Enzyme composition and application thereof in preparation of sialic acid lactose

Abstract

The invention relates to the technical field of biology, in particular to an enzyme composition and application of the enzyme composition in preparation of sialic acid lactose. The enzyme composition is prepared from N-acetylglucosamine-2-epimerase, N-acetylneuraminic acid aldolase, CMP (Chemical Mechanical Polishing) kinase, polyphosphate kinase, CMP-sialic acid synthetase and sialic acid transferase. According to the method, SA is replaced by cheaper N-acetyl-glucosamine and sodium pyruvate, 1 / 2-2 / 3 of substrate cost is successfully saved, the enzyme composition is immobilized by using Ni < 2 + > chelating affinity adsorption type resin, and a preparation process for preparing sialic acid lactose, which is high in yield, good in repeatability, environment-friendly and economical, is successfully established in a semi-automatic column packing flow-through manner.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme engineering, in particular to an enzyme composition and application thereof in the preparation of sialyllactose. Background Art

[0002] Human milk oligosaccharides (HMO) are a general term for all oligosaccharides contained in human milk. They are also called human milk oligosaccharides. They are the second largest carbohydrate component and the third largest nutrient in breast milk after lactose, accounting for 10% of the dry matter of breast milk. Human milk oligosaccharides can be divided into neutral oligosaccharides and acidic oligosaccharides according to the fucose and sialic acid (SA) connected to the core. Sialactose (SL) is an acidic human milk oligosaccharide, which can be divided into 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) according to the position where sialic acid and lactose are combined. Studies have shown that SL has many beneficial effects on infants, including neutralizing toxins produced by intestinal bacteria, preventing bacteria or viruses from adhering to epithelial surfaces, and acting as a prebiotic.

[0003] At present, there are three methods for synthesizing sialyllactose: chemical synthesis, biological fermentation and enzyme catalysis. Due to the electronic hindrance and steric hindrance caused by the carboxyl group on sialic acid and the large number of side reactions, the chemical synthesis of SL has harsh conditions, complex preparation process, high purification cost and is difficult to industrialize. Through genetic engineering and metabolic engineering to regulate and transform engineered bacteria, SL can be synthesized by biological fermentation using SA, glucose and lactose as substrates, but there are problems such as a fermentation cycle of up to 24-90h and low yield, which is not conducive to industrial production.

[0004] Enzymatic synthesis is currently the main production route. Both sialyl esterase and sialyltransferase can be used to synthesize SL, but the sialyl esterase catalytic pathway has more byproducts, which brings great difficulties to purification. Therefore, sialyltransferase is often used in the enzymatic synthesis of SL. In this synthesis pathway, sialic acid must participate in the reaction in its active form, cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-SA). CMP-SA is expensive, but CMP-sialic acid synthetase can be used to catalyze the synthesis of CTP and SA. At present, Michel Gibert et al. have successfully used the liquid enzyme one-pot method to synthesize 3'-SL with SA, lactose, polyphosphate, MgCl2 and CMP as substrates (Nature, 16, (1998)); SA is a substrate with high cost and does not have the advantage of large-scale production.

[0005] Li Zhongkui and others successfully used magnetic nanoparticles to immobilize CMP kinase, polyphosphate kinase, CMP-sialic acid synthase and sialyltransferase, and synthesized 3'-SL and 6'-SL using CMP, polyphosphate, sialic acid and lactose as substrates. This method uses magnetic nanoparticles to immobilize enzymes and SA as a substrate to synthesize SL, but the magnetic nanoparticles used as immobilization carriers are expensive and difficult to recycle, making them unsuitable for industrial production. Summary of the invention

[0006] In view of this, the present invention provides an enzyme composition and an enzyme composition and its application in the preparation of sialyllactose. The present invention uses N-acetyl-glucosamine and sodium pyruvate to replace SA as substrates, and uses an immobilized enzyme composition to catalyze the synthesis of 3'-SL and 6'-SL. The synthesis of sialyllactose has greatly reduced costs, a stable conversion rate of 80-90%, and a high reusability of the immobilized enzyme column, and has a strong continuous production capacity.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] An enzyme composition comprising N-acetylglucosamine-2-epimerase AGE, N-acetylneuraminic acid aldolase NAL, CMP kinase, polyphosphate kinase PPK, CMP-sialic acid synthetase, and sialyltransferase;

[0009] The AGE is derived from Bacteroides fragilis;

[0010] The NAL is derived from Clostridium scindens;

[0011] The CMP kinase is derived from Pusillimonas sp.

[0012] The polyphosphate kinase PPK is derived from Sulfurovum lithotrophicum;

[0013] The CMP-sialic acid synthetase is derived from Idiomarina loihiensis;

[0014] The sialyltransferase includes 2,3-sialyltransferase and / or 2,6-sialyltransferase;

[0015] The 2,3-sialyltransferase is derived from Pasteurella multocida; the 2,6-sialyltransferase is derived from Vibrio alginolyticus.

[0016] In the present invention, the N-acetylglucosamine-2-epimerase (AGE) is derived from Bacteroides fragilis (Uniprot ID: Q5LEN7), and its amino acid sequence is shown in SEQ ID NO: 1;

[0017] The aldolase (NAL) is derived from Clostridium scindens (Uniprot ID: B0NDA1), and the enzyme is modified. The amino acid sequence of the modified NAL is shown in SEQ ID NO: 2;

[0018] The CMP kinase (Cytidylate kinase, CMK) is derived from Idiomarina loihiensis (Uniprot#: Q5QZ51), and its amino acid sequence is shown in SEQ ID NO: 3;

[0019] The polyphosphate kinase (PPK) is derived from Sulfurovum lithotrophicum (Uniprot#: A0A7U4M185), and its amino acid sequence is shown in SEQ ID NO: 4;

[0020] The CMP-Sialyl Synthetase (CMP-Neu5Ac Synthetase, CSS) is derived from Pusillimonassp.ANT_WB101 (Uniprot#A0A5A9Z217, and its amino acid sequence is shown in SEQ ID NO: 5;

[0021] The 2,3-sialyltransferase (2,3-ST) is derived from Pasteurella multocida (Uniprot#: Q9CP67), and its amino acid sequence is shown in SEQ ID NO: 6;

[0022] The 2,6-sialyltransferase (2,6-ST) is derived from Vibrio alginolyticus (Uniprot#: A0A7Y0N06), and its amino acid sequence is shown in SEQ ID NO:7.

[0023] In some embodiments, the enzyme activity ratio of N-acetylglucosamine-2-isomerase AGE, N-acetylneuraminic acid aldolase NAL, CMP kinase, polyphosphate kinase PPK, CMP-sialic acid synthetase, and 2,3-sialyltransferase is (126-350): (200-500): (50-120): (40-80): (185-490): (160-388); the enzyme activity ratio can specifically be 129: 247: 78: 60: 370: 309, 294: 386: 96: 71: 391: 284.

[0024] In some embodiments, the enzyme activity ratio of N-acetylglucosamine-2-isomerase AGE, N-acetylneuraminic acid aldolase NAL, CMP kinase, polyphosphate kinase PPK, CMP-sialic acid synthetase, and 2,6-sialyltransferase is (126-350):(200-500):(50-120):(40-80):(185-490):(120-360); the enzyme activity ratio can specifically be 310:405:88:56:423:310 or 348:482:75:51:399:286.

[0025] The present invention also provides a nucleic acid encoding the enzyme composition, comprising:

[0026] A nucleic acid encoding the AGE, a nucleic acid encoding the NAL, a nucleic acid encoding the CMP kinase, a nucleic acid encoding the polyphosphate kinase PPK, a nucleic acid encoding the CMP-sialic acid synthetase, and a nucleic acid encoding the sialyltransferase;

[0027] The nucleic acid encoding the sialyltransferase includes: a nucleic acid encoding the 2,3-sialyltransferase and / or a nucleic acid encoding the 2,6-sialyltransferase.

[0028] In the present invention, the nucleic acid sequences encoding each enzyme are:

[0029] The sequence of the nucleic acid encoding the AGE is shown in SEQ ID NO: 8;

[0030] The sequence of the nucleic acid encoding the NAL is shown in SEQ ID NO:9;

[0031] The sequence of the nucleic acid encoding the CMK is shown in SEQ ID NO: 10;

[0032] The sequence of the nucleic acid encoding the PPK is shown in SEQ ID NO: 11;

[0033] The sequence of the nucleic acid encoding the CMP-sialic acid synthetase is shown in SEQ ID NO: 12;

[0034] The sequence of the nucleic acid encoding the 2,3-ST is shown in SEQ ID NO: 13;

[0035] The sequence of the nucleic acid encoding the 2,6-ST is shown in SEQ ID NO:14.

[0036] The present invention also provides a plasmid vector comprising the nucleic acid of the present invention.

[0037] The present invention also provides a strain that expresses the enzyme composition of the present invention or is transformed with a plasmid vector containing the nucleic acid of the present invention. In some embodiments, the strain is Escherichia coli. In some specific embodiments, the Escherichia coli BL21 (DE3) strain (purchased from Universal Biotechnology).

[0038] The invention provides an enzyme composition, a nucleic acid or an application of the strain in producing sialyllactose.

[0039] The present invention provides a method for producing sialyllactose, comprising:

[0040] 1) fermenting the strain of the present invention, homogenizing, crushing, and centrifuging to obtain a mixed enzyme solution;

[0041] 2) adding the mixed enzyme solution or the enzyme composition according to any one of claims 1 to 4 to a resin column for immobilization to obtain an immobilized enzyme resin column;

[0042] 3) Adding the substrate reaction solution to the immobilized enzyme resin column described in step 2) to react and obtain the nucleic acid of 2,3-sialyltransferase and / or 2,6-sialyltransferase.

[0043] The substrate reaction solution includes water, N-acetyl-glucosamine, sodium pyruvate, lactose, polyphosphoric acid, MgCl2 and CMP.

[0044] In some embodiments, in step 1), the buffer used for homogenization is 0.02M sodium phosphate buffer with a pH of 8.0.

[0045] In some embodiments, in step 2), before adding the enzyme composition or the mixed enzyme solution, the resin column is also pretreated, and the pretreatment includes: balancing the resin column with 0.1M pH 8.0 sodium phosphate buffer, and then passing the resin column with 0.5 mol / L NiSO4 aqueous solution to chelate Ni. 2+ , control the flow rate to 0.5L / h, flow through 2.5L, after the flow through is completed, wash the resin with deionized water until no Ni is detected in the effluent 2+ Then, the pH inside the resin was neutralized with 0.02 M sodium phosphate buffer (pH 8.0).

[0046] In some embodiments, the concentration of each component in the substrate reaction solution is:

[0047] N-acetyl-glucosamine 0-400mM, sodium pyruvate 0-500mM, lactose 0-600mM, MgCl2 0-400mM, CMP 0-10mM, polyphosphate concentration in terms of phosphate is 0-600mM;

[0048] The concentrations of the above components are not 0.

[0049] In the present invention, the pH of the substrate reaction solution is 7.8 to 8.1, specifically 7.8, 7.9, 8.0, or 8.1. The concentration of each component in the substrate reaction solution is preferably:

[0050] N-acetyl-glucosamine 200-360mM, sodium pyruvate 249-490mM, lactose 240-480mM, MgCl2 300-400mM, CMP 4-10mM, polyphosphoric acid concentration calculated as phosphate is 400-580mM.

[0051] Specifically, the substrate reaction solution includes: 200mM N-acetyl-glucosamine, 250mM sodium pyruvate, 240mM lactose, 4mM CMP, 400mM polyphosphate (in terms of phosphate), 400mM MgCl2, and the balance is water, and the pH is adjusted to 8.0 with aqueous ammonia;

[0052] Or, comprising: 350 mM N-acetyl-glucosamine, 360 mM sodium pyruvate, 400 mM lactose, 6 mM CMP, 510 mM polyphosphoric acid (in terms of phosphate), 320 mM MgCl2, the balance being water, and the pH being adjusted to 8.1 with aqueous ammonia;

[0053] Or, comprising: 300mM N-acetyl-glucosamine, 370mM sodium pyruvate, 380mM lactose, 8mM CMP, 420mM polyphosphoric acid (in terms of phosphate), 350mM MgCl2, the balance being water, and the pH being adjusted to 7.8 with aqueous ammonia;

[0054] Or, comprising: 200 mM N-acetyl-glucosamine, 249 mM sodium pyruvate, 270 mM lactose, 5 mM CMP, 410 mM polyphosphate (in terms of phosphate), 320 mM MgCl2, the balance being water, and the pH being adjusted to 7.9 with aqueous ammonia;

[0055] Or, comprising: 320 mM N-acetyl-glucosamine, 380 mM sodium pyruvate, 410 mM lactose, 4 mM CMP, 500 mM polyphosphoric acid (in terms of phosphate), 350 mM MgCl2, the balance being water, and adjusting the pH to 8.0 with aqueous ammonia.

[0056] Or, comprising: 360 mM N-acetyl-glucosamine, 490 mM sodium pyruvate, 480 mM lactose, 10 mM CMP, 580 mM polyphosphoric acid (calculated as phosphate), 300 mM MgCl2, the balance being water, and adjusting the pH to 8.0 with aqueous ammonia.

[0057] In some embodiments, the resin column is Ni 2+ Chelating affinity adsorption resin, the Ni2+ chelating affinity adsorption resin includes LX-1000IDA resin and / or LX 604 resin.

[0058] The present invention aims to solve the problem of high substrate cost in the existing enzyme catalysis schemes of 3'-SL and 6'-SL, successfully combines the enzyme catalysis synthesis pathway of SA, replaces SA with cheaper N-acetyl-glucosamine and sodium pyruvate, and successfully saves 1 / 2-2 / 3 of the substrate cost. 2+ A 6-enzyme system including N-acetylglucosamine-2-epimerase, N-acetylneuraminic acid aldolase, CMP kinase, polyphosphate kinase, CMP-sialic acid synthetase and sialyltransferase was immobilized on a chelating affinity adsorption resin, and a process scheme for preparing sialyllactose with high yield, good repeatability, environmental protection and economy was successfully established in a semi-automatic column loading and flow-through manner. The present invention has the following beneficial effects:

[0059] ① The present invention uses N-acetyl-glucosamine and sodium pyruvate instead of SA as substrates, screens a 6-enzyme system including N-acetylglucosamine-2-epimerase, N-acetylneuraminic acid aldolase, CMP kinase, polyphosphate kinase, CMP-sialic acid synthetase and sialyltransferase, and successfully synthesizes 3'-SL and 6'-SL.

[0060] ②Use reusable Ni 2+ The chelating affinity adsorption resin LX 1000IDA successfully immobilized the above 6 enzyme systems and successfully catalyzed the synthesis of sialyllactose in a semi-automatic column flow-through manner.

[0061] ③ The immobilized enzyme column prepared by the enzyme combination of the present invention has a high reuse rate. When the substrate concentration is 0-400mM, the resin processing capacity is as high as 0.2L / g, the conversion rate is stable between 80-90%, and it has a strong continuous production capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The HPLC spectrum of the reaction solution before the reaction in Example 2 is shown;

[0063] Figure 2 HPLC spectrum of samples taken after flow-through in Example 2 is shown;

[0064] Figure 3 The figure shows the conversion rate of the immobilized enzyme column after 20 cycles in Example 3;

[0065] Figure 4 The protein gel results of the single enzyme, the mixed enzyme before immobilization, and the mixed enzyme fixed by resin in Example 4 and Example 6 are shown; wherein a is AGE, b is NAL, c is CSS, d is CMK, e is PPK, f is 2,3-ST, and g is 2,6-ST; H is the mixed enzyme before immobilization in Example 1, h is the mixed enzyme fixed by resin in Example 1, I is the mixed enzyme before immobilization in Example 6, and i is the mixed enzyme fixed by resin in Example 6;

[0066] Figure 5 The HPLC spectrum of the reaction solution before the reaction in Example 7 is shown;

[0067] Figure 6 HPLC profile of samples taken after flow-through in Example 7 is shown;

[0068] Figure 7 The figure shows the conversion rate of the immobilized enzyme column after 20 cycles in Example 8;

[0069] Figure 8 The HPLC spectrum of the sample taken after flow-through in Comparative Example 1 is shown;

[0070] Fig. 9 The HPLC spectrum of the sample taken after flow-through in Comparative Example 2 is shown;

[0071] Fig.10 The HPLC spectrum of the sample taken after flow-through in Comparative Example 3 is shown;

[0072] Fig.11 The HPLC spectrum of the sample taken after flow-through in Comparative Example 4 is shown. DETAILED DESCRIPTION

[0073] The present invention provides enzyme compositions and their application in the preparation of sialyllactose. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0074] Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

[0075] The present invention will be further described below in conjunction with embodiments:

[0076] Example 1

[0077] Preparation of immobilized 3'-SL synthesis system enzyme: 300g LX-1000IDA resin was loaded into the column, and the resin was balanced with 1.5L 0.1M pH 8.0 sodium phosphate buffer, and then 0.5mol / L NiSO4 aqueous solution was passed through the column to chelate Ni 2+ , the flow rate was controlled at 0.5 L / h, and a total of 2.5 L flowed through. After the flow through was completed, the resin was washed with deionized water until no Ni was detected in the effluent. 2+ After the resin was washed, 1L of 0.02M pH8.0 sodium phosphate buffer was used to neutralize the pH inside the resin. The fermented bacteria were homogenized and crushed with 0.02M pH8.0 sodium phosphate buffer and then centrifuged to obtain six relatively clear enzyme solutions, which were mixed with N-acetylglucosamine-2-isomerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthase: 2,3-sialyltransferase at an enzyme activity ratio of 430:800:230:210:900:850 to obtain a mixed enzyme solution, and then 1L of the enzyme solution was pumped into the resin column at a flow rate of 0.5L / h at room temperature for solidification. After immobilization, the resin was flushed with 0.02 M sodium phosphate buffer at pH 8.0 at a flow rate of 0.5 L / h until the protein detected in the effluent was less than 1 mg / L, and the immobilized enzyme activity ratio after fixation was N-acetylglucosamine-2-isomerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,3-sialyltransferase = 129:247:78:60:370:309.

[0078] Example 2

[0079] The immobilized enzyme resin column in Example 1 was prepared with a uniform reaction solution, wherein the reaction solution contained 200 mM N-acetyl-glucosamine, 250 mM sodium pyruvate, 240 mM lactose, 4 mM CMP, polyphosphoric acid (400 mM phosphate), 400 mM MgCl2, and the remainder was water. The pH was adjusted to 8.0 with aqueous ammonia, and the reaction solution was subjected to HPLC detection (see Figure 1 ). 3L of reaction solution was pumped into the immobilized enzyme column at room temperature (25°C) at a flow rate of 0.5L / h. 300g of immobilized enzyme made of resin can flow through 3L of reaction solution at one time, that is, one reaction can be completed in 6 hours. The total reaction solution was taken for HPLC detection (see Figure 2 ), 115.93 g / L (91.5% conversion rate) of 3'-SL reaction solution can be obtained.

[0080] Example 3

[0081] After washing the resin column in Example 2 with 1L water at a flow rate of 0.5L / h, 3L of reaction solution was pumped in again. The reaction system contained 350mM N-acetyl-glucosamine, 360mM sodium pyruvate, 400mM lactose, 6mM CMP, polyphosphoric acid (510mM phosphate), 320mM MgCl2, and the pH was adjusted to 8.1 with aqueous ammonia. The reaction solution was pumped into the immobilized enzyme column at a flow rate of 0.5L / h at 30°C, and the total reaction solution was taken for HPLC detection, and 198.46g / L (89.5% conversion rate) of 3'-SL reaction solution was obtained. After washing the immobilized enzyme with 1L water at a flow rate of 0.5L / h, 3L of reaction solution was pumped in again, and the operation was repeated 20 times. The conversion rate of 20 cycles was as follows: Figure 3 As shown, a flow-through liquid with a 3'-SL conversion rate of 81.2% can still be obtained in the 20th time, indicating that the immobilized enzyme flow-through reaction can be used for the production of sialyllactose and has the ability to reuse the biocatalyst.

[0082] Example 4

[0083] The immobilized enzyme used 20 times in Example 3 was rinsed with 1L 0.1M hydrochloric acid at a flow rate of 0.3L / h, and the resin was rinsed with water at 0.5L / h for 2h to wash away the acid therein to obtain a recovered resin. The preparatory steps of activating the immobilized resin and attaching nickel in Example 1 were repeated, and the fermented bacteria were homogenized and crushed with 0.02M pH 8.0 sodium phosphate buffer and then centrifuged to obtain 6 relatively clear enzyme solutions, which were mixed with an enzyme activity ratio of N-acetylglucosamine-2-isomerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,3-sialyltransferase = 612:1200:386:240:1200:961 to obtain a mixed enzyme solution (protein gel results are shown in Figure 4 ), and then pump 1L of enzyme solution into the resin column at a flow rate of 0.5L / h at room temperature for immobilization. After the immobilization is completed, the resin is washed with 0.02M pH8.0 sodium phosphate buffer at a flow rate of 0.5L / h until the protein detected in the effluent is less than 1mg / L, and the immobilized enzyme is obtained (protein gel results are shown in Figure 4 ), the enzyme activity ratio is N-acetylglucosamine-2-epimerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,3-sialyltransferase = 294: 386: 96: 71: 391: 284,

[0084] Example 5

[0085] Take the immobilized enzyme resin column in Example 4, configure a uniform reaction system, the reaction system contains 300mM N-acetyl-glucosamine, 370mM sodium pyruvate, 380mM lactose, 8mM CMP, polyphosphoric acid (420mM phosphate), 350mM MgCl2, and adjust the pH to 7.8 with ammonia water. The reaction solution is pumped into the immobilized enzyme column at a flow rate of 0.5L / h at room temperature. The immobilized enzyme made of 300g resin can flow through 3L of reaction solution once, that is, a reaction can be completed in 6h. The total reaction solution that flows out is taken for HPLC detection, and 181.32g / L (95.4% conversion rate) of 3'-SL reaction solution can be obtained.

[0086] After washing the immobilized enzyme with 1L water at a flow rate of 0.5L / h, 3L of reaction solution was pumped in again, and the operation was repeated 20 times. At the 20th time, a flow-through liquid with a 3'-SL conversion rate of 83.2% was obtained. This shows that the immobilized resin can be recycled repeatedly and the process has good sustainable production capabilities.

[0087] Example 6

[0088] Preparation of immobilized 6'-SL synthesis system enzymes: The immobilized resin was prepared in the same manner as in Example 1. The fermented bacteria were homogenized and broken with 0.02M sodium phosphate buffer at pH 8.0 and then centrifuged to obtain relatively clear 6 enzyme solutions, which were mixed at an enzyme activity ratio of N-acetylglucosamine-2-epimerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthase: 2,6-sialyltransferase = 860:1650:304:180:1533:1190 to obtain a mixed enzyme solution (protein gel results see Figure 4 ), then pump 1 L of enzyme solution into the resin column at a flow rate of 0.5 L / h at room temperature for immobilization. After immobilization is completed, the resin is washed with 0.02 M pH 8.0 sodium phosphate buffer at a flow rate of 0.5 L / h until the protein detected in the effluent is less than 1 mg / L, and the immobilized enzyme is obtained (protein gel results are shown in Figure 4 ), the enzyme activity ratio is N-acetylglucosamine-2-isomerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,6-sialyltransferase = 310:405:88:56:423:310.

[0089] Example 7

[0090] Take the immobilized enzyme resin column in Example 6, configure a uniform reaction system, the reaction system contains 200mM N-acetyl-glucosamine, 249mM sodium pyruvate, 270mM lactose, 5mM CMP, polyphosphoric acid (410mM phosphate), 320mM MgCl2, adjust the pH to 7.9 with ammonia water, and perform HPLC detection on the reaction solution (see Figure 5 The reaction solution was pumped into the immobilized enzyme column at a flow rate of 0.7 L / h. 300 g of the immobilized enzyme made of resin could flow through 4.2 L of reaction solution at one time, that is, one reaction could be completed in 6 h. The total reaction solution was taken for HPLC detection (see Figure 6 ), a 6'-SL reaction solution of 121.76 g / L (96.1% conversion rate) can be obtained.

[0091] Example 8

[0092] After washing the resin column in Example 7 with 1L water at a flow rate of 0.5L / h, 3.5L of reaction solution was pumped in again. The reaction system contained 320mM N-acetyl-glucosamine, 380mM sodium pyruvate, 410mM lactose, 4mM CMP, polyphosphoric acid (500mM phosphate), 350mM MgCl2, and the pH was adjusted to 8.0 with aqueous ammonia. The reaction solution was pumped into the immobilized enzyme column at a flow rate of 0.5L / h at 30°C, and the total reaction solution was taken for HPLC detection, and 182.06g / L (89.8% conversion rate) of 6'-SL reaction solution was obtained. After washing the immobilized enzyme with 1L water at a flow rate of 0.5L / h, 3L of reaction solution was pumped in again, and the operation was repeated 20 times. The conversion rate of 20 cycles was as follows: Figure 7 As shown, a flow-through liquid with a 6'-SL conversion rate of 85.7% can still be obtained in the 20th time, indicating that the immobilized enzyme flow-through reaction can be used for the production of sialyllactose and has the ability to reuse the biocatalyst.

[0093] Example 9

[0094] The immobilized enzyme used 20 times in Example 8 was rinsed with 1L 0.1M hydrochloric acid at a flow rate of 0.3L / h, and then the resin was rinsed with water at 0.5L / h for 1.5h to wash away the acid therein, and the recovered resin was obtained. The preparatory steps of immobilized resin activation and nickel attachment in Example 1 were repeated, and the fermented bacteria were homogenized and crushed with 0.02M pH 8.0 sodium phosphate buffer and then centrifuged to obtain 6 relatively clear enzyme solutions, which were mixed with an enzyme activity ratio of N-acetylglucosamine-2-isomerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,6-sialyltransferase = 1100: 1630: 245: 190: 1360: 1050 to obtain a mixed enzyme solution, and then 1L of the enzyme solution was pumped into the resin column at a flow rate of 0.5L / h at room temperature for 1 hour. Immobilization. After the immobilization is completed, the resin is washed with 0.02M sodium phosphate buffer at pH 8.0 at a flow rate of 0.5 L / h until the protein detected in the effluent is less than 1 mg / L. The immobilized enzyme activity ratio after fixation is N-acetylglucosamine-2-isomerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,6-sialyltransferase = 348:482:75:51:399:286.

[0095] Example 10

[0096] Take the immobilized enzyme resin column in Example 9, and the conversion system contains 360mMN-acetyl-glucosamine, 490mM sodium pyruvate, 480mM lactose, 10mM CMP, polyphosphoric acid (580mM phosphate), 300mM MgCl2, and adjust pH to 8.0 with ammonia. The reaction solution is pumped into the immobilized enzyme column with a flow rate of 0.7L / h. The immobilized enzyme made by 300g resin can flow through 4.2L reaction solution once, that is, 6h can end a reaction, and the outflowing total reaction solution is taken for HPLC detection, and 212.34g / L (93.1% conversion rate) of 6'-SL reaction solution can be obtained. After cleaning the immobilized enzyme with 1L water 0.5L / h flow rate, 4.2L reaction solution is pumped in again, and this operation is repeated 20 times. The flow-through liquid with a 6'-SL conversion rate of 83.9% can still be obtained for the 20th time. This indicates that the immobilized resin can be recycled repeatedly, and the process also has good sustainable production capacity in the production of 6'-SL.

[0097] Comparative Example 1

[0098] The immobilized resin was prepared in the same manner as in Example 1, and the enzyme activity ratio of the flow-through mixed enzyme solution was changed to N-acetylglucosamine-2-epimerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,3-sialyltransferase = 300:400:304:109:1533:1190. After the flow-through immobilization treatment as shown in Example 1, the enzyme activity ratio of the immobilized enzymes obtained after immobilization was N-acetylglucosamine-2-epimerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,3-sialyltransferase = 50:125:92:29:431:308.

[0099] The reaction solution was prepared in the same manner as in Example 2, and the total reaction solution was taken for HPLC detection. The results are shown in Figure 8 , 71.33 g / L (56.3% conversion rate) of 3'-SL reaction solution can be obtained.

[0100] Comparative Example 2

[0101] The immobilized resin was prepared by the same method as in Example 1, and the enzyme activity ratio of the flow-through mixed enzyme solution was changed to N-acetylglucosamine-2-epimerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,6-sialyltransferase = 610:950:129:290:526:466. After the flow-through immobilization treatment as shown in the example, the immobilized enzyme activity ratio after immobilization was N-acetylglucosamine-2-epimerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,6-sialyltransferase = 211:349:32:64:133:98.

[0102] The reaction solution was prepared in the same manner as in Example 7 and the total reaction solution was taken for HPLC detection. The results are shown in Fig. 9 , a 6'-SL reaction solution of 79.19 g / L (62.5% conversion rate) can be obtained.

[0103] Comparative Example 3

[0104] After activation with epoxy resin 1000EP, the mixed enzyme solution was immobilized by column immobilization using the immobilization method shown in Example 1, and the immobilized enzyme activity ratio after immobilization was N-acetylglucosamine-2-epimerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,3-sialyltransferase = 210:230:51:24:160:110.

[0105] A uniform reaction system was prepared for reaction. The reaction system contained 200 mM N-acetyl-glucosamine, 251 mM sodium pyruvate, 260 mM lactose, 5 mM CMP, polyphosphoric acid (290 mM phosphate), and 310 mM MgCl2. The pH was adjusted to 7.9 with aqueous ammonia. The reaction solution was pumped into the immobilized enzyme column at a flow rate of 0.7 L / h, and 3.2 L of the reaction solution was flowed through. The total reaction solution was taken for HPLC detection. The results are shown in Fig.10 , 86.02 g / L (67.9% conversion rate) of 3'-SL reaction solution can be obtained. After 5 cycles, the conversion rate dropped below 50%.

[0106] Comparative Example 4

[0107] After activation with epoxy resin 1000HA, the mixed enzyme solution was immobilized by column immobilization using the immobilization method shown in Example 6, and the immobilized enzyme activity ratio after immobilization was N-acetylglucosamine-2-isomerase: N-acetylneuraminic acid aldolase: CMP kinase: polyphosphate kinase: CMP-sialic acid synthetase: 2,6-sialyltransferase = 190:210:46:24:155:108.

[0108] A uniform reaction system was prepared for reaction. The reaction system contained 200 mM N-acetyl-glucosamine, 251 mM sodium pyruvate, 260 mM lactose, 5 mM CMP, polyphosphoric acid (290 mM phosphate), and 310 mM MgCl2. The pH was adjusted to 7.9 with aqueous ammonia. The reaction solution was pumped into the immobilized enzyme column at a flow rate of 0.7 L / h, and 3.4 L of the reaction solution was flowed through. The total reaction solution was taken for HPLC detection. The results are shown in Fig.11 , 76.41 g / L (60.3% conversion rate) of 6'-SL reaction solution can be obtained. After 4 cycles, the conversion rate dropped below 50%.

[0109] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An enzyme composition, characterized in that Including N-acetylglucosamine-2-epimerase AGE, N-acetylneuraminic acid aldolase NAL, CMP kinase, polyphosphate kinase PPK, CMP-sialic acid synthetase, sialyltransferase; The AGE is derived from Bacteroides fragilis; The NAL is derived from Clostridium scindens; The CMP kinase is derived from Pusillimonassp; The polyphosphate kinase PPK is derived from Sulfurovum lithotrophicum; The CMP-sialic acid synthetase is derived from Idiomarina loihiensis; The sialyltransferase includes 2,3-sialyltransferase and / or 2,6-sialyltransferase; The 2,3-sialyltransferase is derived from Pasteurella multocida; The 2,6-sialyltransferase is derived from Vibrio alginolyticus.

2. The enzyme composition according to claim 1, characterized in that The amino acid sequence of the AGE is shown in SEQ ID NO: 1; The amino acid sequence of NAL is shown in SEQ ID NO: 2; The amino acid sequence of the CMP kinase is shown in SEQ ID NO: 3; The amino acid sequence of the polyphosphate kinase PPK is shown in SEQ ID NO: 4; The amino acid sequence of the CMP-sialic acid synthetase is shown in SEQ ID NO: 5; The amino acid sequence of the 2,3-sialyltransferase is shown in SEQ ID NO:6; The amino acid sequence of the 2,6-sialyltransferase is shown in SEQ ID NO:

7.

3. The enzyme composition according to claim 1, characterized in that The enzyme activity ratios of N-acetylglucosamine-2-isomerase AGE, N-acetylneuraminic acid aldolase NAL, CMP kinase, polyphosphate kinase PPK, CMP-sialic acid synthetase, and 2,3-sialyltransferase are (126-350): (200-500): (50-120): (40-80): (185-490): (160-388).

4. The enzyme composition according to claim 1, characterized in that The enzyme activity ratios of N-acetylglucosamine-2-epimerase AGE, N-acetylneuraminic acid aldolase NAL, CMP kinase, polyphosphate kinase PPK, CMP-sialic acid synthetase, and 2,6-sialyltransferase are (126-350):(200-500):(50-120):(40-80):(185-490):(120-360).

5. A nucleic acid encoding an enzyme composition according to claims 1 to 4, comprising: A nucleic acid encoding the AGE, a nucleic acid encoding the NAL, a nucleic acid encoding the CMP kinase, a nucleic acid encoding the polyphosphate kinase PPK, a nucleic acid encoding the CMP-sialic acid synthetase, and a nucleic acid encoding the sialyltransferase; The nucleic acid encoding the sialyltransferase includes: a nucleic acid encoding the 2,3-sialyltransferase and / or a nucleic acid encoding the 2,6-sialyltransferase.

6. The nucleic acid according to claim 5, characterized in that The sequence of the nucleic acid encoding the AGE is shown in SEQ ID NO: 8; The sequence of the nucleic acid encoding the NAL is shown in SEQ ID NO:9; The sequence of the nucleic acid encoding the CMP kinase is shown in SEQ ID NO: 10; The sequence of the nucleic acid encoding the polyphosphate kinase PPK is shown in SEQ ID NO: 11; The sequence of the nucleic acid encoding the CMP-sialic acid synthetase is shown in SEQ ID NO: 12; The sequence of the nucleic acid encoding the 2,3-sialyltransferase is shown in SEQ ID NO: 13; The sequence of the nucleic acid encoding the 2,6-sialyltransferase is shown in SEQ ID NO:

14.

7. A strain characterized in that The enzyme composition according to any one of claims 1 to 4 is expressed or a plasmid vector containing the nucleic acid according to claim 5 or 6 is transformed therein.

8. Use of the enzyme composition according to any one of claims 1 to 4, the nucleic acid according to claim 5 or 6, or the strain according to claim 7 in producing sialyllactose.

9. A method for producing sialyllactose, characterized in that: include: 1) fermenting the strain according to claim 7, homogenizing, breaking, and centrifuging to obtain a mixed enzyme solution; 2) adding the mixed enzyme solution or the enzyme composition according to any one of claims 1 to 4 to a resin column for immobilization to obtain an immobilized enzyme resin column; 3) Adding the substrate reaction solution to the immobilized enzyme resin column described in step 2) to react and obtain the nucleic acid of 2,3-sialyltransferase and / or 2,6-sialyltransferase. The substrate reaction solution includes water, N-acetyl-glucosamine, sodium pyruvate, lactose, polyphosphoric acid, MgCl2 and CMP.

10. The method according to claim 9, characterized in that The concentration of each component in the substrate reaction solution is: N-acetyl-glucosamine 0-400mM, sodium pyruvate 0-500mM, lactose 0-600mM, MgCl2 0-400mM, CMP 0-10mM, polyphosphate concentration in terms of phosphate is 0-600mM; The concentration of the above components is not 0; The concentration of each component is preferably: N-acetyl-glucosamine 200-360mM, sodium pyruvate 249-490mM, lactose 240-480mM, MgCl2 300-400mM, CMP 4-10mM, polyphosphoric acid concentration calculated as phosphate is 400-580mM.

11. The method according to claim 9, characterized in that The resin column is a Ni2+ chelating affinity adsorption resin, and the Ni2+ chelating affinity adsorption resin includes LX-1000IDA resin and LX 604 resin.

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