CN117625585A - Bifunctional enzyme NagEA and application thereof - Google Patents
Bifunctional enzyme NagEA and application thereof Download PDFInfo
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- CN117625585A CN117625585A CN202311612803.8A CN202311612803A CN117625585A CN 117625585 A CN117625585 A CN 117625585A CN 202311612803 A CN202311612803 A CN 202311612803A CN 117625585 A CN117625585 A CN 117625585A
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 109
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 109
- 230000001588 bifunctional effect Effects 0.000 title abstract description 12
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- CILYIEBUXJIHCO-UITFWXMXSA-N N-acetyl-alpha-neuraminyl-(2->3)-beta-D-galactosyl-(1->4)-beta-D-glucose Chemical compound O1[C@@H]([C@H](O)[C@H](O)CO)[C@H](NC(=O)C)[C@@H](O)C[C@@]1(C(O)=O)O[C@@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)O[C@@H]2CO)O)O[C@H](CO)[C@@H]1O CILYIEBUXJIHCO-UITFWXMXSA-N 0.000 claims abstract description 33
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- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 claims abstract description 26
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Abstract
The invention relates to the technical field of biology, in particular to a bifunctional enzyme NagEA and application thereof. The invention utilizes the combination application of a plurality of enzymes to effectively convert the acetyl glucosamine into the 3' -sialyllactose, and has the characteristics of simplicity, easiness in control, high conversion rate and few byproducts. The fusion expression of isomerase and lyase reduces the dosage of added enzyme. The immobilized mixed enzyme is used, and the immobilized enzyme can be reused, so that the enzyme dosage can be further reduced and the production process can be optimized. Therefore, the 3' -sialyllactose prepared by the method has the advantages of green and environment-friendly property, low cost, easy mass production and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a bifunctional enzyme NagEA and application thereof.
Background
3'-Sialyllactose (3' -SL) is a natural trisaccharide, which is present in human breast milk and is an important component of breast milk oligosaccharides (HMOs); structurally it is formed by two sugars, N-acetyl-D-neuraminic acid (Neu 5 Ac) and beta-D-lactose (Lac), linked by a beta-1, 3-glycosidic bond. Specific effects of 3' -SL include reduced risk of adhesion of harmful bacteria and their proteins, and are thought to play an important role in the development of the infant immune system and intestinal flora; since sialic acid is an important component of neurons, 3' -SL and 6' -SL support infant brain development in particular by providing sialic acid, 3' -SL has a great potential for application in infant formula industry and has a broad market prospect. However, to date, there is still a lack of efficient and inexpensive synthetic methods on the market.
The 3' -sialyllactose preparation method is reported on the market at present mainly by single strain fermentation and multi-strain coupled fermentation, for example, the method for producing 3' -SL by recombinant large intestine fermentation in the university of south China has been developed in 2016, and later, the method for producing 3' -sialyllactose by 3-strain coupled fermentation in the university of Jiangnan in 2021. Meanwhile, other units and individuals also use similar technology to produce 3'-sialyllactose on the market, such as Nantoon, biological engineering Inc., star et al report "Synthesis of 3' -sialyllactose based on a mixed bacteria coupled fermentation strategy".
The 3' -sialyllactose scheme is prepared by fermenting modified single bacteria or complex bacteria, and the production of the product is not carried out by the related technology of an enzyme method. Although the strain is convenient to ferment, the fermentation time is long, and a large amount of heteroenzyme is contained in cells, so that the byproduct is inevitably increased, and the subsequent purification process and the product quality are affected. Meanwhile, the strain fermentation and amplification process is complex and unstable, and the large-scale production difficulty is great.
Therefore, the method for preparing the 3' -sialyllactose has important significance in mass production and product quality.
Disclosure of Invention
In view of this, the present invention provides the bifunctional enzyme NagEA and uses thereof.
The invention provides a bifunctional enzyme NagEA and application thereof. The invention utilizes the combination application of a plurality of enzymes to effectively convert the acetyl glucosamine into the 3' -sialyllactose, and has the characteristics of simplicity, easiness in control, high conversion rate and few byproducts. The fusion expression of isomerase and lyase reduces the dosage of added enzyme. The immobilized mixed enzyme is used, and the immobilized enzyme can be reused, so that the enzyme dosage can be further reduced and the production process can be optimized. Therefore, the 3' -sialyllactose prepared by the method has the advantages of green and environment-friendly property, low cost, easy mass production and the like.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides fusion enzymes, including isomerases, lyases and ligation fragments;
the isomerase is derived from nostoc punctiforme (Nostoc punctiforme);
the lyase is derived from rhizobia medicago (Rhizobium meliloti);
the connecting segment comprises a flexible connecting segment.
In some embodiments of the invention, the flexible connection section has:
(1) An amino acid sequence as shown in SEQ ID No. 11; or (b)
(2) A sequence of 1 or more amino acids substituted, deleted, added and/or substituted on the basis of the amino acid sequence shown in (1); or (b)
(3) An amino acid sequence having at least 70% sequence homology with the amino acid sequence as set forth in (1).
In some embodiments of the invention, the fusion enzyme has:
(4) An amino acid sequence as shown in SEQ ID No. 2; or (b)
(5) A sequence of 1 or more amino acids substituted, deleted, added and/or substituted on the basis of the amino acid sequence shown in (4); or (b)
(6) An amino acid sequence having at least 70% sequence homology with the amino acid sequence as set forth in (4).
The invention also provides nucleic acid molecules encoding the fusion enzymes.
In some embodiments of the invention, the nucleic acid molecule has:
a nucleotide sequence shown as SEQ ID No. 7; or (b)
(II) a nucleotide sequence which encodes the same protein as the nucleotide sequence shown in (I) but which differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (b)
(III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or (b)
(IV) a nucleotide sequence having at least 70% sequence homology with the nucleotide sequence of any one of (I) to (III).
On the basis of the above study, the present invention also provides an enzyme composition comprising the fusion enzyme, PPK, CMPNeuSyn and 3' -SialTrans.
In some embodiments of the invention, the PPK is derived from delftiasp;
the CMPNeuSyn is derived from meningococcus (Neisseria meningitides);
the 3' -SialTrans is derived from Du Kelei haemophilus (Haemophilus ducreyi).
In some embodiments of the invention, the PPK, CMPNeuSyn and 3' -SialTrans have, in order:
(A) Amino acid sequences shown as SEQ ID No.1, SEQ ID No.4 and SEQ ID No. 5; or (b)
(B) A sequence of 1 or more amino acids substituted, deleted, added and/or substituted on the basis of the amino acid sequence shown in (A); or (b)
(C) An amino acid sequence having at least 70% sequence homology with the amino acid sequence as set forth in (A).
In some embodiments of the invention, the enzyme composition comprises an immobilized enzyme.
In some embodiments of the invention, the immobilized enzymes have a fusion enzyme, PPK, CMPNeuSyn, and 3' -SialTrans enzyme activity ratio of 1: (1-2): (1-2): (2-3).
The present invention also provides a genetic element comprising:
(D) The nucleic acid molecule; and
(E) Nucleic acid molecules encoding PPK, CMPNeuSyn and 3' -SialTrans.
In some embodiments of the invention, the PPK, CMPNeuSyn and 3' -SialTrans have, in order:
(V) the nucleotide sequences shown as SEQ ID No.6, SEQ ID No.9 and SEQ ID No. 10; or (b)
A nucleotide sequence which encodes the same protein as the nucleotide sequence of (V) but differs from the nucleotide sequence of (V) due to the degeneracy of the genetic code; or (b)
(VII) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (V) or (VI), and functionally identical or similar to the nucleotide sequence shown in (V) or (VI); or (b)
(VIII) a nucleotide sequence having at least 70% sequence homology with the nucleotide sequence of any one of (V) to (VII).
The invention also provides expression vectors, including the gene elements and acceptable vectors.
In some embodiments of the invention, the acceptable carrier comprises pET-28a.
The invention also provides a host comprising
1) The nucleic acid molecule; and/or
2) The genetic element; and/or
3) And the expression vector.
In some embodiments of the invention, the host comprises E.coli BL21.
The invention also provides the use of any of the following in the synthesis of 3' -sialyllactose:
1) The fusion enzyme; and/or
2) The nucleic acid molecule; and/or
3) The enzyme composition; and/or
4) The genetic element; and/or
5) The expression vector; and/or
6) And the host.
The invention also provides a preparation method of the 3'-sialyllactose, which is catalyzed by the enzyme composition based on N-acetyl glucosamine, sodium pyruvate, cytidine triphosphate CTP, lactose and sodium hexametaphosphate to prepare the 3' -sialyllactose.
In some embodiments of the invention, the means of addition of the enzyme composition comprises stepwise addition and simultaneous addition;
the enzyme composition includes an immobilized enzyme.
In some embodiments of the invention, the preparation method comprises the following specific steps:
step 1: preparing NeuAc by catalyzing N-acetylglucosamine and sodium pyruvate with NagEA-a in Tris-HCl solution;
step 2: in Tris-HCl solution, the NeuAc, cytidine triphosphate CTP, lactose and sodium hexametaphosphate are catalyzed by the CMPNeuSyn, the 3'-SiaTrans and the PPK to prepare the 3' -sialyllactose.
In some embodiments of the invention, the Tris-HCl concentration of step 1 is 50mM and the pH is 7.5.
In some embodiments of the invention, the temperature of the catalysis described in step 1 is 35 ℃, the pH is 6.0-8.5, and the time is 3 hours.
In some embodiments of the invention, the NagEA-a in step 1 has an enzyme activity of 4000U.
In some embodiments of the invention, the catalytic step 1 further comprises centrifugation, removal of phosphoric acid byproduct, desalting and concentration steps.
In some embodiments of the invention, the dephosphorylation by-product is performed using a D201 anion exchange resin; the desalination is performed by a reverse osmosis membrane.
In some embodiments of the invention, the Tris-HCl concentration of step 2 is 100mM and the pH is 7.5.
In some embodiments of the invention, the temperature of the catalysis described in step 2 is 30 ℃, the pH is 7.0 to 9.0, and the time is 4 hours.
In some embodiments of the invention, the CMPNeuSyn has an enzyme activity of 4000U; the enzyme activity of the 3' -SiaTrans is 5000U; the enzyme activity of the PPK is 8000U.
In some embodiments of the invention, step 2 is followed by the steps of removing the phosphoric acid-containing byproduct, desalting, concentrating, and crystallizing.
In some embodiments of the invention, the dephosphorylation by-product is performed using a D201 anion exchange resin; the desalination is performed by a reverse osmosis membrane; the crystallization conditions are ethanol: water = 2:1 (v: v).
In some embodiments of the invention, the method of making: in Tris-HCl solution, N-acetyl glucosamine, sodium pyruvate, cytidine triphosphate CTP, lactose and sodium hexametaphosphate are catalyzed by the NagEA-a, the CMPNeuSyn, the 3'-SiaTrans and the PPK to prepare the 3' -sialyllactose.
In some embodiments of the invention, the Tris-HCl solution has a concentration of 100mM and a pH of 8.0.
In some embodiments of the invention, the catalytic temperature is 30 ℃, the pH is 7.5 to 8.5, and the time is 5 hours.
In some embodiments of the invention, the NagEA-a has an enzyme activity of 2000U; the enzyme activity of the CMPNeuSyn is 3000U; the enzyme activity of the 3' -SiaTrans is 3000U; the enzyme activity of the PPK is 5000U.
In some embodiments of the invention, the post-catalysis further comprises centrifugation, removal of the phosphate byproduct, desalting, concentration, and crystallization steps.
In some embodiments of the invention, the dephosphorylation by-product is performed using a D201 anion exchange resin; the desalination is performed by a reverse osmosis membrane; the crystallization conditions are ethanol: water = 2:1 (v: v).
In some embodiments of the invention, the method of making: in Tris-HCl solution, N-acetyl glucosamine, sodium pyruvate, cytidine triphosphate CTP, lactose and sodium hexametaphosphate are catalyzed by the enzyme composition to prepare the 3' -sialyllactose.
In some embodiments of the invention, the enzyme composition comprises an immobilized enzyme;
the enzyme activity ratio of fusion enzyme, PPK, CMPNeuSyn and 3' -SialTrans in the immobilized enzyme is 1: (1-2): (1-2): (2-3).
In some embodiments of the invention, the Tris-HCl concentration is 50mM and the pH is 8.0.
In some embodiments of the invention, the catalytic temperature is 35 ℃, the pH is 7.5 to 9.0, and the time is 8 hours.
In some embodiments of the invention, the immobilized enzyme has an enzyme activity of 20,000U.
In some embodiments of the invention, the post-catalysis further comprises the steps of filtering, removing the phosphoric acid-containing byproduct, desalting, concentrating, and crystallizing.
In some embodiments of the invention, the dephosphorylation by-product is performed using a D201 anion exchange resin; the desalination is performed by a reverse osmosis membrane; the crystallization conditions are ethanol: water = 2:1 (v: v).
The invention provides a bifunctional enzyme NagEA and application thereof. The preparation route of the invention uses cheap Acetyl glucosamine (N-Acetyl-D-glucamine), pyruvic acid (pyruvic acid) and lactose (lactose) as main raw materials, cytidine triphosphate (cytidine triphosphate, CTP) and polyphosphoric acid (Pi) N as a small amount of auxiliary materials, and the materials are converted into the target product 3'-sialyllactose with high yield under the continuous catalysis of four enzymes NagEA, CMPNeuSyn,3' -SiaTrans and PPK.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a liquid phase detection spectrum of the reaction for 0 hours in example 1;
FIG. 2 shows a spectrum of the results of the liquid phase reaction for 3 hours in example 1;
FIG. 3 shows a liquid phase detection spectrum of the reaction for 5 hours in example 2;
FIG. 4 shows a mass spectrum of the product sialic acid NeuAc;
FIG. 5 shows a liquid phase detection pattern of the reaction for 0 hour in example 3;
FIG. 6 shows a liquid phase result pattern of the reaction for 4 hours in example 3;
FIG. 7 shows a mass spectrum of 3' -sialyllactose in the present invention;
FIG. 8 shows the liquid phase detection of 0 hours of reaction in example 4;
FIG. 9 shows a liquid phase result pattern of the reaction for 5 hours in example 4;
FIG. 10 shows the preparation of 3' -sialyllactose by a multienzyme reaction with NagEA-b for 5 hours in example 5;
FIG. 11 is a graph showing the results of the liquid phase for 8 hours of the reaction in example 6;
FIG. 12 shows PPK I, nagEA-a, nagEA-b, CMPNeuSyn and 3' -sialTrans electrophoreses; wherein, the leftmost lane is Marker.
Detailed Description
The invention discloses a bifunctional enzyme NagEA and application thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention aims to adopt enzymes to efficiently produce 3'-sialyllactose, realize efficient continuous catalysis of a plurality of enzymes by means of enzyme fusion, catalytic space integration and the like, and finally produce 3' -sialyllactose with high yield.
The preparation scheme of the 3' -sialyllactase is as follows:
the route uses cheap Acetyl glucosamine (N-Acetyl-D-glucamine), pyruvic acid (pyruvic acid) and lactose (lactose) as main raw materials, cytidine triphosphate (cytidine triphosphate, CTP) and polyphosphoric acid (Pi) N as a small amount of auxiliary materials, and the materials are converted into a target product 3'-sialyllactose with high yield under the continuous catalysis of four enzymes NagEA, CMPNeuSyn,3' -SiaTrans and PPK.
The invention utilizes the multienzyme complex to continuously convert the acetyl glucosamine into the 3' -sialyllactose, the reaction can be completed step by step or in the same reaction system, the raw materials and the enzyme can be added at one time, and the purification of a reaction intermediate is not needed. In order to reduce the enzyme consumption, a fusion scheme is adopted to realize one-step fermentation production of the bifunctional enzyme, for example, nagEA can carry out two-step isomerization and condensation reactions; meanwhile, the expensive Cytidine Triphosphate (CTP) is regenerated by adopting a circulating system; the reaction system may be catalyzed by a liquid enzyme or an immobilized enzyme.
Information about enzymes used in the present invention:
polyphosphate Kinase (PPK) derived from Delftia sp., uniprot ID:
F6B1D 3), experimental tests have shown that it is not only capable of regenerating AMP to ATP using polyphosphoric acid (Pi) n, but also capable of regenerating CMP to CTP;
bifunctional enzyme NagEA: is the fusion of isomerase (EC 5.1.3.8: N-acylglucosamine 2-epinase) and lyase (EC 4.1.3.3: N-acetylneuraminic acid lyase), which are derived from nostoc punctiforme (Nostoc punctiforme, uniprot ID: B2IWM 7) and Rhizobium meliloti (Rhizobium meliloti, uniprot ID: Q92WP 0), respectively, by ligation of polypeptide fragments to obtain NagEA-a (GGGGSGGGGSGGGGS flexible fragment linked to SEQ ID No. 11) and NagEA-B (AEAAAKEAAAKEAAAKA rigid fragment linked to SEQ ID No. 12);
sialyl CMP activating enzyme CMPNeuSyn: derived from meningococcus (Neisseria meningitides, uniprot ID: P0A0Z 8);
sialyltransferase 3' -sialTrans from Haemophilus ducreyi (Haemophilus ducreyi, uniprot ID: Q7VPL 1)
Enzyme expression and activity data are shown in table 1:
TABLE 1
The sequences involved in the present invention are shown in tables 2 and 3:
TABLE 2
TABLE 3 Table 3
Fermentation production of enzyme and immobilization method of enzyme:
fermentation production of enzyme:
the enzyme required by the invention is prepared by constructing a specific expression plasmid after the company synthesizes corresponding genes and then fermenting and producing the specific expression plasmid by escherichia coli; the method specifically comprises the following steps: the genes corresponding to the enzymes are synthesized in general biological company (Chuzhou Anhui) after sequence optimization, and NdeI/XhoI restriction sites are introduced and subcloned into a pET 28a expression vector. Plasmid with correct sequence is transferred into E.coli (BL 21) competent cells for plate culture (bio-organism) and monoclonal small-volume liquid culture, and bacteria with correct protein expression are finally subjected to gradual amplification liquid culture. Specifically, the single colony is transferred into 5ml LB culture solution (37 ℃) containing 50 mu M kanamycin for culture, and when the cell grows to the logarithmic phase, the cell is inoculated into 250ml LB culture solution containing the same antibiotics, and when the cell grows to the logarithmic phase, the cell is transferred into a 5L culture fermentation tank for culture, and the final protein expression is carried out. In 5L fermentation tank culture, 0.5mM isopropyl-beta-D-thiopyran galactoside (IPTG) is added at 25 ℃ to induce protein expression for 6 hours when the cells OD-20, and finally, high-speed centrifugation is carried out to collect the cells (4000 rpm,20 min) so as to obtain 30-60g of wet cells with over-expressed enzyme. A small amount of cells are firstly mixed with a buffer solution (50 mM, pH 8.0) of tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) on an ice basin uniformly, then the cells are broken by a freeze thawing method, and clear liquid is subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) after cell walls are removed by high-speed centrifugation to determine protein expression. Cells with correct protein expression were used for the next catalytic experiment, specifically, the remaining cells were mixed with Tris.HCl buffer (50 mM, pH 8.0) at low temperature (200 ml buffer mixing with 10 g wet cells), followed by crushing the cell wall at low temperature Gao Yapo and high speed centrifugation (16000 rpm,45 min) to remove the cell wall and obtain enzyme-containing supernatant (the enzyme activity obtained was 500-2500U/ml, U was the amount of enzyme required for converting 1. Mu. Mol of substrate in one minute at room temperature).
LB medium consisted of: 1% tryptone, 0.5% yeast powder, 1% NaCl,1% dipotassium hydrogen phosphate and 5% glycerol.
Immobilization of enzymes:
slowly adding ammonium sulfate solid into the collected crude enzyme clear solution until protein solid is separated out (30-60% w/v ammonium sulfate: buffer solution), collecting the protein solid by high-speed centrifugation (10000 rpm,10 minutes), slowly dissolving the protein solid into 25mM Tris buffer solution (buffer solution A) with pH of 8.0, dialyzing the protein solid in 50 times of the volume of the buffer solution A (twice, each time for 4 hours), removing ammonium sulfate in the enzyme solution, and finally loading the dialysis solution on DEAE Seplite FF (SiAnblue dawn company) anion exchange column (NaCl is eluted in a buffer solution gradient: 0-1 NNaCl) to obtain primarily purified NagEA-a, CMPNeuSyn,3' -SiaTrans and PPK enzyme solution; the above enzyme was prepared using LX-1000EP epoxy resin (Seiyaka blue dawn) in terms of activity units 1 (1-2): (1-2): (2-3) one-time mixing and fixing by the following method: 10 000U of purified mixed enzyme is dissolved in 10L of 50mM potassium phosphate (buffer B) solution with pH of 8.0, 30-50mM phenoxyacetic acid and 5 kg of LX-1000EP epoxy resin are added until the mixture is stirred at 25 ℃ for 12 hours, immobilized enzyme is filtered, and finally the mixture is washed twice by clean water and buffer B and then is preserved at low temperature for standby, wherein the initial activity of the immobilized enzyme is 76-91% of that of liquid enzyme.
The D201 anion exchange resin adopted by the invention, available from Tianjin Baohong resin technologies Co., ltd, product number is Tianjin Baohong D201.
The bifunctional enzyme NagEA provided by the invention and raw materials and reagents used in the application of the bifunctional enzyme NagEA can be purchased from the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of sialic acid NeuAc Using N-acetylglucosamine as starting Material, nagEA-a enzyme
To 1L of 50mM tris (hydroxymethyl) aminomethane hydrochloride (Tris.HCl) solution at pH 7.5 was added 44.2 g of N-acetylglucosamine (200 mM), 66 g of sodium pyruvate (600 mM); then the pH value of the reaction system is adjusted back to 7.5, and the enzyme NagEA-a 4000U is added to start the reaction. The reaction was maintained at 35 ℃ with gentle agitation and at a reaction pH between 6.0 and 8.5, the reaction equilibrated after 3 hours and no more sialic acid NeuAc was formed as measured by HPLC, see liquid phase assay results figures 1, 2. And after the reaction is finished, adding an HCl aqueous solution to adjust the pH value to 1.0, stopping the reaction, precipitating enzyme, then adjusting the pH value back to 7.0, centrifuging to remove solid impurities, purifying supernatant by using a D201 anion exchange resin, removing residual pyruvic acid by using a membrane with the molecular weight of 300Da, desalting by using a reverse osmosis membrane, concentrating for later use, and ensuring that the liquid phase conversion rate is 64%.
EXAMPLE 2 preparation of sialic acid NeuAc Using N-acetylglucosamine as starting Material, nagEA-b enzyme
Similarly to the preparation of NeuAc described above, 44.2 g of N-acetylglucosamine (100 mM), 66 g of sodium pyruvate (300 mM) were added to 1L of 50mM tris (hydroxymethyl) aminomethane hydrochloride (Tris. HCl) solution at pH 7.5; then the pH value of the reaction system is adjusted back to 7.5, and then the enzyme NagEA-b 5000U is added to start the reaction. The reaction was maintained at 35℃with gentle agitation and the pH of the reaction was maintained between 6.0 and 8.5, after 5 hours the product sialic acid NeuAc stopped increasing, see FIG. 3 for liquid phase detection results. And (2) adding an aqueous solution of HCl to adjust the pH value to 1.0 after the reaction is finished to stop the reaction and precipitate enzyme, then adjusting the pH value back to 7.0, centrifuging to remove solid impurities, purifying supernatant fluid by using a D201 anion exchange resin, removing residual pyruvic acid by using a membrane with the molecular weight of 300Da, desalting by using a reverse osmosis membrane, concentrating for later use, and ensuring that the liquid phase conversion rate is 46%.
EXAMPLE 3 preparation of 3' -SL using sialic acid NeuAc as starting material
Using sialic acid NeuAc obtained in example 1 as a starting material, 61.8 g of sialic acid NeuAc (200 mM), 4.8 g of cytidine triphosphate CTP (10 mM), 82.1 g of lactose (240 mM) and 97.8 g of sodium hexametaphosphate (160 mM) were added to 1L of 50mM Tris-aminomethane hydrochloride (HCl) solution at pH 8.0. The pH of the reaction system was adjusted back to 8.0 before the enzyme addition, and 4000U of CMPNeuSyn,3' -SiaTrans 5000U and PPK 8000U were added to initiate the reaction. The reaction was maintained at 30℃with gentle agitation and at a pH of 7.0-9.0, after 4 hours the reaction was complete as determined by HPLC of the starting sialic acid NeuAc, see FIGS. 5, 6 for liquid phase detection results. After the reaction, HCl aqueous solution was added to adjust pH 2.0 to precipitate enzyme, the solids were removed after centrifugation, then pH was adjusted back to 7.0 and the phosphate-containing by-product was removed using D201 anion exchange resin, finally desalting and concentrating and crystallizing using reverse osmosis membrane (crystallization conditions ethanol: water=2:1v:v) to give 116.4 g of 3' -sialyllactose as a white solid product (final yield 92%). The correct structure of the product was confirmed by mass spectrometry (fig. 7).
Example 4 preparation of 3' -SL containing NagEA-a enzyme by multiple liquid enzyme one pot method Using N-acetylglucosamine as raw material
22.1 g of N-acetylglucosamine (100 mM), 12.1 g of sodium pyruvate (110 mM), 2.4 g of cytidine triphosphate CTP (5 mM), 41 g of lactose (120 mM) and 48.9 g of sodium hexametaphosphate (80 mM) were continuously added to 1L of 50mM tris (hydroxymethyl) aminomethane hydrochloride (Tris. HCl) solution at pH 8.0. The pH of the reaction system was then adjusted back to 8.0, and finally 2000U NagEA-a,3000U CMPNeuSyn,3' -SiaTrans 3000U and PPK 5000U were added to initiate the reaction. The reaction was maintained at 30℃with gentle agitation and pH between 7.5 and 8.5, after 5 hours the consumption of N-acetylglucosamine as starting material was detected by HPLC, see FIGS. 8, 9 for liquid phase detection results. As in example 3, aqueous HCl was added to adjust pH 2.0 to precipitate the enzyme and centrifuged to remove solids after completion of the reaction, then the pH of the supernatant was adjusted back to 7.0 and the phosphate-containing byproduct was removed using D201 anion exchange resin, and finally the resulting product was desalted using reverse osmosis membrane and concentrated and crystallized (crystallization conditions ethanol: water=2:1v:v) to yield 54.4 g of a white solid (final yield about 86%). The structure was correct as confirmed by mass spectrometry (compare fig. 7).
Example 5 preparation of 3' -SL containing NagEA-b enzyme by multiple liquid enzyme one pot method Using N-acetylglucosamine as raw material
22.1 g of N-acetylglucosamine (100 mM), 12.1 g of sodium pyruvate (110 mM), 2.4 g of cytidine triphosphate CTP (5 mM), 41 g of lactose (120 mM) and 48.9 g of sodium hexametaphosphate (80 mM) were continuously added to 1L of 100mM tris (hydroxymethyl) aminomethane hydrochloride (Tris. HCl) solution at pH 8.0. The pH of the reaction system was then adjusted back to 8.0, and finally 2000UNagEA-b,3000UCMPNeuSyn,3' -SiaTrans 3000U and PPK 5000U were added to initiate the reaction. The reaction was maintained at 30℃with gentle stirring and pH between 7.5 and 8.5, after 5 hours HPLC detection of the raw material N-acetylglucosamine was not consumed, and also about 30% of glucosamine was not consumed, with an estimated product 3' -sialyllactose yield of 60%. The results of the liquid phase detection reaction are shown in FIG. 10.
Example 6 preparation of 3' -SL by immobilized MixEnzymes one pot method Using N-acetylglucosamine as raw material
Similarly to example 4, 22.1 g of N-acetylglucosamine (100 mM), 12.1 g of sodium pyruvate (110 mM), 2.4 g of cytidine triphosphate CTP (5 mM), 41 g of lactose (120 mM) and 48.9 g of sodium hexametaphosphate (80 mM) were continuously added to 1L of 50mM tris-methylaminomethane hydrochloride (Tris.HCl) solution at pH 8.0. The pH of the reaction system was then adjusted back to 8.0, and finally an immobilized enzyme mixture (containing NagEA-a, CMPNeuSyn,3' -SiaTrans and PPK) was added to the reaction system, and the specific mixing ratio was found in the fermentation production of the enzyme and the immobilization method of the enzyme in the specific embodiment) to total 20,000U of activity to initiate the reaction. The reaction is maintained at 35 ℃ with slight stirring and the pH value is between 7.5 and 9.0, after the reaction is carried out for 8 hours, the consumption of the raw material N-acetyl glucosamine is detected by HPLC, and the liquid phase detection reaction result is shown in figure 11; after the reaction, directly filtering and recovering the immobilized enzyme, regulating the pH value of the supernatant to 7.0, removing a phosphoric acid-containing byproduct by using a D201 anion exchange resin, and finally desalting and concentrating by using a reverse osmosis membrane and crystallizing (the crystallization condition is ethanol: water=2:1v: v) to obtain 51.9 g of white solid product 3' -sialyllactose (the final yield is about 82%), wherein the immobilized enzyme still maintains the original 64% activity after being used for 5 times.
Effect example
Comparison between different enzymatic processes, please see table 4:
table 4 multiple enzyme assay comparison
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (21)
1. A fusion enzyme comprising an isomerase, a lyase and a linker fragment;
the isomerase is derived from nostoc punctiforme (Nostoc punctiforme);
the lyase is derived from rhizobia medicago (Rhizobium meliloti);
the connecting segment comprises a flexible connecting segment.
2. The fusion enzyme of claim 1, wherein the flexible linker fragment has:
(1) An amino acid sequence as shown in SEQ ID No. 11; or (b)
(2) A sequence of 1 or more amino acids substituted, deleted, added and/or substituted on the basis of the amino acid sequence shown in (1); or (b)
(3) An amino acid sequence having at least 70% sequence homology with the amino acid sequence as set forth in (1).
3. The fusion enzyme according to claim 1 or 2, comprising:
(4) An amino acid sequence as shown in SEQ ID No. 2; or (b)
(5) A sequence of 1 or more amino acids substituted, deleted, added and/or substituted on the basis of the amino acid sequence shown in (4); or (b)
(6) An amino acid sequence having at least 70% sequence homology with the amino acid sequence as set forth in (4).
4. A nucleic acid molecule encoding the fusion enzyme of any one of claims 1 to 3.
5. The nucleic acid molecule of claim 4, comprising:
a nucleotide sequence shown as SEQ ID No. 7; or (b)
(II) a nucleotide sequence which encodes the same protein as the nucleotide sequence shown in (I) but which differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or (b)
(III) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (I) or (II), and having the same or similar functions as the nucleotide sequence shown in (I) or (II); or (b)
(IV) a nucleotide sequence having at least 70% sequence homology with the nucleotide sequence of any one of (I) to (III).
6. An enzyme composition comprising the fusion enzyme of any one of claims 1 to 3, PPK, CMPNeuSyn and 3' -SialTrans.
7. The enzyme composition according to claim 6, wherein the PPK is derived from Delftia sp;
the CMPNeuSyn is derived from meningococcus (Neisseria meningitides);
the 3' -SialTrans is derived from Du Kelei haemophilus (Haemophilus ducreyi).
8. The enzyme composition according to claim 6 or 7, wherein the PPK, CMPNeuSyn and 3' -SialTrans sequences have in order:
(A) Amino acid sequences shown as SEQ ID No.1, SEQ ID No.4 and SEQ ID No. 5; or (b)
(B) A sequence of 1 or more amino acids substituted, deleted, added and/or substituted on the basis of the amino acid sequence shown in (A); or (b)
(C) An amino acid sequence having at least 70% sequence homology with the amino acid sequence as set forth in (A).
9. The enzyme composition according to any one of claims 6 to 8, comprising an immobilized enzyme.
10. The enzyme composition according to claim 9, wherein the enzyme activity ratio of fusion enzyme, PPK, CMPNeuSyn and 3' -SialTrans in the immobilized enzyme is 1: (1-2): (1-2): (2-3).
11. A genetic element comprising:
(D) The nucleic acid molecule of claim 4 or 5; and
(E) Nucleic acid molecules encoding PPK, CMPNeuSyn and 3' -SialTrans.
12. The genetic element of claim 11, wherein the PPK, CMPNeuSyn and 3' -SialTrans sequences comprise, in order:
(V) the nucleotide sequences shown as SEQ ID No.6, SEQ ID No.9 and SEQ ID No. 10; or (b)
A nucleotide sequence which encodes the same protein as the nucleotide sequence of (V) but differs from the nucleotide sequence of (V) due to the degeneracy of the genetic code; or (b)
(VII) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in (V) or (VI), and functionally identical or similar to the nucleotide sequence shown in (V) or (VI); or (b)
(VIII) a nucleotide sequence having at least 70% sequence homology with the nucleotide sequence of any one of (V) to (VII).
13. Expression vector comprising a genetic element according to claim 11 or 12 and an acceptable carrier.
14. A host, characterized by comprising
1) The nucleic acid molecule of claim 4 or 5; and/or
2) A genetic element according to claim 11 or 12; and/or
3) The expression vector of claim 13.
15. Use in the synthesis of 3' -sialyllactose of any of the following:
1) A fusion enzyme according to any one of claims 1 to 3; and/or
2) The nucleic acid molecule of claim 4 or 5; and/or
3) An enzyme composition according to any one of claims 6 to 10; and/or
4) The genetic element of claim 11 or 12; and/or
5) The expression vector of claim 13; and/or
6) The host of claim 14.
A process for the preparation of 3'-sialyllactose, characterized in that said 3' -sialyllactose is prepared based on N-acetylglucosamine, sodium pyruvate, cytidine triphosphate CTP, lactose and sodium hexametaphosphate catalyzed by an enzyme composition according to any one of claims 6 to 10.
17. The method of claim 16, wherein the enzyme composition is added in a manner comprising stepwise addition and simultaneous addition;
the enzyme composition includes an immobilized enzyme.
18. The method of manufacturing as claimed in claim 16 or 17, characterized in that the specific steps include:
step 1: preparing NeuAc by catalyzing N-acetylglucosamine and sodium pyruvate with NagEA-a in Tris-HCl solution;
step 2: in Tris-HCl solution, the NeuAc, cytidine triphosphate CTP, lactose and sodium hexametaphosphate are catalyzed by the CMPNeuSyn, the 3'-SiaTrans and the PPK to prepare the 3' -sialyllactose.
19. The method of claim 16 or 17, wherein N-acetylglucosamine, sodium pyruvate, cytidine triphosphate CTP, lactose and sodium hexametaphosphate are catalyzed by said NagEA-a, said CMPNeuSyn, said 3 '-sialacross and said PPK in Tris-HCl solution to produce said 3' -sialyllactose.
20. The method of preparation of claim 16 or 17, wherein the 3' -sialyllactose is prepared in Tris-HCl solution, N-acetylglucosamine, sodium pyruvate, cytidine triphosphate CTP, lactose and sodium hexametaphosphate by catalysis with an enzyme composition according to any one of claims 6 to 10.
21. The method of claim 20, wherein the enzyme composition comprises an immobilized enzyme;
the enzyme activity ratio of fusion enzyme, PPK, CMPNeuSyn and 3' -SialTrans in the immobilized enzyme is 1: (1-2): (1-2): (2-3).
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| WO2023184822A1 (en) * | 2022-04-02 | 2023-10-05 | 深圳瑞德林生物技术有限公司 | Enzyme co-expression system and use thereof in synthesis of sialic acid |
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| JP2006271372A (en) * | 2005-03-01 | 2006-10-12 | Yamasa Shoyu Co Ltd | Method for producing sugar chain |
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