Method for biosynthesis of n-lauroyl glycine and application thereof

Abstract

The application discloses a biosynthesis method and application of N-lauroyl glycine. The biosynthesis method of the N-lauroyl glycine provided by the application comprises the following steps: taking lauric acid and glycine amide as substrates, taking CalB as a catalytic enzyme, taking water as a medium, and reacting and synthesizing under a solvent-free system to obtain the N-lauroyl glycine. The synthesis system of the application does not contain a solvent, the whole preparation process is mild in conditions, simple in process, high in yield and pollution-free, and meets the current green development demand; meanwhile, the obtained product is excellent in performance and reaches the standard of similar chemical synthesis products.

Description

Technical Field

[0001] This invention belongs to the field of surfactant preparation technology, specifically relating to a biosynthesis method and application of N-lauroyl glycine. Background Technology

[0002] The invention of detergent is a double-edged sword. On the one hand, it is a powerful cleaning agent relied upon by the catering industry and households; on the other hand, in the environmental field, it is classified as an endocrine pollutant. Its residue on tableware can enter the human body and interfere with the endocrine system, especially affecting the male reproductive system, posing a significant threat to human health. Therefore, the development of green, non-toxic, biodegradable, and biofriendly biosurfactants is of great significance.

[0003] Sodium lauroyl glycinate is a green, biodegradable biosurfactant mainly used in detergents, cosmetics, and other daily consumer products. It offers deep cleaning and good foaming properties, but currently holds a relatively small market share. Existing sodium lauroyl glycinate is a chemically synthesized product. It is produced by chemically synthesizing and purifying lauroyl chloride from lauric acid and highly toxic and corrosive thionyl chloride, phosphorus trichloride, or phosphorus pentachloride. Lauroyl chloride possesses certain toxicity and corrosiveness. Then, lauroyl chloride and glycine are used as raw materials, and water / acetone is used as a mixed solvent. A condensation reaction is carried out at 75-80℃ under nitrogen protection to generate N-lauroyl glycine, which is then neutralized with sodium hydroxide to form the sodium salt. Since the process of obtaining lauroyl chloride and the reaction byproducts both cause environmental pollution, and toxic byproducts inevitably remain after product purification, finding safe and non-toxic synthetic methods and products will be a future development trend. This invention provides a method and application for synthesizing lauroyl glycine using biological enzymes. The raw materials and synthesis process are green and environmentally friendly, the product is safe and residue-free, and its performance is equivalent to that of similar chemically synthesized products. Summary of the Invention

[0004] The purpose of this invention is to provide a biosynthetic method and application of N-lauroyl glycine. This method does not use solvents during the synthesis process, reducing environmental pollution, increasing reaction yield, simplifying the purification process, and meeting the current needs of green development. At the same time, the performance of the obtained product meets the performance requirements of similar chemically synthesized products, namely, it has excellent calcium soap dispersibility and penetration ability, good surface activity and foaming properties, antibacterial effect and biodegradability, and high safety.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a method for synthesizing N-lauroylglycine, comprising the following steps: using lauric acid and glycine amide as substrates, CalB as a catalyst enzyme, and water as a medium, N-lauroylglycine is synthesized by reaction in a solvent-free system.

[0007] The reaction conditions are: temperature 53°C-65°C, time 25-32h, and pH 7-9.

[0008] The reaction conditions are as follows: the mass ratio of lauric acid to glycine amide is 1:(0.8-1.1).

[0009] The reaction conditions are as follows: the ratio of CalB used is 0.3-0.5 ml CalB for every 4 g of lauric acid.

[0010] Secondly, the present invention also provides N-lauroyl glycine obtained by the above-described synthetic method.

[0011] Thirdly, the present invention also provides a method for extracting sodium N-lauroyl glycinate, wherein the N-lauroyl glycinate is used for extraction to obtain sodium N-lauroyl glycinate.

[0012] The extraction method includes the following steps: adding alkali and ethanol to the N-lauroyl glycine and heating to dissolve it; the heating temperature range is 75-95°C.

[0013] Fourthly, the present invention also provides a GC-MS detection method for N-lauroylglycine, wherein the above-mentioned N-lauroylglycine is subjected to silanization treatment, and the supernatant is taken for GC-MS detection.

[0014] The reagent used in the silanization treatment is MTBSTFA.

[0015] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0016] This invention utilizes natural substrates and biological enzymes as reaction media, with a small amount of water as an auxiliary agent, to synthesize the surfactant N-lauroyl glycine in a solvent-free system, and then extracts sodium N-lauroyl glycine. The entire preparation process is mild, simple, high-yield, and pollution-free, meeting the current needs of green development. Simultaneously, the resulting product exhibits excellent performance, meeting the standards of similar chemically synthesized products, possessing excellent calcium soap dispersibility and penetration; good surface activity and foaming properties; good antibacterial activity; high safety, being gentle and non-irritating to skin and hair; and good biodegradability. It can be used as a main ingredient in compound formulations of food detergents, facial cleansers, hand soaps, shampoos, and shower gels. Attached Figure Description

[0017] Figure 1The total ion chromatogram of the synthesized sample of Example 1 after being silanized by MTBSTFA is shown. In the figure: 1 represents lauric acid silanized by MTBSTFA, with a retention time of 31.688 min; 2 represents N-lauroylglycine silanized by MTBSTFA, with a retention time of 49.054 min.

[0018] Figure 2 This is the mass spectrum of the silanized derivative of lauric acid by MTBSTFA.

[0019] Figure 3 This is the mass spectrum of the N-lauroyl glycine derivative silanized by MTBSTFA.

[0020] Figure 4 The total ion chromatogram is shown for the MSTFA silanization derivative of the synthesized sample in Example 1.

[0021] Figure 5 This is the mass spectrum of the silanized derivative of lauric acid by MSTFA.

[0022] Figure 6 This is the mass spectrum of the derivative of N-lauroyl glycine silanized by MSTFA. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0026] Example 1: Synthesis of N-lauroyl glycine and extraction of sodium N-lauroyl glycine catalyzed by CalB enzyme method

[0027] 1. The CalB enzymatic method for the synthesis of N-lauroyl glycine involves the following steps:

[0028] Add the following substrate amounts to the reactor: 4g lauric acid, 4g glycine amide, and 7-10ml water, maintaining the pH of the aqueous substrate system at 7. Then add 0.4ml CalB enzyme and begin the reaction. The reaction temperature is 53°C. Cover the reactor with perforated plastic wrap with 1-2mm pores to reduce water evaporation. Add water as needed during the reaction to ensure the magnetic stirrer operates at its normal speed, avoiding excessive water causing too high a speed or too little water hindering stirring. Maintain the magnetic stirrer speed at 400-600 rpm to ensure thorough mixing of the reactants. The reaction time is 28 hours, yielding a white, paste-like solid product.

[0029] 2. Extraction of N-lauroyl glycinate sodium, the specific steps are as follows:

[0030] Approximately 30 ml of 6M NaOH and 30 ml of ethanol were added to the reaction mixture (i.e., the reaction product of Example 1). The mixture was heated at 75°C for about half an hour until the reaction mixture was completely dissolved. The mixture was centrifuged at 12000 rpm for 3 minutes, and the supernatant was collected. The pH was adjusted to 7-8, and the mixture was dried under vacuum to obtain a white powder, which was sodium N-lauroyl glycinate. The results showed that approximately 6.5 g of sodium N-lauroyl glycinate powder could be synthesized from one reaction volume of substrate.

[0031] Example 2: Synthesis of N-lauroyl glycine catalyzed by CalB enzyme method

[0032] The difference from Example 1 is that the reaction amounts were changed to 4g of lauric acid and 4.4g of glycine amide.

[0033] Example 3: Synthesis of N-lauroyl glycine catalyzed by CalB enzyme method

[0034] The difference from Example 1 is that the aqueous substrate for the reaction is changed to pH 9.

[0035] Example 4: Synthesis of N-lauroyl glycine catalyzed by CalB enzyme method

[0036] The difference from Example 1 is that the amount of CalB enzyme added for one reaction is changed to 0.5 ml.

[0037] Example 5: Synthesis of N-lauroyl glycine catalyzed by CalB enzyme method

[0038] The difference from Example 1 is that the reaction temperature is changed to 65°C.

[0039] Example 6: Synthesis of N-lauroyl glycine catalyzed by CalB enzyme method

[0040] The difference from Example 1 is that the reaction time is changed to 32 hours.

[0041] Example 7: Synthesis of N-lauroyl glycine catalyzed by CalB enzyme method

[0042] The difference from Example 1 is that the amount of CalB enzyme added for one reaction is changed to 0.3 ml.

[0043] Example 8: Synthesis of N-lauroyl glycine catalyzed by CalB enzyme method

[0044] The difference from Example 1 is that the reaction time is changed to 25 hours.

[0045] Example 9: Synthesis of N-lauroyl glycine catalyzed by CalB enzyme method

[0046] The difference from Example 1 is that the reaction amounts were changed to 4g of lauric acid and 3.3g of glycine amide.

[0047] Comparative Example 1: Synthesis of N-lauroyl glycine catalyzed by CalB enzyme method

[0048] The difference from Example 1 is that the aqueous substrate for the reaction is changed to pH 6.

[0049] GC-MS detection of N-lauroyl glycine synthesized in Test Examples, Examples 1-9 and Comparative Example 1

[0050] Experimental steps:

[0051] (1) Preparation of standard samples: Weigh 0.05 g of lauric acid and 0.06 g of lauroyl glycine, respectively, dissolve them in water, adjust the pH to 7, add water to 5 ml of each, and combine the equal volumes to obtain a mixed mother liquor of 25 mM lauric acid and 25 mM lauroyl glycine; further dilute to obtain equimolar 0, 5, 10, 15, and 20 mM standard lauric acid and lauroyl glycine mixtures, take 50 μl of each, add 400 μl of ethyl acetate, and shake thoroughly to mix. Centrifuge at 12000 rpm for 3 min, transfer the supernatant organic phase to a centrifuge tube, and then evaporate the solvent on a rotary dryer.

[0052] (2) Sample preparation: Take 0.01 g of the product synthesized in Example 1, add 100 μl of ethanol to dissolve it, and 100 μl of 6M NaOH. Heat slightly to dissolve completely, then add about 800 μl of acetone. A white precipitate will appear. Centrifuge at 12000 rpm for 3 min, retain the precipitate, and evaporate to dryness under vacuum. Add 1 ml of ethanol-water mixture to dissolve the precipitate, adjust the pH to 6-7, take 50 μl, add 400 μl of ethyl acetate, and shake thoroughly to mix. Centrifuge at 12000 rpm for 3 min, transfer the supernatant organic phase to a centrifuge tube, and then evaporate the solvent to dryness using a rotary dryer.

[0053] (3) Sample silanization: Add 40 μl of methoxyamine pyridine to the evaporated sample obtained in steps (1) and (2), vortex vigorously for 1 min, and place at 37°C for 1 min; add 50 μl of MTBSTFA, shake at 37°C for 30 min; add 400 μl of n-hexane, mix well, centrifuge at 12000 rpm for 3 min, and take the supernatant to add to the GC-MS sample vial.

[0054] (4) GC-MS analysis:

[0055] Instrument: Gas Chromatography-Mass Spectrometry System (Agilent 6890N GC-5973N MSD);

[0056] Chromatographic column: HP-5MS (60 m × 0.25 mm × 0.25 µm, J&W Scientific, Folsom, CA);

[0057] GC-MS operating conditions:

[0058] Chromatographic conditions: programmed temperature ramp, starting at 90 ℃, holding for 1 min, then ramping to 175 ℃ at a rate of 5 ℃ / min, holding for 3 min, then ramping to 270 ℃ at 3 ℃ / min, then ramping to 310 ℃ at 20 ℃ / min, holding for 15 min; injection port temperature: 280 ℃; carrier gas: high purity He (99.999%), carrier gas flow rate: 1 mL / min; injection volume: 2 μl, splitless injection mode.

[0059] Mass spectrometry conditions: interface temperature 280℃, ionization mode: EI; ionization energy: 70 eV; ion source temperature: 230℃; quadrupole temperature: 150℃; scan mass range: 50-550 amu; scan rate: 1.52 scans / sec. Solvent delay 8 min.

[0060] Qualitative and quantitative methods: Qualitative analysis was performed using the retention time and mass spectrum of the standards, and quantitative analysis was performed using the area response of the standards. The mass spectral library was searched as NIST02 (Rev. D.04.00, Agilent Technologies, Palo Alto, CA, USA).

[0061] Figure 1 The figure shows the GC-MS spectrum of the product synthesized in Example 1. As can be seen from the figure, the silanized derivatives of lauric acid and N-lauroyl glycine are both single peaks in the total ion chromatogram.

[0062] 2. Calculation of Synthesis Rate

[0063] In the total ion chromatogram, lauric acid and N-lauroylglycine in the synthesized sample of Example 1 were quantified using known amounts of lauric acid and N-lauroylglycine standards to determine their molar concentrations. The molar ratio M of lauroylglycine / lauric acid was calculated; the synthesis rate was calculated as M / (1+M)×100 (%), which is the synthesis rate calculated with the equimolar conversion of lauric acid to N-lauroylglycine as a reference.

[0064] The linear formula for the standard curve of lauric acid is: y = 2E+07x - 2E+07, R² = 0.9716; where: x represents the concentration of standard lauric acid; y represents the integral area of ​​the corresponding peak of silanized lauric acid in the total ion chromatogram; and E represents the power of 10.

[0065] The linear formula for the standard curve of lauroylglycine is: y = 227443x - 194744, R² = 0.9798. Where: x represents the concentration of standard lauroylglycine; y represents the integrated area of ​​the corresponding peak of silanized lauroylglycine in the total ion chromatogram.

[0066] The results are as follows.

[0067] Table 1 Calculation of Integral Area and Combination Rate

[0068]

[0069] Example 10: Extraction of N-lauroyl glycinate sodium

[0070] The difference from Example 1 is that the dissolution temperature of the reaction mixture was changed to 80°C. The results showed that it could be completely dissolved.

[0071] Example 11: Extraction of N-lauroyl glycinate sodium

[0072] The difference from Example 1 is that the dissolution temperature of the reaction mixture was changed to 95°C. The results showed that it could be completely dissolved.

[0073] Comparative Example 2: Extraction of N-lauroyl glycinate sodium

[0074] The difference from Example 1 is that the dissolution temperature of the reaction mixture was changed to 70°C. The results showed that it could not be completely dissolved.

[0075] Comparative Example 3: GC-MS Detection of N-Lauroylglycine

[0076] The difference from the GC-MS detection in the test example is that the silanizing reagent is MSTFA. The results show that N-lauroyl glycine exhibits two silanization derivatives with different degrees of degradation. Figure 4 As indicated by the middle arrow, two consecutive peaks appear, indicating that MSTFA is not suitable for this GC-MS detection.

[0077] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for synthesizing N-lauroyl glycine, characterized in that, The process includes the following steps: using lauric acid and glycine as substrates, CalB as the catalytic enzyme, and water as the medium, N-lauroyl glycine is synthesized in a solvent-free system. The mass ratio of lauric acid to glycine amide is 1:(0.8-1.1). The reaction conditions are: temperature 53°C-65°C, time 25-32h, and pH 7-9. The dosage ratio of CalB is: 0.3-0.5 ml of CalB for every 4 g of lauric acid.

2. A method for extracting sodium N-lauroyl glycinate, characterized in that, include: (1) N-lauroyl glycine was synthesized using the method described in claim 1; (2) Add alkali and ethanol to the N-lauroyl glycine, heat to dissolve it, and extract to obtain sodium N-lauroyl glycine; the heating temperature range is 75-95°C.

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