METHOD FOR SYNTHESIZING LACTO-N-BIOSE

Abstract

A method for synthesizing lacto-N-biose and belongs to the technical field of bioengineering and oligosaccharide synthesis. A multi-enzyme catalytic system with good biological safety and wide application, and an ATP regeneration cycle system is introduced into a multi-enzyme reaction system, so that the synthesis of lacto-N-biose and the utilization rate of substrates are improved. A novel lacto-N-biose synthetic route lays a foundation for large-scale industrial production of lacto-N-biose and has important economic values and social benefits. At the same time, the synthetic method is efficient, mild, simple, easy to operate, low in cost and suitable for industrial production, and has a high practical application value.

Claims

1. A method for synthesizing lacto-N-biose, the method comprising: adding galactokinase and lacto-N-biose phosphorylase into a reaction system containing galactose, acetylglucosamine and lactose as substrates to prepare lacto-N-biose; and adding acetyl phosphate and acetate kinase into the above reaction system for in-situ regeneration of ATP.

2. The method for synthesizing lacto-N-biose according to claim 1, wherein the method further comprises: separating the product lacto-N-biose and ATP and ADP present in the reaction system.

3. The method for synthesizing lacto-N-biose according to claim 1, wherein the galactokinase, lacto-N-biose phosphorylase and acetate kinase are all produced by genetically engineered bacteria through genetic recombination.

4. The method for synthesizing lacto-N-biose according to claim 3, wherein the method of the production comprises: cloning expression vectors derived from galactokinase, lacto-N-biose phosphorylase and acetate kinase respectively; and obtaining target enzyme proteins by culturing and inducing hosts of the corresponding expression vectors.

5. The method for synthesizing lacto-N-biose according to claim 1, wherein the amino acid sequence of galactokinase is shown as SEQ ID No. 1; the amino acid sequence of lacto-N-biose phosphorylase is shown as SEQ ID No. 2; and the amino acid sequence of acetate kinase is shown as SEQ ID No. 3.

6. The method for synthesizing lacto-N-biose according to claim 1, wherein the reaction system further contains ATP, and the concentration of ATP is 5-15 mM.

7. The method for synthesizing lacto-N-biose according to claim 6, wherein the concentration of ATP is 7.5 mM.

8. The method for synthesizing lacto-N-biose according to claim 1, wherein the reaction system further contains MgCl.sub.2 and a Tris-HCl buffer; the concentration of MgCl.sub.2 is 1-10 mM; and the concentration of the Tris-HCl buffer is 10-200 mM.

9. The method for synthesizing lacto-N-biose according to claim 8, wherein the concentration of MgCl.sub.2 is 3 mM, and the concentration of the Tris-HCl buffer is 100 mM.

10. The method for synthesizing lacto-N-biose according to claim 1, wherein the reaction temperature of the reaction system is 25-45° C., and the reaction pH is 5.8-7.5.

11. The method for synthesizing lacto-N-biose according to claim 1, wherein the concentrations of the galactose and acetylglucosamine substrates are both 10-20 mM; the concentration of galactokinase is 1-10 U/mL; the enzyme concentration of LNBP is 100-300 U/mL; the concentration of acetate kinase is 1-10 U/mL; and the concentration of acetyl phosphate is 2.5-5 mM.

12. Lacto-N-biose synthesized by the method according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary examples of the present invention and descriptions thereof are used to explain the present invention, and do not constitute an improper limitation of the present invention.

[0022] FIG. 1 is a schematic diagram showing catalytic production of lacto-N-biose in two steps with galactose as a substrate of the present invention.

[0023] FIG. 2 is a synthetic comparison diagram of lacto-N-biose at an initial amount of 5 mM ATP after introduction of ATP regeneration cycle in Example 2 of the present invention.

[0024] FIG. 3 is a synthetic comparison diagram of lacto-N-biose at an initial amount of 7.5 mM ATP after introduction of ATP regeneration cycle in Example 3 of the present invention.

DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are all exemplary and are intended to provide a further description of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0026] It should be noted that terms used herein are only for the purpose of describing specific implementations and are not intended to limit the exemplary implementations of the present invention. As used herein, the singular form is intended to include the plural form, unless the context clearly indicates otherwise. In addition, it should be further understood that terms “comprise” and/or “include” used in this specification indicate that there are features, steps, operations, devices, assemblies, and/or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the following specific implementation solutions. It should further be understood that terms used in the embodiment of this application are only for describing the specific implementation solutions, and are not intended to limit the protection scope of the present invention. In the following specific embodiments, if specific conditions are not indicated, experimental methods usually follow conventional methods and conditions of molecular biology in the art, and such techniques and conditions are fully explained in the literature. For example, Sambrook et al., techniques and conditions in “Molecular Cloning: Laboratory Manual” or conditions recommended by manufacturers are taken as references.

[0027] As mentioned earlier, it is currently difficult to synthesize human milk oligosaccharides on a large scale using chemical methods, and when a biotechnology is used for heterogeneous production of human milk oligosaccharides, the production efficiency is generally low, and food safety problems are likely to be caused.

[0028] In view of this, in a specific embodiment of the present invention, a method for synthesizing lacto-N-biose is provided and includes:

[0029] adding galactokinase and lacto-N-biose phosphorylase into a reaction system containing galactose, acetylglucosamine and lactose as substrates to prepare lacto-N-biose; and

[0030] adding acetyl phosphate and acetate kinase into the above reaction system for in-situ regeneration of ATP. The present invention aims at consuming one molecule of ATP energy in a galactokinase catalytic step, and constructs but is not limited to realization of ATP energy regeneration in acetyl phosphate with acetate kinase. Experimental results of the present invention prove that by introducing energy regeneration cycle, regeneration cycle of ATP and full conversion of substrates can be realized, thereby effectively improving the synthesis efficiency of lacto-N-biose.

[0031] In another specific embodiment of the present invention, the method further includes: separating the product lacto-N-biose and ATP and ADP present in the reaction system.

[0032] In the present invention, sources of enzymes used are not particularly limited, which may be extracted from natural bacteria, yeast or fungi and other microorganisms, produced by genetically engineered bacteria through genetic recombination and extracted from natural plant tissues or animal tissues. The enzyme product forms are also not particularly limited, which may be solid, powder, liquid, or an immobilized enzyme fixed on a carrier by a physical or chemical method. Enzymes may be commercially available products, or self-made products by enterprises or individuals.

[0033] In another specific embodiment of the present invention, the galactokinase, lacto-N-biose phosphorylase (LNBP) and acetate kinase are all produced by genetically engineered bacteria through genetic recombination.

[0034] Specifically, the method of the production includes: cloning expression vectors derived from galactokinase, lacto-N-biose phosphorylase and acetate kinase respectively; and obtaining target enzyme proteins by culturing and inducing hosts of the corresponding expression vectors.

[0035] In another specific embodiment of the present invention, the expression vectors are any one or more of viral vectors, plasmids, phages, phagemids, cosmids, F cosmids, bacteriophages or artificial chromosomes; viral vectors may include adenovirus vectors, retroviral vectors or adeno-associated virus vectors, and artificial chromosomes include bacterial artificial chromosomes (BAC), phage P1 derived carriers (PAC), yeast artificial chromosomes (YAC) or mammalian artificial chromosomes (MAC); further preferably, the expression vectors are plasmids; more preferably, the expression vectors are pET-28a plasmids.

[0036] In another specific embodiment of the present invention, the hosts include but are not limited to bacteria, fungi and eukaryotic cells, and are further selected from Escherichia coli, Bacillus, Bacillus subtilis, Saccharomyces cerevisiae, Trichoderma reesei and Penicillium oxalicum; more preferably, the hosts are Escherichia coli BL21 (DE3).

[0037] In another specific embodiment of the present invention, the method of the production includes: cloning expression plasmids pET28a-galk, pET28a-Lnbp and pET28a-ack derived from galactokinase, LNBP and acetate kinase respectively; and obtaining target enzyme proteins by culturing and inducing BL21(DE3) strains of the corresponding expression plasmids and carrying out purification (preferably by a nickel column).

[0038] Galactokinase source strains include but are not limited to Escherichia coli; LNBP source strains include but are not limited to Bifidobacterium; and ACK source strains include but are not limited to Escherichia coli.

[0039] In another specific embodiment of the present invention,

[0040] the amino acid sequence of galactokinase is shown as SEQ ID No. 1;

[0041] the amino acid sequence of LNBP is shown as SEQ ID No. 2; and

[0042] the amino acid sequence of acetate kinase is shown as SEQ ID No. 3.

[0043] In another specific embodiment of the present invention, the reaction system further contains ATP, and further, the concentration of ATP is 5-15 mM (preferably 7.5 mM).

[0044] The reaction system further contains magnesium ions (preferably MgCl.sub.2) and a Tris-HCl buffer.

[0045] In another specific embodiment of the present invention, the concentration of MgCl.sub.2 is 1-10 mM (preferably 3 mM); and the concentration of the Tris-HCl buffer is 10-200 mM (preferably 100 mM).

[0046] In another specific embodiment of the present invention, the reaction temperature of the reaction system is 25-45° C., and the reaction pH is 5.8-7.5.

[0047] In another specific embodiment of the present invention, the concentrations of the galactose and acetylglucosamine substrates are both 10-20 mM.

[0048] In another specific embodiment of the present invention, the concentration of galactokinase is 1-10 U/mL.

[0049] In another specific embodiment of the present invention, the enzyme concentration of LNBP is 100-300 U/mL.

[0050] In another specific embodiment of the present invention, the concentration of acetate kinase required for in-situ regeneration of ATP is 1-10 U/mL, and the concentration of acetyl phosphate is 2.5-5 mM.

[0051] In another specific embodiment of the present invention, lacto-N-biose synthesized by the synthetic method is provided. In addition, based on the synthetic method of the present invention, the synthesis of all oligosaccharides or polysaccharides with lacto-N-biose as a skeletal structure is also within the protection scope of the present invention.

[0052] The following further explains and describes the present invention through specific embodiments, but does not constitute a limitation on the present invention. It should be understood that these embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention. An experimental method without indicating a specific condition in the following embodiments is generally performed according to a conventional condition.

Example 1: Synthesis of Lacto-N-Biose by Using Galactose as a Substrate

[0053] 10 mM galactose, 10 mM ATP, 10 mM GlcNAc, 3 mM MgCl.sub.2, 100 mM Tris-HCl, 5 U Galk and 168 U lacto-N-biose phosphorylase are added into a 1 mL reaction system. As shown in Table 1, Control is a control group without addition of lacto-N-biose phosphorylase (LNBP). After a reaction at 37° C. for 12 hours, the reaction system is boiled for 5 minutes and centrifuged, a supernatant is sampled, filtered through a filtering membrane and detected through a Biorad-HPX column, RID detection of corresponding substrates and products is carried out, reaction detection map (HPLC) comparison results verify the synthesis of lacto-N-biose.

TABLE-US-00001 TABLE 1 A reaction table showing catalytic production of lacto-N-biose in two steps with galactose as a substrate Gal ATP GalK MgCl.sub.2 LNBP GlcNAc mM mM U/mL mM U/mL mM Control 10 10 5 3 10 1 10 10 5 3 168 10

Example 2: Synthesis of Lacto-N-Biose by Introducing ATP Regeneration Cycle

[0054] 10 mM galactose, 7.5 mM ATP, 2.5 mM acetyl phosphate, 10 mM GlcNAc, 3 mM MgCl.sub.2, 100 mM Tris-HCl, 5 U Galk, 3 U ACK and 168 U LNBP are added into a 1 mL reaction system. As shown in Table 2, the initial concentration of ATP in Control is 10 mM, and an ATP cyclic reaction is not introduced into the reaction system. After a reaction at 37° C. for 12 hours, the reaction system is boiled for 5 minutes and centrifuged, a supernatant is sampled, filtered through a filtering membrane and detected by a Biorad-HPX column, and RID detection of corresponding substrates and products is carried out. As shown in FIG. 2, under ATP-1 reaction conditions, the synthesis of LNB is increased by 1.60 times in comparison with the control group (without addition of ATP).

Example 3

[0055] 10 mM galactose, 5 mM ATP, 5 mM acetyl phosphate, 10 mM GlcNAc, 3 mM MgCl.sub.2, 100 mM Tris-HCl, 5 U Galk, 3 U ACK and 168 U LNBP are added into a 1 mL reaction system. As shown in Table 2, the initial concentration of ATP in Control is 10 mM, and an ATP cyclic reaction is not introduced into the reaction system. After a reaction at 37° C. for 12 hours, the reaction system is boiled for 5 minutes and centrifuged, a supernatant is sampled, filtered through a filtering membrane and detected by a Biorad-HPX column, and RID detection of corresponding substrates and products is carried out. As shown in FIG. 3, under ATP-2 reaction conditions, the synthesis of LNB is increased by 1.49 times in comparison with the control group (without addition of ATP).

TABLE-US-00002 TABLE 2 A reaction table showing catalytic production of lacto-N-biose in two steps with galactose as a substrate after introduction of ATP regeneration cycle Gal ATP GalK MgCl.sub.2 LNBP GlcNAc ACK Reaction mM mM U mM U mM Acetyl-ACP U Control 10 10 5 3 168 10 ATP-1 10 7.5 5 3 168 10 2.5 3 ATP-2 10 5 5 3 168 10 5 3

[0056] It should be noted that the foregoing embodiments are merely intended for describing the technical solutions of the present invention other than limiting the present invention. Although the present invention is described in detail with reference to the foregoing embodiments, a person skilled in the art may make modifications or equivalent substitutions to the technical solutions of the present invention as required, without departing from spirit and scope of the technical solutions of the present invention.