阅读说明：本技术 一种合成乳-n-二糖的方法 (A method of synthesis cream-N- disaccharides ) 是由 方诩 杜志强 于 2019-10-31 设计创作，主要内容包括：本发明提供一种合成乳-<I>N</I>-二糖的方法,属于生物工程及寡糖合成技术领域。本发明利用生物安全性好且应用广泛的多酶催化体系,通过在多酶反应体系中引入ATP再生循环系统,提高乳-<I>N</I>-二糖合成及底物利用率。本发明提供了一个新的乳-<I>N</I>-二糖合成途径,为乳-<I>N</I>-二糖的大规模工业生产奠定了基础,具有重要的经济价值和社会效益。同时本发明合成方法高效温和、简便易行,成本低、适合产业化生产,具有良好的实际应用之价值。(The present invention provides a kind of synthesis cream- N The method of disaccharides belongs to bioengineering and oligosaccharides synthesis technical field.The present invention good and widely used multienzyme catalyst system using biological safety improves cream-by introducing ATP regeneration cycle system in multienzymatic reaction system N Disaccharides synthesis and substrate utilization ratio.The present invention provides a new creams- N Disaccharides route of synthesis, for cream- N The large-scale industrial production of disaccharides is laid a good foundation, and has important economic value and social benefit.Synthetic method of the present invention is efficiently mild, simple and easy to do simultaneously, at low cost, suitable industrialization production, the value with good practical application.)

1. a kind of synthesis cream-NThe method of disaccharides, which is characterized in that the described method includes:

In containing galactolipin, acetylglucosamine, the reaction system that lactose is substrate, galactokinase, cream-is addedNDisaccharides Phosphorus solution enzyme preparation cream-NDisaccharides；

In above-mentioned reaction system, it is additionally added acetyl phosphate, acetokinase carries out the in-situ regeneration of ATP.

2. synthesis cream-as described in claim 1NThe method of disaccharides, which is characterized in that the method also includes: separation product Cream-NATP, ADP present in disaccharides and reaction system.

3. synthesis cream-as described in claim 1NThe method of disaccharides, which is characterized in that the galactokinase, cream-N- two Sugared phosphorus solution enzyme and acetokinase are all made of genetic recombination and produce acquisition by genetic engineering bacterium.

4. synthesis cream-as claimed in claim 3NThe method of disaccharides, which is characterized in that production method includes: to clone to come respectively Derived from galactokinase, cream-NDisaccharides phosphorus solution enzyme and acetokinase expression vector；By cultivating, inducing corresponding expression vectors Host obtains target zymoprotein.

5. synthesis cream-as described in claim 1NThe method of disaccharides, which is characterized in that

The amino acid sequence of the galactokinase is as shown in SEQ ID No.1；

The cream-NThe amino acid sequence of disaccharides phosphorus solution enzyme is as shown in SEQ ID No.2；

The amino acid sequence of the acetokinase is as shown in SEQ ID No.3.

6. synthesis cream-as described in claim 1NThe method of disaccharides, which is characterized in that also contain ATP in reaction system；Institute Stating ATP concentration is 5-15mM.

7. synthesis cream-as claimed in claim 6NThe method of disaccharides, which is characterized in that the ATP concentration is 7.5mM.

8. synthesis cream-as described in claim 1NThe method of disaccharides, which is characterized in that also contain MgCl in reaction system₂With Tris-HCl buffer；

The MgCl₂Concentration is 1-10 mM；The concentration of the Tris-HCl buffer is 10-200mM.

9. synthesis cream-as claimed in claim 8NThe method of disaccharides, which is characterized in that the MgCl₂Concentration is 3 mM；It is described The concentration of Tris-HCl buffer is 100 mM.

10. synthesis cream-as described in claim 1NThe method of disaccharides, which is characterized in that the reaction temperature of reaction system is 25-45 DEG C, pH value in reaction 5.8-7.5.

11. synthesis cream-as described in claim 1NThe method of disaccharides, which is characterized in that the galactolipin, acetyl glucosamine Amine concentration of substrate is 10-20 mM；

The galactokinase enzyme concentration is 1-10U/mL；

The LNBP enzyme concentration is 100-300U/mL；

Acetokinase concentration is 1-10 U/mL；

Acetyl phosphate concentration is 2.5-5 mM.

12. the cream-of any one of the claim 1-11 synthetic method synthesisNDisaccharides.

Technical field

The invention belongs to bioengineering and oligosaccharides synthesis technical fields, and in particular to a kind of synthesis cream-NThe method of disaccharides.

Background technique

Disclosing the information of the background technology part, it is only intended to increase understanding of the overall background of the invention, without certainty It is considered as recognizing or implying in any form that information composition has become existing skill well known to persons skilled in the art Art.

The matter and energy tie that breast milk is uniquely contacted with parent after being born as newborn, it is considered to be infant nutrition Gold standard.Other than providing the nutriments such as protein, fat and carbohydrate necessary to infant physical growth, breast milk is also Provide the enzyme that can promote infantile health and development, antibody, growth factor and oligosaccharides isoreactivity component.Further research hair Existing, compared with cattle and sheep cream, maximum difference is functionality human milk oligosaccharides rich in and its derivative (Human in breast milk Milk Oligosaccharides, HMOs), content is up to 12-24 g/L, is only second to lactose and lipid components, is in breast milk The third-largest nutriment.HMOs is perfect especially for the digestive system, intestinal health and immune system of infant for human body There is special effect.Recent studies have found that HMOs acts not only as the prebiotics of beneficial bacteria of intestinal tract, baby's intestines are adjusted and promoted The maturation of road immunization barrier, and can be used as the bait molecule of pathogen, there is inhibition bacterium and antifungic action, protect baby Infection of the child from pathogen.Therefore for HMOs research for infantile health important in inhibiting, and by HMOs is added in milk powder and simulates breast milk composition, for some battalion that can not carry out breast-fed babies and post-weaning baby It is particularly important to support health care.

However, limited source and chemical method scale is difficult to be utilized being combined to, the research of its biological function is constrained And extensive use.In order to break through this bottleneck, because its available sources is limited, need to establish it is succinct, efficiently, economically largely obtain cores Core structure cream-NThe route of synthesis of disaccharides (LNB, Gal β 1-3GlcNAc, chemical structural formula are as follows).Therefore, bioanalysis synthesizes Cream-NThe technical costs of disaccharides (LNB) is low, and synthesis process is simple, and separation method is easy.

Have multinomial patent at present to concentrate on using human milk oligosaccharides biosynthesis technology is produced in Escherichia coli, such as:

Proprietary term: " a kind of fucosyltransferase and its genetic engineering bacterium and application " (CN201611147477.8)

Proprietary term: " generations of human milk oligosaccharides in the microbial hosts with modified input/output " (CN201680052611.8)

Proprietary term: " a kind of fucosyltransferase and its application " (CN201611147478.2)

Proprietary term: " method for being used to prepare the tetrose lacto-N-neotetraose (LNNT) of the acetyl lactosamine containing N- " (CN201510751641.5).

It is all made of in above-mentioned patent using Escherichia coli as host, the technology of heterologous production human milk oligosaccharides.But large intestine bar The endotoxic removal of bacterium is a significant challenge in large-scale industrial production.Endotoxin is in gram-negative bacterial cell wall A kind of ingredient, also referred to as lipopolysaccharides is that a kind of pair of human body can generate toxicant.However, for especially as baby For the additive human milk oligosaccharides of milk powder, foodsafety is clearly extremely important.Therefore find safety is good, stable yield, High production efficiency is particularly important to the synthetic method for being allowed to be more suitable for the human milk oligosaccharides of technical scale metaplasia production.

Summary of the invention

In view of the above shortcomings of the prior art, the present invention utilizes biological safety good and widely used multienzyme catalytic body Cream-improves by introducing ATP regeneration cycle system in multienzymatic reaction system in systemNDisaccharides synthesis and substrate utilization ratio.This hair It is bright to provide a new cream-NDisaccharides route of synthesis, for cream-NThe large-scale industrial production of disaccharides is laid a good foundation, and is had Important economic value and social benefit.

The first aspect of the invention provides a kind of synthesis cream-NThe method of disaccharides, which comprises

In containing galactolipin (Gal), acetylglucosamine (GlcNAc), the reaction system that lactose (Lac) is substrate, it is added half Galactokinase (GalK), cream-NDisaccharides phosphorus solution enzyme (LNBP) preparation cream-NDisaccharides；

In above-mentioned reaction system, it is additionally added acetyl phosphate, acetokinase (ACK) carries out the in-situ regeneration of ATP.

Further, the method also includes: separation product creams-NATP, ADP present in disaccharides and system.

Composition principle of the invention is as follows: present invention building is using galactolipin as the multienzyme catalyst system of substrate synthesis cream-N- Disaccharides.Reaction substrate galactolipin generates galactose-1-phosphate under galactokinase enzymatic, while ATP is degraded to ADP；Into one The galactose-1-phosphate of step is in cream-NUnder disaccharides phosphorus solution enzyme (LNBP) effect, cream-is generatedNDisaccharides.At acetokinase (ACK) Under effect, ADP combination acetyl phosphate obtained above is regenerated as ATP, realizes the regeneration of ATP.It is only needed in reactant of the present invention The ATP for putting into low concentration, substantially reduces reaction cost and improves substrate conversion efficiency, avoids high concentration ATP and inhibits gala The effect of sugared kinases, so that substrate realizes conversion completely, and the reaction time shortens.

The second aspect of the invention provides the cream-of above-mentioned synthetic method preparation synthesisNDisaccharides.In addition, being based on this hair Bright synthetic method synthesizes all containing and/or with cream-NThe oligosaccharides or polysaccharide that disaccharides is skeleton are also in guarantor of the invention Within the scope of shield.

Advantageous effects of the present invention: the present invention passes through oligosaccharides synthesized by two-step catalysis using galactolipin as raw material substrate Structure includes cream-NDisaccharides, it is all with cream-at the same time it can also synthesizeNDisaccharides is the oligosaccharides or polysaccharide of skeleton, product system For at low cost, practical value is high.The present invention good and widely used multienzyme catalyst system using biological safety, by multienzyme ATP regeneration cycle system is introduced in reaction system, establishes simplified and economic enzymatic clarification cream-NDisaccharides is regenerated with ATP Coupling system improves cream-NDisaccharides synthesis and substrate utilization ratio.

To sum up, the present invention provides a new creams-NDisaccharides route of synthesis, for cream-NDisaccharides and the like it is big Technical scale production is laid a good foundation, and has important economic value and social benefit.The raw materials used in the present invention is easy to get simultaneously, closes It is efficiently mild, simple and easy to do at method, at low cost, environmentally protective, suitable industrialization production, the valence with good practical application Value.

Detailed description of the invention

The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.

Fig. 1 is the present invention with galactose substrate two-step catalysis generation cream-NThe schematic diagram of disaccharides.

Fig. 2 is cream-under 5 mM ATP initial amounts after introducing ATP regeneration cycle in the embodiment of the present invention 2NDisaccharides synthesis Compare figure.

Fig. 3 is cream-under 7.5 mM ATP initial amounts after introducing ATP regeneration cycle in the embodiment of the present invention 3NDisaccharides synthesis Compare figure.

Specific embodiment

It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field The identical meanings of understanding.

It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.It should be understood that protection model of the invention It encloses and is not limited to following specific specific embodiments；It is also understood that term used in the embodiment of the present invention is to retouch Specific specific embodiment is stated, rather than limiting the scope of protection of the present invention.If not in following detailed description The experimental method of actual conditions is indicated, it is this usually according to the conventional method and condition of the molecular biology in art technology Technology and condition have complete explanation in the literature.See, for example, Sambrook et al., described in " molecular cloning: laboratory manual " Technology and condition, or according to the normal condition proposed by manufacturer.

As previously mentioned, current human milk oligosaccharides are difficult with chemical method scale and are combined to, and use the heterologous life of biotechnology Human milk oligosaccharides are produced, in addition to universal production efficiency is not high, and are also easy to produce food safety question.

In view of this, providing a kind of synthesis cream-in the specific embodiment of the present inventionNThe method of disaccharides, it is described Method includes:

In containing galactolipin, acetylglucosamine, the reaction system that lactose is substrate, galactokinase, cream-is addedNDisaccharides Phosphorus solution enzyme preparation cream-NDisaccharides；

In above-mentioned reaction system, it is additionally added acetyl phosphate, acetokinase carries out the in-situ regeneration of ATP.The present invention is directed to gala Consume a molecule ATP energy in the kinase catalytic step of sugar, building with but do not limit to and acetokinase realizes ATP energy in acetyl phosphate Amount regeneration.The results show of the present invention can be realized ATP regeneration cycle and substrate by introducing energy regeneration circulation Full conversion, to effectively improve cream-NThe combined coefficient of disaccharides.

In still another embodiment of the invention, the method also includes: separation product cream-NDisaccharides and reaction system Present in ATP, ADP.

In the present invention, the source of used enzyme is not particularly limited, and be can be the bacterium of nature, is also possible to It extracts and obtains in the microbial cells such as yeast or fungi；It can be and obtained by genetic recombination by genetic engineering bacterium production；It can be with It is to be obtained by being extracted in the plant tissue of nature or animal tissue.The product form of enzyme is also not particularly limited, can be solid, Powder, liquid or the immobilised enzymes being fixed on by method physically or chemically on carrier；Enzyme can be commercially available commodity, It can be the product of enterprise or custom setup.

In still another embodiment of the invention, the galactokinase, cream-NDisaccharides phosphorus solution enzyme (LNBP) and acetic acid Kinases is all made of genetic recombination and produces acquisition by genetic engineering bacterium.

Specifically, the production method includes: difference Clone Origin in galactokinase, cream-NDisaccharides phosphorus solution enzyme and second Acid kinase expression vector；By cultivating, inducing the host of corresponding expression vectors, target zymoprotein is obtained.

In still another embodiment of the invention, the expression vector be viral vectors, plasmid, bacteriophage, phasmid, Any one or more in sticking grain, F sticking grain, bacteriophage or artificial chromosome；Viral vectors may include adenovirus vector, reverse Viral vectors or adeno-associated virus vector are recorded, artificial chromosome includes bacterial artificial chromosome (BAC), the derivative load of bacteriophage P1 Body (PAC), yeast artificial chromosome (YAC) or artificial mammalian chromosome (MAC)；Further preferably plasmid；More into one Step is preferably pET-28a plasmid；

In still another embodiment of the invention, the host includes but is not limited to bacterium, fungi and eukaryocyte, further Selected from Escherichia coli, bacillus, bacillus subtilis, saccharomyces cerevisiae, trichoderma reesei and penicillium oxalicum；Further Preferably e. coli bl21 (DE3).

In still another embodiment of the invention, the production method include: respectively Clone Origin in galactokinase, LNBP and acetokinase expression plasmid pET28a-galk, pET28a-Lnbp and pET28a-ack；By cultivating, inducing respective table Target zymoprotein is obtained by purifying (preferably using ni-sepharose purification) up to BL21 (DE3) bacterial strain of plasmid.

Wherein, galactokinase source strain includes but is not limited to Escherichia coli；The source LNBP strain includes but not It is limited to Bifidobacterium；The source ACK strain includes but is not limited to Escherichia coli.

In still another embodiment of the invention,

The amino acid sequence of the galactokinase is as shown in SEQ ID No.1；

The amino acid sequence of the LNBP is as shown in SEQ ID No.2；

The amino acid sequence of the acetokinase is as shown in SEQ ID No.3.

In still another embodiment of the invention, ATP is also contained in reaction system, ATP concentration described further is 5- 15mM(is preferably 7.5mM).

Also containing magnesium ion, (it is preferable to use MgCl in reaction system₂) and Tris-HCl buffer.

In still another embodiment of the invention, the MgCl₂Concentration is that 1-10 mM(is preferably 3 mM)；；It is described The concentration of Tris-HCl buffer is that 10-200mM(is preferably 100 mM).

In still another embodiment of the invention, the reaction temperature of reaction system is 25-45 DEG C, pH value in reaction 5.8- 7.5。

In still another embodiment of the invention, the galactolipin, acetylglucosamine concentration of substrate are 10-20 mM。

In still another embodiment of the invention, the galactokinase enzyme concentration is 1-10U/mL.

In still another embodiment of the invention, the LNBP enzyme concentration is 100-300U/mL.

In still another embodiment of the invention, acetokinase concentration needed for ATP in-situ regeneration is 1-10 U/mL, Acetyl phosphate concentration is 2.5-5 mM.

In still another embodiment of the invention, the cream-of above-mentioned synthetic method synthesis is providedNDisaccharides.In addition, being based on Synthetic method of the invention synthesizes all containing with cream-NThe oligosaccharides or polysaccharide that disaccharides is skeleton are also in guarantor of the invention Within the scope of shield.

Explanation is further explained to the present invention by the following examples, but is not construed as limiting the invention.It should be understood that These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.Actual conditions are not specified in the following example Test method, carry out usually according to normal condition.