Method for catalytic production of lauroyl glycine by immobilized cells and application

Abstract

The invention belongs to the fields of biosynthesis and enzymology, and discloses a method for efficiently producing lauroyl glycine by immobilized cells. According to the method disclosed by the invention, saccharides and alcohols are added as enzyme stabilizers in the process of immobilizing the acylase, so that the utilization rate and the stability of acylase cells in the process of producing the lauroyl glycine are improved. According to the method disclosed by the invention, the lauroyl glycine is produced by utilizing the immobilized cells, so that the production efficiency is improved, the production cost is reduced, and the enzyme activity is improved by 3.1 times compared with that of free enzyme.

Description

Technical Field

[0001] The invention belongs to the fields of biosynthesis and enzymology, and specifically relates to the technical field of lauroylglycine preparation, and more specifically to a method for efficiently producing lauroylglycine by using immobilized cells. Background Art

[0002] Sodium lauroyl glycinate (2-(dodecanoylamino)aceticacid), chemical formula is C 14 H 26 NO3Na is an amino acid surfactant, which can be obtained by one-step saponification of lauroyl glycine. The structural formula of lauroyl glycine is:

[0003]

[0004] Amino acid surfactants are an important class of natural surfactant molecules, which are amphiphilic molecules with high surface activity formed by the condensation of an amino acid as an acyl acceptor and one or more non-polar molecules such as long-chain fatty acids as acyl donors. Amino acid surfactants have the characteristics of low toxicity, high biodegradability, high eco-compatibility and antibacterial properties, so they are widely used in the food, pharmaceutical and cosmetic industries.

[0005] The most common method for producing amino acid surfactants is through Schotten-Baumann condensation reaction. It is a process in which fatty acyl chloride and amino acid react in alkaline aqueous solution or other organic reagents to obtain fatty acyl amino acid salts, and then the crude fatty acyl amino acid separated by inorganic acid neutralization is neutralized with alkali to obtain purer fatty acyl amino acid salts. The process and equipment of this chemical method are not complicated, the raw materials are easy to obtain, the reaction temperature is not high, the reaction conditions are mild, and it has a high synthesis yield, but the production on an industrial scale leads to a large amount of wastewater, and the post-treatment cost is very expensive.

[0006] Nowadays, more and more industries are trying to develop sustainable technologies to reduce waste generation, environmental problems and post-processing costs. One of the most effective ways to achieve these goals is to use biologically mediated reactions, generally known as bioconversion.

[0007] Enzymatic synthesis is the most efficient and rapid method in biotransformation. It mainly uses enzymes to catalyze the amidation reaction of fatty acids and their derivatives with amino acids. It has received extensive research attention due to its greenness and safety. The main challenges faced by the enzyme catalytic pathway are that the known enzymes are not very active and have poor stability when catalyzing acylation reactions. The enzyme cost is high and the product yield is low. These are the main factors restricting the enzymatic synthesis of amino acid surfactants.

[0008] Cells containing acylase activity can catalyze lauric acid and glycine to produce lauroylglycine, but due to the complexity of the reaction solution, the stability of the cells containing acylase is greatly affected, which can easily lead to cell lysis and enzyme loss, reduce the efficiency of the use of acylase, and increase the steps and costs of post-treatment. Fixing cells containing acylase in a certain carrier can significantly improve the stability of cells containing acylase in the reaction solution, improve the catalytic efficiency of the enzyme, and reduce the cost of lauroylglycine purification. Common cell immobilization methods include adsorption, embedding, covalent and cross-linking. The cross-linking method is to connect the microbial cells to each other into a network structure through the reaction of microorganisms with reagents having two or more functional groups to achieve the purpose of fixation. A common cross-linking agent is glutaraldehyde. This technology is usually used in conjunction with other methods.

[0009] In the enzyme-catalyzed production of lauric acid to generate lauroylglycine, free enzyme cells are often used, which has low enzyme stability and leads to a complicated extraction process of lauroylglycine in the enzyme reaction solution, which affects the purity of the finished lauroylglycine and is not conducive to industrial production. Therefore, a method is needed that is simpler and more economical and can improve the reuse efficiency of acylase. Summary of the invention

[0010] In view of the above problems, the present invention provides a method for efficiently producing lauroylglycine using immobilized cells. In the method, sugars and / or alcohols are added as enzyme stabilizers during the immobilization process, thereby improving the utilization rate and stability of recombinant acylase cells in the process of producing lauroylglycine.

[0011] The invention provides a method for preparing immobilized cells containing acylase, wherein sugars and alcohols are added as enzyme stabilizers during the immobilization process.

[0012] Specifically, the sugar is one or more of fructose, mannose and trehalose; the alcohol is one or more of glycerol, inositol or propylene glycol;

[0013] Preferably, 1 wt% to 5 wt% of sugars and 1 wt% to 5 wt% of alcohols are added during the immobilization process.

[0014] In a specific implementation, the following steps are included:

[0015] The wet bacterial bodies containing the recombinant bacteria expressing acylase are obtained by fermentation culture, agar is added to resuspend the bacterial sludge, sugars and / or alcohols are added to oscillate and mix, and the mixture is dropped into a container with an upper layer of liquid wax and a lower layer of water to fix the cells. After hardening, the primary immobilized cells are obtained by filtering, a physiological saline solution containing glutaraldehyde is added for cross-linking, and the immobilized cells are obtained by filtering.

[0016] Specifically, the steps of obtaining the wet cells are as follows:

[0017] (1) fermenting and culturing recombinant bacteria expressing acylase; (2) collecting the bacteria by centrifugation to obtain bacterial cells, and washing the bacterial cells with physiological saline to obtain wet bacterial cells.

[0018] Preferably, the step of adding agar to resuspend the bacterial sludge comprises adding 10-30 times the volume of 1-3% agar to resuspend the bacterial sludge at 40-50°C.

[0019] More specifically, the hardening is carried out at 3-5°C for 1-3 hours; the concentration of the glutaraldehyde-based saline solution is 0.05-0.5% (V / V); and the cross-linking is carried out at 3-5°C.

[0020] The present invention provides immobilized cells obtained by the method.

[0021] The present invention also provides application of the immobilized cells in preparing lauroylglycine.

[0022] The present invention provides a method for preparing lauroylglycine, which comprises adding the immobilized cells as claimed in claim 7 into a reaction solution for catalytic reaction, wherein the reaction solution comprises lauric acid and glycine and uses water as a solvent.

[0023] Preferably, the reaction solution is stirred at 40-50°C, and after adding the immobilized cells, the solution is sealed and shaken for reaction; the reaction solution includes 50-150 mM lauric acid and 50-90% (V / V) 2-4 M (preferably 3 M) glycine, and the pH is adjusted to 7.5-8.5 after the volume is supplemented with water.

[0024] In a specific embodiment, the specific operation steps include:

[0025] (1) Pick a single BL21(DE3)_pET28a_EcACY colony and inoculate it into a test tube of LB medium containing 50 ug / mL kanamycin at 30-37°C, 200 rpm, and culture for 12-16 h to obtain primary seeds;

[0026] (2) The primary seeds were inoculated into a shake flask containing a fermentation medium, cultured at 30-37° C., 200-250 rpm, for 2-5 h, and IPTG was added to a final concentration of 1 mM, cultured at 20-25° C., 180-220 rpm, for 12-16 h; the fermentation medium components are as follows:

[0027] Tryptone 12g / L, yeast extract 24g / L, glycerol 4g / L, potassium dihydrogen phosphate 2.5g / L, potassium dihydrogen phosphate trihydrate 16g / L.

[0028] (3) Collect the bacteria by centrifugation to obtain bacterial cells, wash the bacterial cells twice with physiological saline, add 20 times the volume of 2% agar to resuspend the bacterial mud at 45°C, add 1wt%-5wt% sugar and 1wt%-5wt% alcohol, shake and mix, drop into a container with upper liquid wax and lower water to form fixed cells with a diameter of 2-4mm, harden at 4°C for 2h, filter to obtain fixed cells, and wash with physiological saline for many times. Add to physiological saline solution containing 0.15% (V / V) glutaraldehyde, crosslink at 4°C for 1h, filter, wash with physiological saline for many times to obtain fixed cells.

[0029] (4) The reaction solution was stirred at 45° C., 160 g of immobilized cells were added, and the reaction was sealed and shaken; the reaction solution included 100 mM lauric acid and 80% (V / V) 3 M glycine, and the pH was adjusted to 7.5-8.5 after the volume was made up with water.

[0030] The beneficial effects of the present invention are as follows: the present invention fixes the recombinant acylase Escherichia coli cells with an agar solution and cross-links them with glutaraldehyde, which can effectively improve the stability of the cells, especially the thermal stability and the stability of tolerance to lauroyl glycine, and can prolong the time for converting and synthesizing lauroyl glycine and increase the substrate concentration when relevant substrates are provided. The process is simple, the cost is low, the yield is high, and the application value of industrial production is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Comparison of the ratio of enzyme activity of immobilized cells prepared with different enzyme stabilizer combinations to that of free enzyme.

[0032] Figure 2 This is the relationship between the number of repeated uses of immobilized cells No. 2 and the specific enzyme activity.

[0033] Figure 3 Comparison of enzyme activity of immobilized cells prepared with different addition amounts of enzyme stabilizer.

[0034] Figure 4 The relationship between the number of reuses and enzyme activity of immobilized cells prepared by adding 4 wt.% glycerol.

[0035] Figure 5 This is a curve of the number of times the immobilized cells were reused and the reaction end time. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0037] Example 1: Fermentation of recombinant acylase in Escherichia coli

[0038] LB medium: tryptone 10 g / L, yeast extract 0.5 g / L, sodium chloride 10 g / L, purified water.

[0039] Fermentation medium: tryptone 12g / L, yeast extract 24g / L, glycerol 4g / L, potassium dihydrogen phosphate 2.5g / L, potassium dihydrogen phosphate trihydrate 16g / L, purified water;

[0040] 1) Pick a single BL21(DE3)_pET28a_EcACY colony and inoculate it into a 4 mL LB medium test tube, add 4 uL of 50 mg / mL kanamycin (final concentration 50 ug / mL), culture at 37°C, 200 rpm, for 12 h to obtain primary seeds;

[0041] 2) The primary seeds were inoculated into a shake flask containing 100 ml of fermentation medium, cultured at 37°C, 200 rpm for 4 h, IPTG was added to a final concentration of 1 mM, cultured at 20°C, 200 rpm for 12 h;

[0042] 3) The bacterial liquid in step (2) was centrifuged to collect the bacterial cells and placed in a -20°C refrigerator.

[0043] Example 2: Preparation of immobilized cells with different enzyme stabilizer combinations

[0044] Weigh the bacterial sludge in Example 1, wash the bacterial cells twice with physiological saline, add 20 times the volume of 2% agar at 45°C to resuspend the bacterial sludge, add 2wt% sugars (sugars are fructose, mannose and trehalose) and use no sugar as a control, 2wt% alcohols (alcohols are glycerol, inositol, propylene glycol) and use no alcohol as a control, shake and mix, drop into a container with upper liquid wax and lower water, form fixed cells with a diameter of 2-4mm, harden at 4°C for 2h, filter to obtain fixed cells, and wash with physiological saline for many times. Add to a physiological saline solution containing 0.15% (V / V) glutaraldehyde, crosslink at 4°C for 1h, filter, wash with physiological saline for many times, and obtain fixed cells.

[0045] According to the different enzyme stabilizer combinations mentioned above, 16 kinds of immobilized cells were obtained. Here, different enzyme stabilizer combinations are represented by the form of (sugars, alcohols), where "without" means that there is no corresponding sugar or alcohol in this enzyme stabilizer combination. The corresponding combinations are numbered as follows: 1 (without, without), 2 (without, glycerol), 3 (without, inositol), 4 (without, propylene glycol), 5 (fructose, without), 6 (fructose, glycerol), 7 (fructose, inositol), 8 (fructose, propylene glycol), 9 (mannose, without), 10 (mannose, glycerol), 11 (mannose, inositol), 12 (mannose, propylene glycol), 13 (trehalose, without), 14 (trehalose, glycerol), 15 (trehalose, inositol), 16 (trehalose, propylene glycol).

[0046] These immobilized cells were tested for activity according to the following reaction system:

[0047] 1) Substrate solution: 100 mM lauric acid and 80% (V / V) 3 M glycine, make up to volume with water, stir at 45°C, and adjust pH to 7.5-8.5.

[0048] 2) Add 0.1L substrate solution to 12-20g of immobilized cells, stir well, seal and shake at 45°C;

[0049] 3) Samples were taken at 2, 4, and 6 hours after the reaction. The reaction was stopped when the product was no longer generated. The reaction data were as follows: Figure 1 As shown, the activities of immobilized cells and free enzymes were similar after 2 hours of reaction, with little difference; the ratio of immobilized cell to free enzyme activities increased after 4 and 6 hours of reaction, indicating that after immobilization, the enzyme stability in the reaction was enhanced and the sustained reaction effect was better. Among them, the immobilized cell enzyme activity prepared with enzyme stabilizer combination No. 2 was the highest.

[0050] 4) filtering the reaction liquid to separate the immobilized enzyme and the reaction liquid and recovering the immobilized cells;

[0051] 5) The immobilized enzyme corresponding to the selected enzyme stabilizer combination is recovered, and the immobilized cells are recovered and reacted again to test the activity. The reaction data is as follows: Figure 2 As shown in the figure, compared with the data of 2h reaction, the activity of the immobilized cells used 20 times was 70% of the free enzyme, indicating that the immobilized cells had good stability in repeated use; the activity ratio of the immobilized enzyme and the free enzyme increased after 4h and 6h of reaction, indicating that the free enzyme was not stable enough in long-term reactions, and the activity of the immobilized cells decreased significantly after being reused 10 times under the condition of longer reaction time, and was lower than the activity of the free enzyme after 20 times.

[0052] Wherein, the product detection method in the present invention is as follows:

[0053] Instrument: Thermo U3000 system HPLC

[0054] Column: Thermo C18, 4.6mm×150mm, 5μm

[0055] Detection wavelength: ultraviolet (UV) = 210nm;

[0056] Flow rate: 1mL / min

[0057] Column temperature: 30°C

[0058] Injection volume: 10 μL

[0059] Mobile phase: acetonitrile / water with 0.5% trifluoroacetic acid added (V / V) = 85:15.

[0060] The corresponding product concentration was calculated based on the product standard curve, and the ratio of product concentrations was used as the ratio of immobilized cell to free enzyme activity.

[0061] Example 3: Preparation and screening of immobilized cells with different enzyme stabilizer addition amounts

[0062] The bacterial sludge of Example 1 was weighed, and the bacterial bodies were washed twice with physiological saline. 20 times the volume of 2% agar was added to resuspend the bacterial sludge at 45° C., 1-5wt% glycerol was added and shaken to mix, and the mixture was dropped into a container with an upper layer of liquid wax and a lower layer of water to form immobilized cells with a diameter of 2-4 mm. The cells were hardened at 4° C. for 2 h, filtered to obtain the immobilized cells, and washed several times with physiological saline.

[0063] The cells were added to a physiological saline solution containing 0.15% (V / V) glutaraldehyde, cross-linked at 4°C for 1 hour, filtered, and washed with physiological saline for multiple times to obtain immobilized cells. There are five types of immobilized cells obtained this time (1, 2, 3, 4, and 5 wt% glycerol). According to the reaction method and detection method of Example 3, the time gradient sampling detection results are as follows: Figure 3 As shown in FIG. 1 , the activity of the immobilized cells prepared when the glycerol addition amount was 4 wt % was the best. A group of immobilized cells were recovered from this, and the activity of the recovered immobilized cells was tested. The data are shown in FIG. Figure 4 As shown. Figure 2 In comparison, under the same number of uses, the activity of immobilized cells was significantly improved, and the activity decreased significantly after 15 times. The recommended number of uses should be around 15 times.

[0064] Example 4: Plotting of the immobilized cell recovery times versus reaction time curve

[0065] The type and amount of enzyme stabilizer for acylase immobilized cells were determined from Example 4 and Example 5, and the final preparation plan for immobilized cells was determined as follows: weigh the bacterial sludge, wash the bacteria twice with physiological saline, add 20 times the volume of 2% agar at 45°C to resuspend the bacterial sludge, add 4wt% glycerol and shake to mix, drop into a container with an upper layer of liquid wax and a lower layer of water to form immobilized cells with a diameter of 2-4mm, harden at 4°C for 2h, filter to obtain immobilized cells, and wash with physiological saline for many times. Add to a physiological saline solution containing 0.15% (V / V) glutaraldehyde, cross-link at 4°C for 1h, filter, wash with physiological saline for many times to obtain immobilized cells. The immobilized cells obtained according to this method produce lauroylglycine according to the reaction method of Example 3, and detect the reaction activity according to the detection method of Example 3. After the reaction is completed, the precipitate is collected by centrifugation to recover the immobilized cells. The relationship between the reaction time and the number of times each immobilized enzyme reaction is determined based on the reaction data, and a curve is drawn. Figure 5As shown in the figure, as the number of times the immobilized cells are reused increases, the rate of product generation decreases, which prolongs the reaction time. The reaction time within 10 reuses is within 13 hours, and the enzyme is still active after 10-15 reuses, with the longest reaction time being 22 hours. After 20 uses, the product is no longer generated after 6 hours of reaction, and it is judged that the enzyme activity has been lost. Therefore, the immobilized cells can be reused repeatedly within 15 uses.

Claims

1. A method for preparing immobilized cells containing acylase, characterized in that: Sugars and / or alcohols are added as enzyme stabilizers during the immobilization process.

2. The method according to claim 1, characterized in that The sugar is one or more of fructose, mannose and trehalose; the alcohol is one or more of glycerol, inositol or propylene glycol; Preferably, 1 wt% to 5 wt% of sugars and 1 wt% to 5 wt% of alcohols are added during the immobilization process.

3. The method according to claim 1, characterized in that The steps include: The wet bacterial bodies containing the recombinant bacteria expressing acylase are obtained by fermentation culture, agar is added to resuspend the bacterial sludge, sugars and / or alcohols are added to oscillate and mix, and the mixture is dropped into a container with an upper layer of liquid wax and a lower layer of water to fix the cells. After hardening, the primary immobilized cells are obtained by filtering, a physiological saline solution containing glutaraldehyde is added for cross-linking, and the immobilized cells are obtained by filtering.

4. The method according to claim 1, characterized in that The wet cell obtaining steps are as follows: (1) fermenting and culturing recombinant bacteria expressing acylase; (2) collecting the bacteria by centrifugation to obtain bacterial cells, and washing the bacterial cells with physiological saline to obtain wet bacterial cells.

5. The method according to claim 1, characterized in that The step of adding agar to resuspend the bacterial sludge is to add 10-30 times the volume of 1-3% agar to resuspend the bacterial sludge at 40-50°C.

6. The method according to claim 1, characterized in that The hardening is carried out at 3-5°C for 1-3h; the concentration of the glutaraldehyde-containing physiological saline solution is 0.05-0.5% (V / V); and the cross-linking is carried out at 3-5°C.

7. Immobilized cells obtained by the method according to any one of claims 1 to 6.

8. Use of the immobilized cells according to claim 7 in the preparation of lauroylglycine.

9. A method for preparing lauroyl glycine, characterized in that: The immobilized cells as claimed in claim 7 are added to a reaction solution for catalytic reaction, wherein the reaction solution comprises lauric acid and glycine and uses water as a solvent.

10. The method according to claim 9, characterized in that The reaction solution is stirred at 40-50°C, and after adding the immobilized cells, the solution is sealed and shaken for reaction; the reaction solution includes 50-150 mM lauric acid and 50-90% (V / V) 2-4 M (preferably 3 M) glycine, and the pH is adjusted to 7.5-8.5 after the volume is supplemented with water; Optionally, the immobilized cells are recovered and reused after the reaction is completed, and the number of reuses is within 15 times.

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