrase is characterized by comprising the following steps: adding spherical immobilized cells containing amylosucrase into sucrose and hydroquinone, and converting the mixed solution at 28-35 ℃ for 3-6 h to obtain alpha-arbutin; the concentration of each substance in the mixed solution is as follows: 180-300 g/L of sucrose and 5-20 g/L of hydroquinone; 260-300 g/L of spherical immobilized cells containing amylosucrase;
the preparation method of the spherical immobilized cell containing the amylosucrase comprises the following steps:
(1) adding a buffer solution and distilled water into the thalli containing the amylosucrase to prepare a bacterial suspension, then adding a carrier, and uniformly stirring;
(2) adding sodium alginate and polyvinyl alcohol, and stirring uniformly to obtain a mixed solution;
(3) dropping the mixed solution into CaCl2Curing the mixture in the boric acid solution for 5 to 15 hours to obtain spherical immobilized cells containing amylosucrase;
the amylosucrase-containing thalli is obtained by performing slant culture, seed culture and fermentation culture on recombinant engineering bacteria containing amylosucrase encoding genes;
the buffer solution in the step (1) is a Tris-HCl solution with the concentration of 0.01-1 mol/L;
the carrier in the step (1) is one of diatomite, activated carbon and perlite;
the concentration of each substance in the mixed solution in the step (2) is as follows: 8-12 OD of amylosucrase-containing thalli, 15-25 g/L of carrier, 2-5 wt% of sodium alginate and 8-15 wt% of polyvinyl alcohol;
CaCl of step (3)2In boric acid solution of (1), CaCl2The concentration of the boric acid is 5-15 g/L, the concentration of the boric acid is 8-12 g/L, and the pH value of the solution is 6.5-7.5;
the dropping speed of the mixed solution in the step (3) is 70-120 drops/min;
the diameter of the spherical immobilized amylosucrase-containing cell in the step (3) is 2-5 mm, and the recovery rate of enzyme activity is 80-91.5%.
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| CN112725327A (en) * | 2021-02-26 | 2021-04-30 | 华东理工大学 | Preparation method and application of cell immobilization carrier |
| CN114058660B (en) * | 2021-10-15 | 2023-06-27 | 安徽华恒生物科技股份有限公司 | Immobilized enzyme conversion synthesis method of alpha-arbutin and application |
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| CN101358185A (en) * | 2008-09-19 | 2009-02-04 | 哈尔滨理工大学 | Method for fixing nitrile hydratase strain by sodium alginate-polyvinyl alcohol |
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