PLD immobilized enzyme, carrier as well as preparation and application of PLD immobilized enzyme

Abstract

The invention belongs to the field of biological enzyme immobilization, and particularly relates to a PLD immobilized enzyme, a carrier and preparation and application of the PLD immobilized enzyme, a preparation method of the carrier comprises the steps that a natural polysaccharide material is dissolved in advance to form a water phase, then the water phase is mixed with an oil phase, and a polysaccharide emulsion is obtained; then adding silicon dioxide, and mixing to obtain a silicon dioxide-polysaccharide emulsion; and adding a modifier in a formula 1 # imgabs0 #, mixing, and carrying out cross-linking modification under an alkaline condition, so as to obtain the composite material. A natural polysaccharide material and silicon dioxide are compounded in advance on the basis of an emulsion mode, and then the compound is cured and modified by adopting the formula 1, so that the adaptability of the prepared carrier to PLD enzyme can be enhanced, the shielding problem to the active structural domain of the PLD enzyme in the curing process can be reduced, the enzyme activity can be improved, and in addition, the immobilization stability and the mechanical strength can be effectively improved.

Description

Technical Field

[0001] The present invention belongs to the field of enzymes, and in particular relates to the field of enzyme immobilization. Background Art

[0002] Phosphatidylserine (PS) is a member of the phospholipid family. It is the only phospholipid that can regulate the functional state of key cell membrane proteins and is an indispensable substance for the human body. Studies have shown that PS has a variety of physiological functions that are beneficial to the human body: (1) Improving brain function and assisting in the treatment of Alzheimer's disease. PS is an active substance in the cell membrane, especially in brain cells. Its main function is to improve the function of nerve cells, regulate the conduction of nerve impulses, and enhance brain memory function. Due to its strong lipophilicity, it can quickly pass through the blood-brain barrier into the brain after absorption, soothing vascular smooth muscle cells and increasing blood supply to the brain; (2) Promoting brain development and enhancing memory. The lack of neurotransmitters can cause difficulty in concentration and even memory loss. This is because this phenomenon will lead to a decrease in enzyme activity and cell transport capacity in brain cells, and the number of synapses between cells will also show a trend of reduction, ultimately leading to memory loss. Therefore, oral administration of PS can increase the number of brain spikes and the fluidity of brain cell membranes, restore the release of neurotransmitters, thereby making brain cells more active and ultimately increasing the activity of active enzymes in the brain, thereby enhancing memory. (3) Repairing damage and treating children's ADHD. PS can activate various enzymes in brain cells, replenish the number of brain neurotransmitters, and reduce the symptoms of ADHD. (4) Assisting in the treatment of depression. Relieve brain fatigue and mental stress, promote recovery from brain fatigue, regulate emotions, and treat depression. (5) In terms of sports nutrition, PS has the function of preventing exercise tension, promoting physical recovery, and regulating emotions.

[0003] Among the currently reported PS production technologies, extraction and enzymatic synthesis are the most common. Enzymatic synthesis uses phosphatidylcholine and serine as substrates for catalytic synthesis, and has the advantages of mild reaction conditions and high catalytic synthesis efficiency, and is known as a "green synthesis" technology. Among the technologies for enzymatic synthesis of PS, liquid phospholipase D is often used for catalytic synthesis. Since the substrate PC is insoluble in water, while phospholipase D dissolves in the aqueous phase, the biocatalytic synthesis of PS usually uses an interfacial reaction between the oil-water phase. However, due to the large mass transfer resistance between the two phases, the contact area between the phospholipase and the substrate PC is very limited. At the same time, the contact between the enzyme and the organic solvent easily leads to structural changes and loss of activity. This leads to problems such as low conversion efficiency and high enzyme usage costs, which are not conducive to industrial-scale production.

[0004] After the enzyme is immobilized, the multi-batch catalytic synthesis of the product is achieved, which is an efficient and economical biocatalytic synthesis method. Chitosan is one of the most abundant natural polysaccharides in nature. Compared with existing commercial carriers, it has significant advantages as a carrier for enzyme immobilization, such as being non-toxic, odorless, safe, environmentally friendly, and low cost. Using it as an immobilized carrier to immobilize the enzyme and achieve multi-batch catalytic synthesis of PS has the prospect of industrial-scale application.

[0005] For example, patent publication No. US9303256A2 discloses an enzyme immobilization particle, wherein the carrier described therein is selected from at least one of silica, zeolite, alumina, diatomaceous earth, and kaolin. In another example, patent publication No. WO2013151757A1 discloses a polysilicic acid-polysilicone enzyme immobilization material. Patent publication No. JP2001506499A discloses a scheme using sugars, starches, glucans, water-soluble cellulose derivatives, and fermentation residues as carriers for immobilized enzymes.

[0006] In summary, although some enzyme immobilization methods have been reported in the prior art, different enzymes have different two-dimensional and three-dimensional structures and different requirements for carrier adaptation. There are still few immobilization methods in the prior art that can effectively adapt to PLD and can take into account the activity, stability and mechanical strength of PLD-immobilized enzymes. Summary of the Invention

[0007] In response to the problems of existing enzyme immobilization, the first purpose of the present invention is to provide a method for preparing a carrier for PLD immobilized enzyme, aiming to obtain a carrier that is adapted to the two-dimensional and spatial characteristics of PLD enzyme and can prepare a carrier that can improve enzyme activity, stability and mechanical strength.

[0008] The second purpose of the present invention is to provide a carrier prepared by the preparation method and its application in PLD enzyme immobilization.

[0009] The third object of the present invention is to provide the carrier-immobilized PLD-immobilized enzyme and its preparation and application.

[0010] Different proteins have varying secondary and tertiary structures. This can lead to varying degrees of shielding of the enzyme's active domain during solidification, as well as differences in enzyme-support binding and stability. Existing technologies lack effective solutions tailored to the characteristics of PLD enzymes and improving their immobilization performance and mechanical strength. To address this issue, the present invention provides the following improved solutions:

[0011] A method for preparing a carrier for immobilizing a PLD enzyme comprises dissolving a natural polysaccharide material in advance to form an aqueous phase, which is then mixed with an oil phase to obtain a polysaccharide emulsion; subsequently adding silicon dioxide and mixing to obtain a silicon dioxide-polysaccharide emulsion; then adding a modifier of formula 1, mixing, and cross-linking (curing) under alkali to obtain the carrier;

[0012]

[0013] The R1 is hydrogen, hydroxyl, or alkoxy; and the R2 is H or an ester group.

[0014] The present invention pre-compounds a natural polysaccharide material and silica in an emulsion manner, and then solidifies and modifies them using Formula 1. This can enhance the adaptability of the prepared carrier to the PLD enzyme, reduce the shielding problem of the active domain of the PLD enzyme during the solidification process, improve enzyme activity, and effectively improve the immobilization stability and mechanical strength.

[0015] In the present invention, the natural polysaccharide material includes at least one of agarose, dextran and chitosan.

[0016] The aqueous phase is a solution formed by mixing a natural polysaccharide material and an acid aqueous solution; wherein the concentration of the natural polysaccharide material is 1 to 3% (w / v);

[0017] The oil phase is a solution containing at least one of Span 80, Tween 80, sucrose fatty acid ester, and sorbitan oleate.

[0018] In the present invention, there is no particular requirement for the amount of the oil phase added. For example, the weight ratio of the oil phase to the natural polysaccharide material can be 1:10 to 50; further, it can be 1:20 to 40.

[0019] In the present invention, the weight ratio of natural polysaccharide material to silicon dioxide is 10 to 50: 1, further 15 to 25: 1. Under the preferred ratio, the components can be further synergistically strengthened, and the strength of the carrier and the solidification effect of the enzyme can be improved.

[0020] In the present invention, in Formula 1, R2 is H or an ester group. The ester group is, for example,

[0021]

[0022] The weight ratio of the formula 1 to the natural polysaccharide material is 1:10 to 70, and further, can be 1:20 to 50. Under the preferred ratio, the synergy of the components can be further enhanced, and the enzyme curing effect can be improved.

[0023] The pH during the cross-linking modification process is 7.0 to 9.0, and can further be 7.5 to 8.5.

[0024] The present invention also provides a carrier prepared by the preparation method.

[0025] The present invention also includes the use of the carrier prepared by the preparation method to immobilize the PLD enzyme.

[0026] The present invention also provides a PLD immobilized enzyme, which comprises the carrier and the PLD enzyme loaded thereon.

[0027] In the present invention, the PLD enzyme is a PLD mutant enzyme, which has an amino acid sequence having at least one mutation among T346V, K653L, S697Y, T346H, S697P, T346L, and K653A in the wild amino acid sequence shown in SEQ ID NO.1.

[0028] The present invention studies show that the innovative use of the carrier to immobilize the special mutant PLD mutant enzyme can obtain better adaptability, better immobilization mechanical strength and enzyme activity.

[0029] In the present invention, the loading amount of the PLD enzyme in the PLD-immobilized enzyme is 10 to 100 U / g, further 20 to 50 U / g.

[0030] In the present invention, the PLD immobilized enzyme can be prepared based on known means. For example, an optional method for preparing the PLD immobilized enzyme of the present invention is to activate the carrier and then mix it with the PLD enzyme for loading treatment.

[0031] Preferably, the activator used in the activation process is a dialdehyde compound, for example, glutaraldehyde. The amount of the activator can be adjusted reasonably as needed, for example, it can be 1-10% of the weight of the support before activation, and further can be 3-6%.

[0032] The present invention also provides an application of the PLD immobilized enzyme, which is used as an enzyme catalyst for synthesizing at least one phosphatidyl compound selected from phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol.

[0033] Beneficial effects

[0034] The present invention pre-compounds a natural polysaccharide material and silica in an emulsion manner, and then solidifies and modifies them using Formula 1. This can enhance the adaptability of the prepared carrier to the PLD enzyme, reduce the shielding problem of the active domain of the PLD enzyme during the solidification process, improve enzyme activity, and effectively improve the immobilization stability and mechanical strength.

[0035] The present invention innovatively adopts the carrier to immobilize the special PLD mutant enzyme, which can improve the physicochemical compatibility between the carrier and the mutant enzyme and synergistically enhance the mechanical strength, activity and stability of the immobilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a physical picture of the carrier prepared in Example 1;

[0037] Figure 2 This is a micrograph of the carrier prepared in Example 1. DETAILED DESCRIPTION

[0038] A more specific method for preparing a carrier of the present invention mainly comprises the following steps:

[0039] Step 1: Preparation of chitosan emulsion: chitosan is added to acetic acid solution and stirred thoroughly until the chitosan is completely dissolved to form a chitosan-acetic acid solution; then, an oil phase is added to obtain a polysaccharide emulsion;

[0040] Step 2: Based on step 1, a certain amount of silicon dioxide is added and the mixture is thoroughly stirred to obtain a chitosan-silicon dioxide emulsion;

[0041] Step 3: Based on step 2, a certain amount of formula 1 is added and stirred thoroughly to obtain a silica-chitosan-formula 1 emulsion.

[0042] Step 4: The obtained chitosan-silica-Formula 1 emulsion is fully stirred, and the fully dissolved chitosan-silica-Formula 1 emulsion is pumped into the NaOH solution at a certain flow rate using a peristaltic pump device, and then filtered to obtain chitosan composite microspheres after standing.

[0043] In the present invention, the aqueous phase is a solution formed by mixing a natural polysaccharide material and an acid aqueous solution; wherein the concentration of the natural polysaccharide material is 1 to 3% (w / v);

[0044] The oil phase is a hydrophobic solution in which at least one emulsifier selected from the group consisting of Span 80, Tween 80, sucrose fatty acid ester, and sorbitan oleate is dissolved;

[0045] In the present invention, the weight ratio of the natural polysaccharide material to silicon dioxide is 10 to 50:1.

[0046] In the present invention, in Formula 1, R2 is H or an ester group. The ester group is, for example,

[0047]

[0048] There are no particular requirements for the configuration and conformation of Formula 1. For example, it can be at least one of Formula 1A and Formula 1B.

[0049] In the present invention, the formula 1 can be a formula 1A and a formula 1B in a weight ratio of 0.5 to 2:1. Studies have shown that the preferred formula 1 can further optimize the carrier structure and help further improve the enzyme immobilization effect and activity.

[0050] The weight ratio of the formula 1 to the natural polysaccharide material is 1:10-70;

[0051] The concentration of NaOH during the cross-linking modification process is 0.5-2.0 mol / L.

[0052] In order to achieve effective immobilization of phospholipase D, the technical solution adopted by the present invention is as follows: activating the prepared chitosan microsphere carrier and then covalently binding it to the enzyme protein;

[0053] The activation reagent selected in the present invention is glutaraldehyde, and its dosage can be 1-10% of the weight of the carrier before activation, and the activation time is 12-16 hours;

[0054] In the present invention, the phospholipase D protein is effectively immobilized, and the dosage ratio of the enzyme activity to the carrier weight is 10 to 100 U / g, further 20 to 50 U / g.

[0055] In order to achieve efficient multi-batch synthesis of phosphatidylserine, the technical solution adopted by the present invention is:

[0056] Phosphatidylserine is synthesized in a fully aqueous phase using the immobilized phospholipase D on the chitosan composite carrier. The synthesis conditions can be reasonably adjusted according to conventional needs. For example, as an optional scheme, the following can be specifically employed:

[0057] The substrates are soybean lecithin and serine (the mass ratio can be 1:0.8-1.2), anhydrous CaCl2 (which is 0.05-0.2 times the weight of soybean lecithin; further can be 0.1-0.15 times), the reaction temperature is 30°C-50°C, the reaction pH is 4.5-5.5, and the reaction time is 3-6h.

[0058] The present invention innovatively discovered that the use of composite microspheres can effectively enhance the mechanical strength of the chitosan carrier, significantly increasing the loading capacity and enzyme activity recovery rate of phospholipase D. The reaction under fully aqueous conditions can effectively increase the enzyme batch size, reduce costs, and be more suitable for industrial-scale production applications.

[0059] The PLD enzyme is the wild-type amino acid sequence shown in SEQ ID NO. 1, or a PLD mutant enzyme containing at least one of the following mutations: T346V, K653L, S697Y, T346H, S697P, T346L, or K653A. The mutant enzyme can be prepared using conventional methods (e.g., first cloning the wild-type PLD enzyme gene into the expression vector pET30a to obtain a recombinant expression vector and a recombinant Escherichia coli strain containing the PLD gene; designing mutagenesis primers (containing the target mutation site) and introducing the mutation using PCR (e.g., whole-plasmid PCR); transforming the mutant plasmid into host cells, E. coli BL21 (DE3); screening positive clones using the kanamycin resistance marker; verifying the mutation accuracy by sequencing; and performing subsequent mutant enzyme verification analysis). The mutant enzyme can be obtained using known methods, for example, see 2025106384287 for details.

[0060] In the present invention, as an optional solution, the PLD enzyme can specifically be selected from the wild amino acid sequences shown in SEQ ID NO.1 to NO.7.

[0061] Table 1 PLD enzymes and their corresponding sequences

[0062] Mutant name Corresponding amino acid sequence Wild type WT SEQ ID NO.1 SrPLD-T346V SEQ ID NO.2 SrPLD-K653L SEQ ID NO.3 SrPLD-S697Y SEQ ID NO.4 SrPLD-K653L-T346H SEQ ID NO.5 SrPLD-K653L-S697P SEQ ID NO.6 SrPLD-K653L-T346H-S697P SEQ ID NO.7

[0063] In the present invention, the T346V refers to the mutation of T at position 346 of the wild amino acid sequence shown in SEQ ID NO.1 to V; the K653L refers to the mutation of K at position 653 of the wild amino acid sequence to L; the S697Y refers to the mutation of S at position 697 of the wild amino acid sequence to Y; the T346H refers to the mutation of T at position 346 of the wild amino acid sequence to H; the S697P refers to the mutation of S at position 697 of the wild amino acid sequence to P; the T346L refers to the mutation of T at position 346 of the wild amino acid sequence shown in SEQ ID NO.1 to L; and the K653A refers to the mutation of K at position 653 of the wild amino acid sequence to A.

[0064] Example 1: Preparation of modified chitosan microspheres

[0065] Chitosan-silica-Formula 1 composite microspheres:

[0066] Step 1: Weigh 2 g of chitosan and add it to 100 mL of 1% acetic acid solution. Stir thoroughly until the chitosan is completely dissolved to form a 2% (w / v) chitosan-acetic acid solution. Filter to remove undissolved particles. Set the temperature to 40°C and slowly add Span 80 solution (the weight ratio of chitosan to Span 80 is 30±1:1) to the system. Stir thoroughly to form a chitosan emulsion.

[0067] Step 2: Add silica to the chitosan emulsion (chitosan / silicon dioxide weight ratio is 20:1), and stir thoroughly to obtain a chitosan-silicon dioxide emulsion;

[0068] Step 3: Add Formula 1 (specifically Formula 1A in this case, whose structure is The weight ratio of chitosan to Formula 1 was 20:1); after each group was added, the mixture was thoroughly stirred and mixed at 40° C. to obtain an emulsion of chitosan-silica-Formula 1;

[0069] Step 4:

[0070] The fully dissolved chitosan-silica-Formula 1 emulsion was pumped into the NaOH solution at a certain flow rate using a peristaltic pump device, the pH of the system was controlled to 8±0.5, and stirred at this pH for 24 hours. After standing, the chitosan microspheres were filtered and used for later use.

[0071] Step 5: Slowly add 25% glutaraldehyde solution (wherein the glutaraldehyde is 5% by weight of the chitosan carrier) to the beaker containing the chitosan carrier. Stir at 150 rpm / min for 14 ± 1 h to activate the chitosan carrier. After activation, filter the resulting product, wash it repeatedly with water, and set aside.

[0072] Comparative Example 1

[0073] Compared with Example 1, the only difference is that steps 2 and 3 are not performed, and the chitosan solution is directly solidified and activated. The difference is that the chitosan-silica emulsion in step 1 is directly subjected to the alkaline cross-linking reaction in step 4. The other operations and parameters are the same as those in Example 1 (that is, silica and Formula 1 are not added to the carrier).

[0074] Comparative Example 2

[0075] Compared with Example 1, the only difference is that step 3 is not performed, and the chitosan-silica solution in step 2 is directly subjected to the alkali crosslinking and curing treatment in step 4. Other operations and parameters are the same as those in Example 1 (that is, Formula 1 is not added to the carrier).

[0076] Comparative Example 3

[0077] Compared with Example 1, the only difference is that step 2 is not performed, and the solution of step 1 is directly subjected to step 3 and subsequent treatments. The other operations and parameters are the same as those of Example 1 (that is, Formula 1 is not added to the carrier) (that is, silica is not added to the carrier).

[0078] Example 2

[0079] Compared with Example 1, the only difference is that in step 1, the amount of silicon dioxide is changed. The experimental groups are:

[0080] Group A: chitosan / silica weight ratio of 10:1;

[0081] Group B: chitosan / silica weight ratio 40:1;

[0082] Other operations and parameters are the same as in Example 1.

[0083] Example 3

[0084] Compared with Example 1, the only difference is that the addition amount of Formula 1A in step 3 is changed. The experimental groups are:

[0085] Group A: chitosan / Formula 1 weight ratio was 10:1;

[0086] Group B: The weight ratio of chitosan to Formula 1 was 50:1.

[0087] Other operations and parameters are the same as in Example 1.

[0088] Example 4

[0089] Compared with Example 1, the only difference is that in step 3, the components of Formula 1 are changed. The experimental groups are:

[0090] Group A: The components of Formula 1 are replaced with Formula 1B, whose structure is:

[0091] Group B: The components of Formula 1 are Formula 1A and Formula 1B in a weight ratio of 1:1, and the total amount of Formula 1 is the same as in Example 1;

[0092] Other operations and parameters are the same as in Example 1.

[0093] Mechanical strength determination of chitosan microspheres

[0094] The mechanical strength of the chitosan microspheres of the above different cases was measured. The specific method was as follows: 50 g of the carrier finally prepared in each of the above cases was taken, 200 mL of water was added, the stirring speed was turned on at 500 rpm / min, and the stirring was continued for 10 hours. The turbidity of the clear liquid was measured with a turbidity meter.

[0095] Example 5: Immobilization of Phospholipase D on Different Chitosan Carriers

[0096] The PLD enzyme used in this case has the wild amino acid sequence shown in SEQ ID NO.1.

[0097] Weigh 20 g of chitosan microsphere carriers from different cases and add them to a pH 8.0 phosphate buffer solution. Add the liquid enzyme at a dosage of 50 U / g (enzyme activity / carrier weight) to the carrier solution. Stir at 120 rpm for 24 hours at 25°C to obtain immobilized phospholipase D enzyme. The immobilized phospholipase D enzyme was then washed 5–8 times with 25 mM pH 8.0 phosphate buffer, drained, and refrigerated. The activity and recovery of the immobilized phospholipase D enzyme were measured.

[0098] Table 2: Enzyme activity and enzyme activity recovery of phospholipase D immobilized on different types of chitosan carriers

[0099]

[0100] As can be seen from Table 2, the strength of the carrier according to the method of the present invention is 70-100 NTU, which has good strength. In addition, it can also obtain better immobilized enzyme activity.

[0101] Example 6

[0102] Compared with the immobilization method of Example 5 (the carrier is the carrier of Example 1), the only difference is that the type of PLD enzyme is changed. Specifically, SEQ ID NO. 2 to NO. 7 are selected, and the structures respectively have the following mutations in the wild amino acid sequence shown in SEQ ID NO. 1:

[0103] SEQ ID NO. 2: T346V;

[0104] SEQ ID NO.3: K653L;

[0105] SEQ ID NO.4: S697Y;

[0106] SEQ ID NO.5: K653L-T346H;

[0107] SEQ ID NO.6: K653L-S697P;

[0108] SEQ ID NO.7: K653L-T346H-S697P;

[0109] Table 3: Immobilized enzyme activity and enzyme activity recovery rate of different phospholipase D mutants

[0110]

[0111] It can be seen from Examples 5 and 6 that the free mutant enzyme can obtain better enzyme activity than the wild enzyme, but the enzyme and the carrier need to have good adaptability. Studies have shown that the use of preferred mutant enzymes (such as SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.7; especially SEQ ID NO.5) has better immobilization adaptation advantages, and unexpectedly better immobilized enzyme activity and stability can be obtained.

[0112] Example 7: Synthesis of phosphatidylserine catalyzed by chitosan microspheres immobilized enzyme

[0113] The immobilized enzyme of Example 6 (wherein the amino acid sequence of the PLD enzyme is SEQ ID NO. 5) was subjected to the following cyclic enzyme catalytic synthesis, the steps being:

[0114] Substrate A: Weigh 48g of soybean lecithin (50%) and dissolve it in 160mL of 0.3M pH5.0 acetic acid-sodium acetate buffer with stirring until it is emulsified. Add 10g of immobilized enzyme. Substrate B: Weigh 50g of serine and 6g of anhydrous CaCl2 and dissolve them in 400mL of 0.3M pH5.0 acetic acid-sodium acetate buffer with stirring. Set the temperature to 40°C. Add substrate B to substrate A by feeding. Start stirring at 150rpm / min. Time the reaction for 4-5h and measure the yield of the product phosphatidylserine. After the reaction is completed, separate the immobilized enzyme and repeat the second batch of reaction according to the above reaction system. The cycle effect is as follows:

[0115] Table 4: Synthesis of phosphatidylserine catalyzed by chitosan microsphere-immobilized PLD enzyme (amino acid sequence is SEQ ID NO.5)

[0116]

[0117] As can be seen from Table 4, the prepared chitosan microsphere immobilized enzyme can be used for the preparation of phosphatidylserine for at least 10 reactions, and the conversion reaction yield is >70%.

[0118] SEQ ID NO.1

[0119] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVA

[0120] ATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI

[0121] AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFT

[0122] WPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADT

[0123] DYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVG

[0124] VLSAGINAASPNKELAKEFLENYLDEGLEAVNKDKPLGAVALKSYEEELAK

[0125] DPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQ

[0126] TGSGGSGASPTPHLDSVEQTLRQVSPGLEGSVWERTAGNSLGASAPGGSDWL

[0127] LQTPGCWGDPSCTDRPGSRRLLDKTRQDIAQARQSVDISTLAPFPNGGFQDAV

[0128] VAGLKEAVAKGNRLQVRILVGAAPIYHANVIPSSYRDEMVARLGPAAANVTL

[0129] NVASMTTSKTGFSWNHSKLVVVDGGSVITGGINSWKDDYLDTAHPVNDVDL

[0130] ALGSPAAGSAGRYLDTLWDWTCRNKSSWSSVWFASSNNAGCMPTLPPRPAAP

[0131] AGGGDVPALAVGGLGVGIRQSDPASAFKVPLPTAPDTKCGIGVHDNTNADRD

[0132] YDTVNPEESALRALVASANSHVEISQQDLNATCPPPRYDIRLYDTLAAKLAA

[0133] GVKVRIVVSDPANRGAVGSDGYSQIKSLNEVSDALRGRLTALTGDERTSKAA

[0134] MCQNLQLATFRASDKATWADGKPYAQHHKLVSVDDSAFYIGSKNLYPSWLQ

[0135] DFGYVVESPAAANQLKDSLLAPQWKYSQATATYDYARGLCQA

[0136] SEQ ID NO.2

[0137] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRVAVINAASGRQTVDEALKDAQTGSGGSGASPTPHLDSVEQTLRQVSPGLEGSVWERTAGNSLGASAPGGSDWLLQTPGCWGDPSCTDRPGSRRLLDKTRQDIAQARQSVDISTLAPFPNGGFQDAVVAGLKEAVAKGNRLQVRILVGAAPIYHANVIPSSYRDEMVARLGPAAANVTLNVASMTTSKTGFSWNHSKLVVVDGGSVITGGINSWKDDYLDTAHPVNDVDLALSGPAAGSAGRYLDTLWDWTCRNKSSWSSVWFASSNNAGCMPTLPRPAAPAGGGDVPALAVGGLGVGIRQSDPASAFKPVLPTAPDTKCGIGVHDNTNADRDYDTVNPEESALRALVASANSHVEISQQDLNATCPPLPRYDIRLYDTLAAKLAAGVKVRIVVSDPANRGAVGSDGYSQIKSLNEVSDALRGRLTALTGDERTSKAAMCQNLQLATFRASDKATWADGKPYAQHHKLVSVDDSAFYIGSKNLYPSWLQDFGYVVESPAAANQLKDSLLAPQWKYSQATATYDYARGLCQA

[0138] SEQ ID NO.3

[0139] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGSGGSGASPTPHLDSVEQTLRQVSPGLEGSVWERTAGNSLGASAPGGSDWLLQTPGCWGDPSCTDRPGSRRLLDKTRQDIAQARQSVDISTLAPFPNGGFQDAVVAGLKEAVAKGNRLQVRILVGAAPIYHANVIPSSYRDEMVARLGPAAANVTLNVASMTTSKTGFSWNHSKLVVVDGGSVITGGINSWKDDYLDTAHPVNDVDLALSGPAAGSAGRYLDTLWDWTCRNKSSWSSVWFASSNNAGCMPTLPRPAAPAGGGDVPALAVGGLGVGIRQSDPASAFLPVLPTAPDTKCGIGVHDNTNADRDYDTVNPEESALRALVASANSHVEISQQDLNATCPPLPRYDIRLYDTLAAKLAAGVKVRIVVSDPANRGAVGSDGYSQIKSLNEVSDALRGRLTALTGDERTSKAAMCQNLQLATFRASDKATWADGKPYAQHHKLVSVDDSAFYIGSKNLYPSWLQDFGYVVESPAAANQLKDSLLAPQWKYSQATATYDYARGLCQA

[0140] SEQ ID NO.4

[0141] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGSGGSGASPTPHLDSVEQTLRQVSPGLEGSVWERTAGNSLGASAPGGSDWLLQTPGCWGDPSCTDRPGSRRLLDKTRQDIAQARQSVDISTLAPFPNGGFQDAVVAGLKEAVAKGNRLQVRILVGAAPIYHANVIPSSYRDEMVARLGPAAANVTLNVASMTTSKTGFSWNHSKLVVVDGGSVITGGINSWKDDYLDTAHPVNDVDLALSGPAAGSAGRYLDTLWDWTCRNKSSWSSVWFASSNNAGCMPTLPRPAAPAGGGDVPALAVGGLGVGIRQSDPASAFKPVLPTAPDTKCGIGVHDNTNADRDYDTVNPEESALRALVASANYHVEISQQDLNATCPPLPRYDIRLYDTLAAKLAAGVKVRIVVSDPANRGAVGSDGYSQIKSLNEVSDALRGRLTALTGDERTSKAAMCQNLQLATFRASDKATWADGKPYAQHHKLVSVDDSAFYIGSKNLYPSWLQDFGYVVESPAAANQLKDSLLAPQWKYSQATATYDYARGLCQA

[0142] SEQ ID NO.5

[0143] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRHAVINAASGRQTVDEALKDAQTGSGGSGASPTPHLDSVEQTLRQVSPGLEGSVWERTAGNSLGASAPGGSDWLLQTPGCWGDPSCTDRPGSRRLLDKTRQDIAQARQSVDISTLAPFPNGGFQDAVVAGLKEAVAKGNRLQVRILVGAAPIYHANVIPSSYRDEMVARLGPAAANVTLNVASMTTSKTGFSWNHSKLVVVDGGSVITGGINSWKDDYLDTAHPVNDVDLALSGPAAGSAGRYLDTLWDWTCRNKSSWSSVWFASSNNAGCMPTLPRPAAPAGGGDVPALAVGGLGVGIRQSDPASAFLPVLPTAPDTKCGIGVHDNTNADRDYDTVNPEESALRALVASANSHVEISQQDLNATCPPLPRYDIRLYDTLAAKLAAGVKVRIVVSDPANRGAVGSDGYSQIKSLNEVSDALRGRLTALTGDERTSKAAMCQNLQLATFRASDKATWADGKPYAQHHKLVSVDDSAFYIGSKNLYPSWLQDFGYVVESPAAANQLKDSLLAPQWKYSQATATYDYARGLCQA

[0144] SEQ ID NO.6

[0145] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGSGGSGASPTPHLDSVEQTLRQVSPGLEGSVWERTAGNSLGASAPGGSDWLLQTPGCWGDPSCTDRPGSRRLLDKTRQDIAQARQSVDISTLAPFPNGGFQDAVVAGLKEAVAKGNRLQVRILVGAAPIYHANVIPSSYRDEMVARLGPAAANVTLNVASMTTSKTGFSWNHSKLVVVDGGSVITGGINSWKDDYLDTAHPVNDVDLALSGPAAGSAGRYLDTLWDWTCRNKSSWSSVWFASSNNAGCMPTLPRPAAPAGGGDVPALAVGGLGVGIRQSDPASAFLPVLPTAPDTKCGIGVHDNTNADRDYDTVNPEESALRALVASANPHVEISQQDLNATCPPLPRYDIRLYDTLAAKLAAGVKVRIVVSDPANRGAVGSDGYSQIKSLNEVSDALRGRLTALTGDERTSKAAMCQNLQLATFRASDKATWADGKPYAQHHKLVSVDDSAFYIGSKNLYPSWLQDFGYVVESPAAANQLKDSLLAPQWKYSQATATYDYARGLCQA

[0146] SEQ ID NO.7

[0147] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRHAVINAASGRQTVDEALKDAQTGSGGSGASPTPHLDSVEQTLRQVSPGLEGSVWERTAGNSLGASAPGGSDWLLQTPGCWGDPSCTDRPGSRRLLDKTRQDIAQARQSVDISTLAPFPNGGFQDAVVAGLKEAVAKGNRLQVRILVGAAPIYHANVIPSSYRDEMVARLGPAAANVTLNVASMTTSKTGFSWNHSKLVVVDGGSVITGGINSWKDDYLDTAHPVNDVDLALSGPAAGSAGRYLDTLWDWTCRNKSSWSSVWFASSNNAGCMPTLPRPAAPAGGGDVPALAVGGLGVGIRQSDPASAFLPVLPTAPDTKCGIGVHDNTNADRDYDTVNPEESALRALVASANPHVEISQQDLNATCPPLPRYDIRLYDTLAAKLAAGVKVRIVVSDPANRGAVGSDGYSQIKSLNEVSDALRGRLTALTGDERTSKAAMCQNLQLATFRASDKATWADGKPYAQHHKLVSVDDSAFYIGSKNLYPSWLQDFGYVVESPAAANQLKDSLLAPQWKYSQATATYDYARGLCQA。

Claims

1. A method for preparing a carrier for PLD immobilized enzyme, characterized in that: The natural polysaccharide material is dissolved in advance to form an aqueous phase, which is then mixed with an oil phase to obtain a polysaccharide emulsion; silicon dioxide is then added and mixed to obtain a silicon dioxide-polysaccharide emulsion; a modifier of formula 1 is then added, mixed, and cross-linked and modified under alkaline conditions to obtain the product; The R1 is hydrogen, hydroxyl, or alkoxy; and the R2 is H or an ester group.

2. The method for preparing a PLD enzyme-immobilized carrier according to claim 1, wherein: The natural polysaccharide material includes at least one of agarose, dextran and chitosan; The aqueous phase is a solution formed by mixing a natural polysaccharide material and an acid aqueous solution; wherein the concentration of the natural polysaccharide material is 1 to 3% (w / v); Preferably, the oil phase is a solution comprising at least one of Span 80, Tween 80, sucrose fatty acid ester, and sorbitan oleate; Preferably, the weight ratio of the oil phase to the natural polysaccharide material may be 1:10-50.

3. The method for preparing a carrier for PLD immobilized enzyme according to claim 1, wherein: The weight ratio of the natural polysaccharide material to silicon dioxide is 10 to 50:

1.

4. The method for preparing a PLD enzyme-immobilized carrier according to claim 1, wherein: In formula 1, R2 is H or an ester group; Preferably, the weight ratio of the formula 1 to the natural polysaccharide material is 1:10-70; Preferably, the pH during the cross-linking modification process is 7.0 to 9.

0.

5. A carrier prepared by the preparation method according to any one of claims 1 to 4.

6. A PLD immobilized enzyme, characterized in that The invention comprises the carrier according to claim 5 and the PLD enzyme loaded thereon.

7. The PLD immobilized enzyme according to claim 6, wherein The PLD enzyme is a PLD mutant enzyme, which has an amino acid sequence having at least one mutation among T346V, K653L, S697Y, T346H, S697P, T346L, and K653A in the wild amino acid sequence shown in SEQ ID NO.

1.

8. The PLD immobilized enzyme according to claim 6 or 7, wherein The loading amount of the PLD enzyme in the PLD-immobilized enzyme is 10 to 100 U / g, further 20 to 50 U / g.

9. A method for preparing the PLD immobilized enzyme according to any one of claims 6 to 8, characterized in that: The carrier is activated and then mixed with the PLD enzyme for loading treatment to obtain; Preferably, the activator used in the activation process is a dialdehyde compound.

10. Use of the PLD immobilized enzyme according to any one of claims 6 to 8, characterized in that: It is used as an enzyme catalyst for the synthesis of at least one phosphatidyl compound among phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylglycerol.

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