An enzyme catalyzing the production of octanoylalanine and its immobilization method and application

Abstract

The present invention belongs to the technical field of biocatalytic synthesis, and discloses an enzyme for catalyzing the generation of octanoylalanine, and an immobilization method and application thereof. The present invention selects a suitable epoxy resin carrier to immobilize an acylase or a lysine-modified acylase using an immobilization technology. The immobilized enzyme has increased stability and can be recycled and reused up to 20 times. The method of the present invention has a simple synthesis process, mild reaction conditions, less organic reagents, low pollution, reduced impact on the product purification process, and realizes the recycling of bioenzyme catalysts, simplifies the post-processing process, and improves the yield.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to an enzyme for catalyzing the generation of octanoylalanine and an immobilization method and application thereof. Background Art

[0002] N-(1-oxooctyl)alanine is an amino acid surfactant with the structural formula:

[0003]

[0004] Amino acid surfactants are an important class of natural surface-active molecules. They 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. This property, coupled with their low toxicity, good biodegradability, high eco-compatibility, antibacterial, corrosion-resistant and anti-disease properties, makes them the molecules of choice in the food, pharmaceutical and cosmetic industries.

[0005] The fatty acyl amino acid salt is obtained by reacting in an organic reagent, and then the crude fatty acyl amino acid is separated by neutralization with an inorganic acid, and then neutralized with an alkali to obtain a relatively pure fatty acyl amino acid salt. This chemical method is difficult to synthesize, has a low yield, and is not a green synthesis process. The byproducts and environmental pollution problems are difficult to solve.

[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 biotransformation. Enzymatic synthesis mainly uses bioenzymes to catalyze the amidation reaction of fatty acids and their derivatives with amino acids, which has received extensive research attention due to its greenness and safety. The main challenge facing the enzyme catalytic pathway is that the known enzymes have low activity and poor stability when catalyzing acylation reactions, high enzyme costs and low product yields, which are the main factors restricting the enzymatic synthesis of amino acid surfactants.

[0007] Acylase (EC 3.5.1.4) catalyzes the acylation reaction of amino groups. It is an enzyme used for the reverse reaction of amide hydrolysis. It can catalyze the removal of a molecule of water from the carboxyl group of octanoic acid and the amino group of alanine to generate octanoyl alanine. Naturally occurring acylases have a narrow substrate spectrum, low catalytic activity, and poor stability. Wild-type plant-derived acylases ( Vigna umbellata , XP_047162094) has high activity towards octanoylalanine, but its stability and utilization rate are still insufficient for industrial application.

[0008] One of the most effective strategies for the successful use of enzymes in biotechnology processes is their immobilization. Compared with free enzymes, immobilized enzymes not only maintain their high efficiency, specificity and mild enzyme catalytic reaction characteristics, but also overcome the shortcomings of free enzymes. They have a series of advantages such as high storage stability, easy separation and recovery, multiple reuse, continuous and controllable operation and simple process. Traditional enzyme immobilization methods include adsorption, embedding, cross-linking and covalent bonding. Each method has its own advantages and disadvantages. Among them, the covalent bonding method has great advantages over other methods in preparing immobilized enzymes with strong binding.

[0009] Epoxy resin is a synthetic resin with epoxy groups on the surface. This type of carrier has strong resistance to microbial and acid-base corrosion and strong mechanical properties over a long period of time, making it very suitable as a carrier for industrial enzyme preparations. The epoxy groups on the surface can covalently bind to the inactive groups such as -NH2 and -HS on the surface of the enzyme molecules under very mild conditions, thereby fixing the enzyme molecules on the carrier surface. The reaction conditions of the epoxy group are mild, and the enzyme molecules are generally not destroyed after binding. After proper treatment, an immobilized enzyme with good stability can be obtained. Summary of the invention

[0010] The object of the present invention is to provide a fixed Vigna umbellata The immobilized acylase obtained above is used to transform and produce octanoyl alanine in the presence of a substrate solution.

[0011] By using enzyme immobilization technology to immobilize acylase, the enzyme's tolerance to mechanical shear force, temperature changes, and substrate octanoic acid in the production process can be improved, and the stability of the acylase can be enhanced, the recycling of bio-enzyme catalysts can be improved, the post-processing process can be simplified, and the yield can be improved. Therefore, it is necessary to find different immobilization carriers to immobilize the acylase, find the best carrier for immobilized acylase, and achieve the maximum number of applications of the immobilized enzyme without affecting its catalytic activity.

[0012] The present invention adopts the following technical solutions to achieve the above-mentioned purpose:

[0013] The present invention provides a method for immobilizing an enzyme that catalyzes the production of octanoylalanine, comprising the following steps:

[0014] S1. Add epoxy resin carrier to the enzyme solution for catalyzing the production of octanoylalanine, and shake and mix at 20-30°C for 12-24h;

[0015] S2 then collects the immobilized enzyme by filtration, and rinses and filters it with potassium phosphate buffer to obtain the immobilized enzyme.

[0016] Preferably, the enzyme catalyzing the production of octanoylalanine is from Vigna umbellata or an enzyme obtained by adding 1-50 lysines to its C-terminus. More preferably, the number of lysines is 5-30.

[0017] Further preferably, the epoxy resin carrier is activated before use, and the epoxy resin carrier is placed in a pH 7.0-8.5 buffer and shaken at 18-25° C. for 0.5 to 2 h, and then filtered and washed with deionized water until the water is clear.

[0018] Specifically, the model of the epoxy resin carrier is EP109, and the dosage of the epoxy resin carrier is 50-150% of the mass of the free enzyme.

[0019] In a specific embodiment, the enzyme that catalyzes the production of octanoylalanine is expressed and purified by recombinant bacteria.

[0020] The invention provides an immobilized enzyme for catalyzing the generation of octanoyl alanine obtained by the immobilization method.

[0021] The invention further provides the use of the enzyme in preparing octanoyl alanine.

[0022] The present invention also provides a method for preparing octanoyl alanine, which comprises the following steps: adding the enzyme to a substrate solution containing n-octanoic acid for shaking reaction.

[0023] Preferably, the substrate solution is a saturated alanine solution containing n-octanoic acid, with a pH of 7.5 to 8.5; the n-octanoic acid concentration is 10-100 g / L; and the shaking reaction is carried out at 45-65° C. for 12-24 hours.

[0024] More preferably, after the reaction is completed, the immobilized enzyme and the reaction solution are separated, and the immobilized enzyme is recovered and used for the next batch of reactions, and is reused 1 to 20 times.

[0025] The beneficial effects of the present invention are as follows: the present invention covalently fixes the acylase and EP109, which can effectively improve the stability of the immobilized enzyme, especially the thermal stability and the stability of the substrate n-octanoic acid, and can be repeatedly applied 20 times. The process is simple, the cost is low, the yield is high, and it has application value for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Comparison of enzyme activity immobilized on different types of epoxy resins.

[0027] Figure 2 Comparison of enzyme activity after adding different amounts of carrier to the enzyme solution.

[0028] Figure 3 Comparison of enzyme activity before and after enzyme molecule modification.

[0029] Figure 4 The temperature tolerance of the immobilized enzyme is compared with that of the free enzyme.

[0030] Figure 5 The substrate tolerance of the immobilized enzyme was compared with that of the free enzyme. DETAILED DESCRIPTION

[0031] Main experimental materials:

[0032] Acylase ( Vigna umbellate , XP_047162094) Escherichia coli recombinant expression strain: The acylase gene was cloned into the vector pET-28a by homologous recombination, named VuGH-pET28a plasmid, and transformed into the Escherichia coli expression strain BL21 (DE3) to obtain the recombinant strain, hereinafter referred to as VuGH-pET28a-BL21 (DE3).

[0033] Immobilized carrier: LX-1000EP (Xi'an Lanxiao Technology New Materials Co., Ltd.), ES-108 (Tianjin Nankai Hecheng Technology Co., Ltd.), ES-109 (Tianjin Nankai Hecheng Technology Co., Ltd.), ES-103B (Tianjin Nankai Hecheng Technology Co., Ltd.), EP107 (Ningbo Zhengguang Resin Co., Ltd.), EP109 (Ningbo Zhengguang Resin Co., Ltd.), LX-103B (Xi'an Lanxiao Technology New Materials Co., Ltd.), LX-109S (Xi'an Lanxiao Technology New Materials Co., Ltd.)

[0034] Example 1: Fermentation to produce acylase

[0035] LB medium: tryptone 10 g / L, yeast extract 0.5 g / L, sodium chloride 10 g / L, purified water.

[0036] Fermentation medium: tryptone 12 g / L, yeast extract 24 g / L, glycerol 4 g / L, potassium dihydrogen phosphate 2.5 g / L, potassium dihydrogen phosphate trihydrate 16 g / L, purified water.

[0037] 1) Pick a single colony VuGH-pET28a-BL21 (DE3) and inoculate it into a 4 ml LB medium test tube, add 4 uL kanamycin (original concentration 50 mg / mL), and culture at 37°C, 200 rpm for 12 h to obtain primary seeds.

[0038] 2) The primary seeds are inoculated into a shake flask with 100 ml of fermentation medium, cultured at 37°C, 200 rpm for 3-4 h, IPTG is added to a final concentration of 0.05-0.5 mM (preferably 0.2 mM), and cultured at 18-25°C (preferably 20°C), 200 rpm for 12-16 h (preferably 15 h).

[0039] 3) The bacterial solution in step (2) was centrifuged at 6000×g for 20 min to collect the bacterial cells and stored in a -20°C refrigerator.

[0040] Example 2: Extraction and immobilization of acylase

[0041] 1) Add 0.1 M sodium phosphate buffer according to the bacterial concentration (200 g / L) and crush with a high-pressure homogenizer. The parameters are 500 bar once and 800 bar twice.

[0042] 2) Add polyethyleneimine with a molecular weight of 25,000 at a final concentration of 0.3 wt.% and flocculate for 30 minutes. Centrifuge at high speed to obtain the supernatant enzyme solution.

[0043] 3) Carrier activation: Place the epoxy resin carrier in a shaker at 20°C with pH 7.0-8.5 buffer for 1 hour, filter and wash the carrier with deionized water until the water is clear.

[0044] 4) Add 50% of the mass of free enzyme to the epoxy resin immobilized carrier into the enzyme solution, and shake and mix at 20-30°C for 12-24h.

[0045] 5) After centrifugation at 12000 rpm for 10 min in a high-speed centrifuge, the supernatant was discarded, the immobilized enzyme was collected, and it was rinsed several times with potassium phosphate buffer (20-100 mM, pH 7-8.5), and filtered to obtain the immobilized enzyme.

[0046] Example 3: Screening of epoxy resin immobilization carriers

[0047] There are various types of commercial epoxy resin carriers. At present, there is no obvious rule for the selection of carriers for different types of enzymes. Different types of carriers should be selected according to the different properties of the enzymes to obtain the best effect. Therefore, it is necessary to screen the carrier. According to the immobilization method in Example 2, 8 different types of epoxy resins were used to prepare immobilized enzymes, and the enzyme solution was added with 50% of the mass of the free enzyme epoxy resin immobilization carrier. The immobilized enzyme activity was detected, and the free enzyme was used as a control to screen the carrier with the highest immobilized enzyme activity. The reaction system for detecting the activity of the immobilized enzyme is:

[0048] Preparation of 1 mL substrate solution: add 14.4 g / L octanoic acid to 900 uL saturated alanine solution, add water to 1 ml, stir at 50 °C, and adjust pH to 8.0;

[0049] Take 20 mg of immobilized enzyme (40 mg of free enzyme in the control group), add 1 mL of substrate solution, stir well, and react at 50°C for 18 hours. Detection method: dilute the above reaction system 100 times with methanol, shake and mix thoroughly, centrifuge at 12000 rpm for 3 minutes, take the supernatant, and detect the formation of the product octanoyl alanine by HPLC.

[0050] like Figure 1As shown, the EP109 immobilized enzyme has the highest enzymatic activity, which is 0.84 times that of the free enzyme, and EP109 is determined to be the optimal carrier.

[0051] Example 4: Optimizing the ratio of enzyme solution to carrier

[0052] In the study of immobilized enzymes, the amount of carriers must be consistent with the protein content in the immobilized enzyme solution to achieve the desired effect. If the carrier content is too low, some enzyme proteins will not have the opportunity to bind to the carrier, resulting in unnecessary enzyme activity loss. In Example 3, the amount of carriers using 50% of the free enzyme mass did not reach the enzyme activity of the free enzyme, indicating that the amount of carriers is insufficient. If the carrier content is too high, the carrier will not be able to give full play to its ability to bind to enzyme proteins, which will increase the cost of immobilization to a certain extent. Therefore, controlling the amount of carriers is conducive to improving the benefits of immobilized enzymes.

[0053] In order to determine the optimal ratio of enzyme solution to carrier, 0%, 50%, 100%, 150%, and 200% of the carrier by mass of the free enzyme were added to 10 mL of 20% crude enzyme solution to prepare immobilized enzymes. The activity of the immobilized enzyme was detected according to the method in Example 3. The reaction system for detecting the activity of the immobilized enzyme is detailed in Example 3.

[0054] The results are as follows Figure 2 The results showed that the conversion rate of immobilized enzyme increased significantly when the carrier with a mass of 100% of the free enzyme was added, while the conversion rate did not increase significantly when the amount of carrier was further increased. Considering the cost factor, the optimal amount of carrier added was determined to be 100% of the bacterial cell mass.

[0055] Example 5: Enzyme molecule modification

[0056] The principle of epoxy resin immobilization is mainly that the lysine on the surface of the enzyme molecule forms a chemical covalent bond with the epoxy group of the epoxy resin, thereby effectively connecting the carrier and the enzyme. Increasing the number of lysine on the surface of the enzyme molecule is conducive to strengthening the connection, thereby improving the stability and activity of the immobilized enzyme.

[0057] A certain number of lysine sequences were added directly before the C-terminal stop codon of the target enzyme to construct sequences with lengths of 5, 10, 15, 20, 25, and 30 lysines, respectively. The construction process is as follows:

[0058] 1) Design upstream and downstream primers with lysine lengths of 5, 10, 15, 20, 25, and 30, respectively, as shown in Table 1.

[0059] 2) Using VuGH-pET28a plasmid as template (20 ng / uL), PCR was performed using PrimerStar Max enzyme (Takara). The PCR system is shown in Table 2.

[0060] 3) After PCR, the gel was cut and a band of about 6 kb was recovered.

[0061] 4) The recovered product was treated with T4 ligase (Biyuntian) and Ligation high Ver.2 (TOYOBO). The ligation system is shown in Table 3.

[0062] 5) Transform into Escherichia coli BL21 (DE3) and verify by sequencing.

[0063] VuGH-pET28a-BL21 (DE3) with different lysine lengths was added to prepare the immobilized enzyme, and the immobilized enzyme activity was detected according to the carrier amount of 20% free enzyme mass and the method in Example 3. The immobilized enzyme activity detection reaction system is shown in Example 3. Wherein, since the carrier amount of 50% bacterial mass has been added to reach 84% free enzyme activity, if the carrier is added according to 50% bacterial mass or higher 100% after the transformation, the transformation result will not be significant, and it is not easy to see the difference. Reduce the amount of carrier added to better reflect that the sequence after the transformation is easier to bind to the carrier, and the enzyme activity shown is higher, so the carrier amount of 20% free enzyme mass is selected for comparative experiments.

[0064] The results are as follows Figure 3 As shown, adding lysine can improve the activity of the immobilized enzyme, among which the activity of the immobilized enzyme with 20 lysines at the C-terminus was significantly improved after the modification, which was twice that before the modification.

[0065] Table 1. Primer list

[0066]

[0067] Table 2. PCR system

[0068]

[0069] Table 3. T4 ligase system

[0070]

[0071] Example 6: Immobilized acylase conversion to produce octanoylalanine and reuse of batches

[0072] 1) 1L substrate solution: 14.4g / L octanoic acid, 90% saturated alanine solution, stir at 50℃, adjust pH to 7.5-8.5.

[0073] 2) 10-30 g of the immobilized enzyme in Example 5 (immobilized enzyme with 20 lysine residues at the C-terminus) was added to 1 L of substrate solution, stirred evenly, and then sealed and shaken for reaction at 50°C.

[0074] 3) The reaction was allowed to proceed for 16-20 h. During the reaction, the pH of the reaction was adjusted to 8.0 with 5 mol / L NaOH solution. The reaction was stopped when the substrate was no longer consumed.

[0075] 4) The reaction liquid was filtered to separate the immobilized enzyme from the reaction liquid, the immobilized enzyme was recovered, and the immobilized enzyme was washed with 20 mM PB8.0 for use in the next batch of reactions.

[0076] 5) The immobilized enzyme used in the 1L scale reaction can be recycled and applied up to 20 times. Each time the immobilized enzyme is applied, its activity is 0.95 times that of the free enzyme. The activity of the immobilized acylase applied 20 times is 0.81 times that of the free enzyme for the 21st time. The activity further decreases with further application. The specific activity per application is shown in Table 4 below.

[0077] From the data in the table below, it can be seen that the immobilized acylase can be reused up to 20 times. Although the conversion rate is different each time it is reused, there is no significant difference compared with the immobilized acylase that has not been reused. In comparison, the conversion rate is significantly reduced in the 21st application, so the number of applications of the immobilized enzyme is 20 times.

[0078] Table 4. Immobilized acylase application times and conversion rates

[0079]

[0080] Example 7: Thermal stability of immobilized enzyme

[0081] The bacterial cells collected in Example 1 were used, 0.1M sodium phosphate buffer was added according to the bacterial cell concentration (200 g / L), and the cells were crushed by a high-pressure homogenizer with the parameters of 500 bar once and 800 bar twice, and the supernatant was taken as a control group after low-temperature high-speed centrifugation at 12000 rpm for half an hour. The immobilized enzyme obtained in Example 5 (immobilized enzyme with 20 lysine residues at the C-terminus) was used as an experimental group.

[0082] The control group and the experimental group set up 6 groups of reactions according to the temperature gradient: 50℃, 53℃, 56℃, 59℃, 62℃, 65℃. The reaction system is as follows: 1mL substrate solution: 14.4mg / mL of n-octanoic acid is added to 900uL of saturated alanine solution, and 1ml is added with water, then stirred at 50℃, and the pH is adjusted to 8.0; 20mg of immobilized enzyme (the amount of free enzyme added in the control group is 10uL), 1mL of substrate solution is added, stirred, and shaken at different temperatures for 18h. The detection conditions are shown in Example 3, and the enzyme activity detection results are shown in Figure 4 From the range of 50-65℃, the ratio of enzyme activity of immobilized enzyme to free enzyme continues to rise. Because the stability of free enzyme is poor, the activity decreases sharply when the temperature rises, while the stability is greatly improved after immobilization, which meets the requirements of industrial applications.

[0083] Example 8: Immobilized enzyme substrate tolerance

[0084] The bacterial cells collected in Example 1 were used, 0.1M sodium phosphate buffer was added according to the bacterial cell concentration (200 g / L), and the cells were crushed by a high-pressure homogenizer with the parameters of 500 bar once and 800 bar twice, and the supernatant was taken as a control group after low-temperature high-speed centrifugation at 12000 rpm for half an hour. The immobilized enzyme obtained in Example 5 (immobilized enzyme with 20 lysine residues at the C-terminus) was used as an experimental group.

[0085] The control group and the experimental group set up 7 groups of reactions according to the concentration gradient of substrate n-octanoic acid: 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 60g / L, 100g / L, and the substrate mass was calculated according to the concentration and added to the reaction system. The reaction system is as follows: 1mL substrate solution: different masses of n-octanoic acid are added to 900uL of saturated alanine solution, supplemented with water to 1ml and stirred at 50℃, and the pH is adjusted to 7.5-8.5; 20mg of immobilized enzyme (the amount of free enzyme added in the control group is 10uL), add 1mL of substrate solution, stir well, and react at 50℃ for 18h. The detection conditions are shown in Example 3, and the enzyme activity detection results are shown in Figure 5 The substrate tolerance of free enzyme is poor. Under the premise of increasing substrate concentration, the ratio of immobilized enzyme activity to free enzyme activity gradually increases, but decreases after 60g / L, because the activity of immobilized enzyme also decreases significantly at this time, but it is still greater than that of free enzyme. Therefore, immobilized enzyme significantly improves the substrate tolerance of enzyme.

Claims

1. A method for immobilizing an enzyme that catalyzes the production of octanoylalanine, characterized in that: The steps include: S1. Add an epoxy resin carrier to the enzyme solution that catalyzes the production of octanoylalanine, and shake and mix at 20-30°C for 12-24h; wherein the epoxy resin carrier is LX_100EP, ES-108, ES-103B, EP107, EP109, LX-103B, LX-109S, and its dosage is 50-150% of the mass of the free enzyme; S2 then collects the immobilized enzyme by filtration, and washes and filters it with potassium phosphate buffer to obtain the immobilized enzyme; The enzyme catalyzing the production of octanoyl alanine is Vigna umbellata The enzyme obtained by adding 5-30 lysines directly before the C-terminal terminator codon of the source acylase XP_047162094; The epoxy resin carrier is activated before use. The epoxy resin carrier is placed in a pH 7.0-8.5 buffer and shaken at 18-25° C. for 0.5 to 2 hours. After suction filtration, the carrier is washed with deionized water until the water is clear.

2. The immobilization method according to claim 1, characterized in that: The number of the lysines is 20.

3. The immobilization method according to claim 1 or 2, characterized in that: The enzyme catalyzing the production of octanoylalanine is obtained by expression and purification of recombinant bacteria.

4. The immobilized enzyme catalyzing the production of octanoylalanine obtained by the immobilization method according to claim 1 or 2.

5. Use of the enzyme as claimed in claim 4 in the preparation of octanoylalanine.

6. A method for preparing octanoyl alanine, characterized in that: The method comprises the following steps: adding the enzyme as claimed in claim 4 into a substrate solution containing n-octanoic acid for shaking reaction.

7. The method according to claim 6, characterized in that The substrate solution is a saturated alanine solution containing n-octanoic acid, with a pH of 7.5 to 8.5; the n-octanoic acid concentration is 10-100 g / L; and the shaking reaction is carried out at 45-65° C. for 12-24 hours.

8. The method according to claim 7, characterized in that After the reaction is completed, the immobilized enzyme and the reaction solution are separated, and the immobilized enzyme is recovered and used for the next batch of reactions, and can be reused 1 to 20 times.

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