阅读说明：本技术 一种生物制备甘露寡糖的方法 (Method for biologically preparing mannan oligosaccharide ) 是由 杨建刚 孙媛霞 田朝玉 曾艳 马延和 于 2020-06-19 设计创作，主要内容包括：本发明公开了一种生物制备甘露寡糖的方法,涉及构建体外多酶催化反应体系,实现转化甘露糖合成甘露寡糖；本发明还公开了重组谷氨酸棒杆菌的构建方法,并将重组菌株应用于甘露寡糖的发酵合成,实现了以甘露糖为原料发酵法合成甘露二糖和甘露三糖产物,并且实现发酵生产单一产物甘露三糖,便于产物分离纯化,所获得的甘露寡糖可应用于食品、医药、化妆品等领域。(The invention discloses a method for biologically preparing mannan oligosaccharide, which relates to the construction of an in vitro multi-enzyme catalytic reaction system to realize the synthesis of mannan oligosaccharide by converting mannose; the invention also discloses a construction method of the recombinant corynebacterium glutamicum, and the recombinant strain is applied to fermentation synthesis of mannooligosaccharide, so that a method for synthesizing mannobiose and mannotriose products by taking mannose as a raw material through a fermentation method is realized, single product mannotriose is produced through fermentation, separation and purification of the product are facilitated, and the obtained mannooligosaccharide can be applied to the fields of food, medicine, cosmetics and the like.)

1. A method of making a mannooligosaccharide, the method comprising:

1) taking mannose as a substrate, and adopting mannose kinase catalysis to convert the mannose into mannose-1-phosphate;

2) mannose and mannose-1-phosphate are converted into mannose and mannose triose by catalysis of mannose disaccharide phosphorylase.

2. The method of claim 1, wherein the substrate mannose is derived from konjac, starch, cellulose, sucrose, maltose, fructose, glucose, mannitol, or the like.

3. The method according to claim 1 or 2, wherein the substrate mannose is present at a concentration of 1-200g/L, more preferably 5-50g/L, more preferably 8-20g/L, most preferably 20 g/L.

Preferably, the temperature of the catalytic reaction is 10 to 95 ℃, more preferably 20 to 80 ℃, more preferably 30 to 60 ℃, and most preferably 37 ℃.

Preferably, the time for the catalytic reaction is 0.5 to 150 hours, more preferably 1 to 60 hours, more preferably 6 to 48 hours, most preferably 30 hours.

Preferably, the catalytic reaction further comprises buffer, phosphate and metal ions.

4. The method of any one of claims 1 to 3, wherein the mannose kinase comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID No. 1 and having the function of catalyzing the conversion of mannose to mannose-1-phosphate.

5. The method of any one of claims 1 to 3, wherein the mannose disaccharide phosphorylase comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID NO. 2 and having the function of catalyzing the conversion of mannose and mannose-1-phosphate to mannobiose and mannotriose.

6. The method for preparing the mannooligosaccharide is characterized in that mannose kinase and mannose disaccharide phosphorylase are over-expressed in an original strain to construct and obtain engineering bacteria, and the engineering bacteria are used for producing mannose disaccharide and mannose trisaccharide.

7. The method of claim 6, wherein the starting strain simultaneously over-expresses mannose-6-phosphoglucomutase to construct an engineering bacterium, and the engineering bacterium is used for producing mannotriose.

8. The method of claim 6 or 7, wherein the starting strain is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, lactic acid bacteria, Saccharomyces cerevisiae. Preferably, the starting strain is corynebacterium glutamicum.

9. The method of any one of claims 6 to 8, wherein the mannose kinase is encoded by a polynucleotide comprising a polynucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID No. 4; the mannobiose phosphorylase enzyme is encoded by a polynucleotide comprising a polynucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to SEQ ID No. 5; the mannose 6-phosphate mutase is encoded by a polynucleotide comprising a polynucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID No. 6.

10. Use of the engineered bacteria constructed according to the method of any one of claims 6 to 9 in the preparation of mannooligosaccharides.

Technical Field

The invention relates to the technical field of biology, in particular to a method for biologically preparing mannan oligosaccharide, and belongs to the field of biological manufacturing.

Background

Mannooligosaccharides (MOS) and their derivatives are a series of functional compounds found in nature, such as low-degree polymeric saccharides consisting of 2 to 10 mannoses, and because these oligosaccharides have diverse structures, they derive diverse biological functions and play an important role in the industries of food, pharmaceutical and animal feed. Firstly, MOS can also selectively promote the growth of beneficial flora in human intestinal tract, and can be used as a non-nutritive food additive. In addition, the mannobiose can obviously improve and enhance the immunity of the chicken and enhance the lethality to the salmonella, so that the mannobiose can be used as feed to control the infection of the salmonella in poultry.

Mannan from konjac, carob bean gum, guar gum and the like can be used for preparing mannan oligosaccharide by hydrolyzing mannan enzyme, and the method is the main preparation way of mannan oligosaccharide at present. However, the method simply depends on enzymatic hydrolysis or extraction of the mannooligosaccharides and the derivatives thereof, on one hand, products with uniform polymerization degree cannot be obtained, and a complex separation and purification process is often required; on the other hand, the production method relies on natural polysaccharide resources, limiting the diversity of oligosaccharide products. Therefore, a new environment-friendly preparation technology of the mannooligosaccharides is urgently needed to be developed, the mannooligosaccharide products with single products, various configurations and controllable structures are obtained, a low-cost and high-efficiency biosynthesis technology is established, and a foundation is provided for the application of the mannooligosaccharides in a wider range.

Disclosure of Invention

The invention aims to provide a method for preparing mannan oligosaccharide, which takes mannose as a substrate and produces mannan oligosaccharide through in vitro multi-enzyme catalytic reaction catalysis, and the method has the advantages of high yield, environmental protection, single product configuration and the like.

It is characterized in that mannose is taken as a substrate, mannose kinase and mannose phosphorylase are added to carry out multi-enzyme catalytic reaction. In the invention, mannose is taken as a substrate, mannose is catalyzed and converted into mannose-1-phosphate by mannose kinase, and the mannose-1-phosphate and the mannose are catalyzed by mannose phosphorylase to generate mannose disaccharide and mannose trisaccharide.

Preferably, the mannose raw material may be pure mannose or mannose obtained by converting a raw material such as konjac, starch, cellulose, sucrose, maltose, fructose, glucose, mannitol, or the like by an acidolysis or enzymatic conversion method.

Preferably, the concentration of mannose in the multi-enzyme catalyzed reaction is 1 to 200g/L, more preferably 5 to 50g/L, even more preferably 8 to 20g/L, and most preferably 20 g/L.

Preferably, the reaction temperature for the multi-enzyme catalyzed reaction is 10 to 95 ℃, more preferably 20 to 80 ℃, more preferably 30 to 60 ℃, and most preferably 37 ℃.

Preferably, the reaction time of the multi-enzyme catalyzed reaction is 0.5 to 150 hours, more preferably 1 to 60 hours, more preferably 6 to 48 hours, and most preferably 30 hours.

Preferably, buffer solution, phosphate and metal ions are also added in the multi-enzyme catalytic reaction.

Preferably, the mannose kinase is derived from Bifidobacterium longum (SEQ ID NO:1), or the amino acid sequence thereof has at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identity to the mannose kinase from the above source; the mannobiose phosphorylase is derived from Thermoanaerobacterium (SEQ ID NO:2), or has an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the mannobiose phosphorylase from the above source.

The invention also aims to provide a construction method of the microbial recombinant strain for producing the mannooligosaccharide by fermentation.

The construction method of the recombinant strain comprises the following steps:

1) introducing a mannooligosaccharide synthesis pathway consisting of mannose kinase and mannose phosphorylase into a recombinant strain of a microorganism.

2) Enhances mannose 6-phosphate mutase in microorganism body, and promotes intracellular mannose 6-phosphate to be converted into mannose 1-phosphate precursor.

The construction method of the microbial recombinant strain is suitable for various microbial model strains, including microorganisms such as escherichia coli, corynebacterium glutamicum, bacillus subtilis, lactic acid bacteria and saccharomyces cerevisiae.

The third purpose of the invention is to provide a construction method of a corynebacterium glutamicum recombinant strain for fermentation production of mannan-oligosaccharide.

The construction method of the recombinant strain comprises the following steps:

1) introducing mannose kinase MK (SEQ ID NO:1) derived from bifidobacterium longum and mannose phosphorylase MBP (SEQ ID NO:2) derived from thermoanaerobacterium into corynebacterium glutamicum to obtain a corynebacterium glutamicum recombinant strain named as strain MOS 1;

2) introducing mannose-6-phosphoglucomutase ManB (SEQ ID NO:3) derived from Escherichia coli into recombinant Corynebacterium glutamicum MOS1 to obtain recombinant Corynebacterium glutamicum, which is named as MOS 2;

the specific method for recombining the Corynebacterium glutamicum MOS1 in the step 1) comprises the following steps:

a gene encoding mannose kinase MK derived from Bifidobacterium longum (SEQ ID NO:4) and a gene encoding mannose phosphorylase MBP derived from Thermoanaerobacterium (SEQ ID NO:5) were synthesized by Kingsu Jinzhi Biotechnology, Inc., and they were requested to provide a vector pUC57-MK containing the mannose kinase gene and a vector pUC57-MBP containing the mannose phosphorylase gene; PCR amplification of mannose kinase gene with plasmid pUC57-MK as template, PCR amplification of mannose phosphorylase MBP gene with plasmid pUC57-MBP as template, and connection to vector pEC-XK99E to obtain recombinant vector carrying MK gene and MBP gene, named pE-MK-MBP; the recombinant plasmid pE-MK-MBP is introduced into the corynebacterium glutamicum through an electrotransformation mode to obtain a corynebacterium glutamicum recombinant strain MOS 1.

The specific method for recombining the Corynebacterium glutamicum MOS2 in the step 2) comprises the following steps:

the mannose 6-phosphoglucomutase ManB gene (SEQ ID NO:6) derived from Escherichia coli is amplified by PCR, and is connected into a vector pE-MK-MBP to obtain a recombinant vector carrying the mannose kinase MK gene, the mannose disaccharide phosphorylase MBP gene and the mannose 6-phosphoglucomutase ManB gene, which is named as pE-MK-MBP-ManB, and a recombinant plasmid pE-MK-MBP-ManB is introduced into Corynebacterium glutamicum by an electrotransformation mode to obtain a Corynebacterium glutamicum recombinant strain MOS 2.

The fourth object of the present invention is to provide a method for producing mannooligosaccharides by fermentation, which is characterized by culturing the recombinant strains MOS1 and MOS2 of Corynebacterium glutamicum obtained by the third object of the present invention, and producing mannooligosaccharides such as mannobiose, mannotriose, mannotetraose, etc. by fermentation using mannose as a substrate.

In a preferred embodiment, the method is characterized by initial cell density (OD) under fermentation conditions₆₀₀) 0.5 to 60; the temperature is 30-37 ℃; the mannose content is 20-100 g/L.

The corynebacterium glutamicum recombinant strain can be applied to the field of synthesis of mannosyl oligosaccharide and derivatives thereof, and the obtained mannosyl oligosaccharide has wide application prospects in the food and medicine industries.

The present invention will be described in further detail with reference to specific examples.

Drawings

FIG. 1 is a technical scheme for synthesizing mannooligosaccharide.

FIG. 2 shows the HPLC analysis results of the recombinant strain of Corynebacterium glutamicum to synthesize mannooligosaccharides.

Detailed Description

The present invention will be described in further detail with reference to examples.

The percentage concentrations mentioned in the present invention and examples are mass/mass (W/W, unit g/100g) percentage concentrations, mass/volume (W/V, unit g/100mL) percentage concentrations or volume/volume (V/V, unit mL/100mL) percentage concentrations unless otherwise specified.

The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold spring harbor).

Materials or reagents having the same names used in the respective examples are the same unless otherwise specified. The various biological material access approaches described in the examples are provided for the purpose of specific disclosure, and should not be construed as limiting the source of biological material in practicing the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

The primers used in the present invention were synthesized by Jiangsu Jinzhi Biotechnology GmbH.