阅读说明：本技术 复杂序列壳二糖的制备方法 (Preparation method of chitobiose with complex sequence ) 是由 于华华 郝文桐 李克成 李荣锋 马玉珍 邢荣娥 刘松 李鹏程 于 2020-12-11 设计创作，主要内容包括：本发明属于海洋生物工程技术领域,具体涉及一种复杂序列壳二糖的制备方法。具有特定脱乙酰基模式的几丁质脱乙酰酶与几丁二糖于缓冲体系内进行酶反应,而后脱盐、浓缩冻干,即得复杂序列壳二糖；其中,具有特定脱乙酰基模式的几丁质脱乙酰酶为通过重组表达获得的NodB或VcCOD。本发明使用具有单点脱乙酰模式的几丁质脱乙酰酶对几丁二糖进行温和的酶解反应,避免了副产物的产生和对环境的污染,该方法简便快速。(The invention belongs to the technical field of marine bioengineering, and particularly relates to a preparation method of chitobiose with a complex sequence. Performing enzyme reaction on chitin deacetylase with a specific deacetylation mode and chitobiose in a buffer system, desalting, concentrating and freeze-drying to obtain chitobiose with a complex sequence; wherein the chitin deacetylase with a specific deacetylation pattern is NodB or VcCOD obtained by recombinant expression. The invention uses chitin deacetylase with single-point deacetylation mode to carry out mild enzymolysis reaction on the chitobiose, thereby avoiding the generation of byproducts and the pollution to the environment.)

1. A preparation method of chitobiose with complex sequence is characterized in that: performing enzyme reaction on chitin deacetylase with a specific deacetylation mode and chitobiose in a buffer system, desalting, concentrating and freeze-drying to obtain chitobiose with a complex sequence; wherein the chitin deacetylase with a specific deacetylation pattern is NodB or VcCOD obtained by recombinant expression.

2. The method of preparing chitobiose having a complex sequence according to claim 1, wherein: performing enzyme reaction on the chitin deacetylase NodB and the chitobiose with the specific deacetylation mode in a buffer system, reacting for 10-24h at 30-40 ℃, desalting, concentrating and freeze-drying to obtain chitobiose GlcN-GlcNAc with a complex sequence, wherein the structural formula is as follows:

wherein the mass ratio of NodB to chitobiose in the buffer system is 1:20-1: 30;

the buffer system is 15-25mM of Mops, 8-10mM of DTT and 0.5-1.5mM of MnSO₄，pH 6-8。

3. The process for the preparation of chitobiose having a complex sequence according to claim 1 or 2, wherein: respectively mixing the chitin deacetylase VcCOD with the specific deacetylation mode with chitobiose or complex sequence chitobiose GlcN-GlcNAc, reacting in 10mM-20mM ammonium bicarbonate buffer solution with pH of 6-8 at 30-40 ℃ for 10-24h, desalting, concentrating and freeze-drying to obtain complex sequence chitobiose;

the raw material is chitobiose to obtain chitobiose GlcNAc-GlcN with a complex sequence, and the structural formula is as follows:

the raw material is complex sequence chitobiose GlcN-GlcNAc to obtain complex sequence chitobiose GlcN-GlcN, the structural formula is as follows:

wherein the mass ratio of VcCOD in the buffer system to chitobiose or chitobiose GlcN-GlcNAc with complex sequence is 1:20-1: 30.

4. The method of preparing chitobiose having a complex sequence according to claim 1, wherein: the chitin deacetylase with the specific deacetylation mode is specifically a gene plasmid for synthesizing deacetylase NodB or VcCOD through recombinant expression, separation and purification, is transferred into competent cells, is subjected to shake culture, and is subjected to OD (OD)₆₀₀The value is 0.4-1.2, the temperature of the system is reduced to 15-18 ℃, an inducer IPTG is added for induction for 15-24h, thalli are collected by centrifugation, cells are broken, supernatant is separated by centrifugation, and the supernatant is collected and is used for purifying protein by a nickel column for later use.

5. The method of preparing chitobiose having a complex sequence according to claim 1, wherein: the desalting is to process reactants through a desalting column; the eluent is purified water.

6. The method of preparing chitobiose having a complex sequence according to claim 5, wherein: the desalting column chromatographic packing is gel exclusion packing.

Technical Field

The invention belongs to the technical field of marine bioengineering, and particularly relates to a preparation method of chitobiose with a complex sequence.

Background

Chitosan oligosaccharide is a linear oligosaccharide formed by connecting D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) through beta-1, 4 glycosidic bonds. Chitosan oligosaccharide is found to have various physiological activities, such as anti-tumor, antibacterial, anti-inflammatory, antioxidant, growth promotion, blood sugar and blood fat regulation, intestinal flora activation and the like. The activity of the chitosan oligosaccharide is closely related to the structure of the chitosan oligosaccharide, such as the degree of polymerization DP, the degree of acetylation DA, the acetylation pattern PA and the like. At present, the chitosan oligosaccharide activity research generally adopts mixtures, and it is difficult to know which molecule or molecules are in effect, and further difficult to research the biological mechanism. Moreover, the biological activity of the chitosan oligosaccharide is not only dependent on the polymerization degree and the acetyl degree but also influenced by the sequence of the chitosan oligosaccharide. It has been reported that different isomers have different effects on promoting angiogenesis with the same degree of polymerization. Therefore, the research of chitosan oligosaccharide is no longer limited to polymerization degree and acetyl degree, and the acquisition of chitosan oligosaccharide with determined sequence is necessary for researching the biological activity mechanism of chitosan oligosaccharide. At present, chitobiose and chitobiose can be obtained by separation and purification, the complex sequence chitobiose can only obtain the activity by a chemical synthesis method, but the sugar chain synthesis needs a large amount of hydroxyl and amino protection and deprotection reactions, the steps are very complex, and the yield is very low.

Disclosure of Invention

The invention aims to provide a preparation method of chitobiose with a complex sequence.

In order to achieve the purpose, the invention adopts the technical scheme that:

a preparation method of chitobiose with complex sequence comprises performing enzyme reaction on chitin deacetylase with a specific deacetylation mode and chitobiose in a buffer system, desalting, concentrating, and lyophilizing to obtain chitobiose with complex sequence; wherein the chitin deacetylase with a specific deacetylation pattern is NodB or VcCOD obtained by recombinant expression.

Performing enzyme reaction on the chitin deacetylase NodB and the chitobiose with the specific deacetylation mode in a buffer system, reacting for 10-24h at 30-40 ℃, desalting, concentrating and freeze-drying to obtain chitobiose GlcN-GlcNAc with a complex sequence, wherein the structural formula is as follows:

wherein the mass ratio of NodB to chitobiose in the buffer system is 1:20-1: 30;

the buffer system is 15-25mM of Mops, 8-10mM of DTT and 0.5-1.5mM of MnSO₄，pH 6-8。

Respectively mixing the chitin deacetylase VcCOD with the specific deacetylation mode with chitobiose or complex sequence chitobiose GlcN-GlcNAc, reacting in 10mM-20mM ammonium bicarbonate buffer solution with the pH of 6-8 at the temperature of 30-40 ℃ for 10-24h, desalting, concentrating and freeze-drying to obtain complex sequence chitobiose;

the raw material is chitobiose to obtain chitobiose GlcNAc-GlcN with a complex sequence, and the structural formula is as follows:

the raw material is complex sequence chitobiose GlcN-GlcNAc to obtain complex sequence chitobiose GlcN-GlcN, the structural formula is as follows:

wherein the mass ratio of VcCOD in the buffer system to chitobiose or chitobiose GlcN-GlcNAc with complex sequence is 1:20-1: 30.

Wherein NodB acts on the acetyl group at the first position of the non-reducing end of the chitosan oligosaccharide chain, and VcCOD acts on the acetyl group at the second position of the non-reducing end of the chitosan oligosaccharide chain. And then, allowing two deacetylases to act on the raw material chitobiose GlcNAc-GlcNAc, allowing NodB to act independently to obtain GlcN-GlcNAc, allowing VcCOD to act independently to obtain GlcNAc-GlcN, allowing the VcCOD to act simultaneously to obtain GlcN-GlcN, desalting the obtained sample by a desalting column, and concentrating.

The chitin deacetylase with the specific deacetylation mode is specifically a gene plasmid for synthesizing deacetylase NodB or VcCOD through recombinant expression, separation and purification, is transferred into competent cells, is subjected to shake culture, and is subjected to OD (OD)₆₀₀The value is 0.4-1.2, the temperature of the system is reduced to 15-18 ℃, an inducer IPTG is added for induction for 15-24h, thalli are collected by centrifugation, cells are broken, supernatant is separated by centrifugation, and the supernatant is collected and is used for purifying protein by a nickel column for later use.

The competent cells were BL21(DE3) and the antibiotic was ampicillin sodium.

The desalting is to carry out desalting column treatment on the reactant through eluent; the eluent is purified water. The desalting column Sephadex G10, the flow rate is 0.2-0.4 ml/min.

The desalting column chromatography packing is a gel exclusion packing, such as Sephadex G10, Bio gel P2, and the like.

The invention has the advantages that:

1. the invention uses chitin deacetylase with single-point deacetylation mode to carry out mild enzymolysis reaction on the chitobiose, thereby avoiding the generation of byproducts and the pollution to the environment.

2. The disaccharide prepared by the invention can obtain chitosan oligosaccharide monomers with higher polymerization degree and different sequences by a method for recombinant expression of protein, and the method has important significance for further activity screening and activity mechanism clarification of chitosan oligosaccharide; wherein, the recombinant expression protein adopts low-temperature induction, avoids the existence of an inclusion body of the protein, purifies the pure protein and ensures the activity and reaction conditions of the pure protein.

Drawings

FIG. 1 is a mass spectrum diagram of a complex sequence chitobiose GlcNAc-GlcN obtained by the embodiment of the invention; wherein, A is a primary mass spectrum, B is a secondary cleavage fragment of the molecular ion peak 576 after AMAC labeling, GlcN-AMAC: 179+194+1 ═ 374.

FIG. 2 is a mass spectrum diagram of the obtained complex sequence chitobiose GlcN-GlcNAc; wherein, A is a primary mass spectrum, B is a secondary cleavage fragment of an AMAC-labeled molecular ion peak 576, GlcNAc-AMAC: 221+194+1 is 416.

FIG. 3 is a mass spectrum diagram of GlcN-GlcN of the complex-sequence chitodisaccharide obtained by the embodiment of the invention.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The present invention is further described with reference to the drawings attached to the specification, and the scope of the present invention is not limited to the following examples.

Example 1

Construction of chitin deacetylase with a specific deacetylation pattern:

construction of NodB plasmid:

carrying out enzyme digestion on rhizobium GRH2(NCBI acc. No. AJW76244.1) and a cloning site NdeI/XhoI of a basic vector pET-22b (+), and then connecting the two through T4 to obtain a chitin deacetylase NodB plasmid; the expressed protein can act on the acetyl at the first position of the non-reducing end of the chitosan oligosaccharide chain.

Construction of the VcCDA plasmid:

carrying out enzyme digestion on the cloning sites SalI/Xho of vibrio cholerae (NCBI acc. No. AAF94439.1) and a basic vector pET-22b (+), and then connecting the cloning sites SalI/Xho through T4 to obtain a chitin deacetylase VcCDA plasmid; the expressed protein can act on acetyl at the second position of the non-reducing end of the chitosan oligosaccharide chain.

Both plasmids were synthesized by Biotechnology engineering (Shanghai) GmbH.

Example 1

The NodB plasmid (NCBI acc. No. AJW76244.1, cloning site NdeI/XhoI, vector pET-22b (+), synthesized by Biotechnology engineering (Shanghai) Co., Ltd.) 3. mu.L was added to BL21(DE3) competent cells, and shake culture was performed at 37 ℃Culturing for more than 2h, and measuring OD₆₀₀The value was around 0.6, after which the temperature was lowered to 16 ℃ and the rotation speed was reduced to 120rpm, IPTG was added to a final concentration of 0.1mM, and induction was carried out for 15 h. And (3) centrifuging to collect thalli, breaking cells, centrifuging for 20min at 4 ℃ and 12000rpm, collecting supernatant, and purifying by a nickel column to obtain the purified NodB protein.

And (3) passing through a nickel column: before purification, filtering the supernatant by using a 0.22uM filter, injecting a Binding buffer balanced nickel column with 10 times of column volume, injecting the supernatant filtered by the filter, injecting a Washing buffer with 10 times of column volume to wash and remove foreign proteins, injecting an Elution buffer with 2.5 times of column volume, and collecting the effluent, namely the protein.

Wherein:

Binding buffer：50mM Tris，150mM NaCl,pH 8.0；

washing Buffer: 50mM Tris, 150mM NaCl, 20mM imidazole, pH 8.0;

elution Buffer: 50mM Tris, 150mM NaCl, 250mM imidazole, pH 8.0.

1mg of chitobiose and 50ug of the purified NodB protein obtained above were reacted in 300ul of buffer system at 37 ℃ for 20 hours. Desalting with desalting column Sephadex G10 after reaction, collecting purified water with flow rate of 0.5ml/min for 10min, collecting sugar component in 45-52 tubes, collecting salt component in 52 tubes, and vacuum freeze drying to obtain chitobiose GlcN-GlcNAc.

The buffer system is 20mM of Mops, 10mM of DTT and 1mM of MnSO₄，pH 6-8。

Example 2

Adding 3 μ L of the above VcCOD plasmid into BL21(DE3) competent cells, shake-culturing at 37 deg.C for more than 2h, and measuring OD₆₀₀The value was around 0.6, after which the temperature was lowered to 16 ℃ and the rotation speed was reduced to 120rpm, IPTG was added to a final concentration of 0.1mM, and induction was carried out for 15 h. And (3) centrifugally collecting thalli, ultrasonically breaking cells for 20min, separating supernatant, centrifugally separating at 4 ℃ and 12000rpm for 20min, collecting supernatant, purifying protein by a nickel column, and obtaining purified VcCOD protein.

1mg of chitobiose and 50ug of the obtained purified VcCOD protein react in 500ul of ammonium bicarbonate buffer solution at 37 ℃ for 20 hours, after the reaction is finished, the mixture is desalted by a desalting column Sephadex G10, the eluent is purified water, the flow rate is 0.5ml/min, one tube is collected for 10min, the sugar component is placed in a 45-52 tube, the salt component is placed in a 52 tube, and the obtained sugar component is concentrated, frozen and dried by a vacuum freeze dryer to obtain the chitobiose GlcNAc-GlcN.

The ammonium bicarbonate buffer solution is 10mM ammonium bicarbonate buffer solution.

Example 3

1mg of chitobiose GlcN-GlcNAc obtained in example 1 and 50ug of the purified VcCOD protein obtained above were reacted in 500ul of ammonium bicarbonate buffer at 37 ℃ for 20 hours, desalted by desalting column Sephadex G10 after the reaction was finished, the eluent was purified water at a flow rate of 0.5ml/min, one tube was collected for 10min, the sugar fraction was placed in 45-52 tubes, the salt fraction was placed in 52 tubes, and the sugar fraction obtained above was concentrated, freeze-dried by a vacuum freeze-dryer to obtain chitobiose GlcN-GlcN.

Respectively adopting mass spectrum AMAC to mark and detect a molecular weight sequence of the chitobiose obtained in the embodiment; and combines an AMAC labeling method and a secondary mass spectrum to read the sugar chain sequence, which specifically comprises the following steps:

1mg of the sugar obtained in the above embodiment is respectively dissolved in 20ul of AMAC solution and mixed evenly, 20ul of sodium cyanoborohydride solution is added, shaking is carried out evenly, the reaction solution is subjected to water bath at 90 ℃, reaction is carried out for 30min in a dark place, freeze-drying is carried out, and primary mass spectrum characterization and molecular ion peak secondary mass spectrum analysis are carried out (see figure 1-3).

As can be seen from FIGS. 1-3, in the mass spectrum of the complex sequence chitobiose GlcNAc-GlcN of FIG. 1, A is the first-order mass spectrum, and the molecular ion peak of AD is 383. It is proved that the chitobiose does remove an acetyl group, B is a secondary cleavage fragment of the molecular ion peak 576 after the AMAC is marked, the AMAC is marked at the reducing end of the sugar chain, the molecular weight of the AMAC is 194Da, and the labeled molecular ion peak 576(382+194) is cleaved. The fragment after GlcNAc-GlcN cleavage is GlcN-amac: 179+194+1 ═ 374. Indicating that the reducing end is a GlcN, it can be concluded that the other fragment is a GlcNAc at the non-reducing end. I.e. the sequence is AD.

In the mass spectrum of the complex sequence chitobiose GlcN-GlcNAc in FIG. 2, A is the first-order mass spectrum, and the molecular ion peak of DA is 383. It is proved that the chitobiose does remove an acetyl group, B is a secondary cleavage fragment of the molecular ion peak 576 after the AMAC is marked, the AMAC is marked at the reducing end of the sugar chain, the molecular weight of the AMAC is 194Da, and the labeled molecular ion peak 576(382+194) is cleaved. The fragment after GlcN-GlcNAc cleavage is GlcNAc-amac: 221+194+1 is 416. Indicating that the reducing end is a GlcNAc, it can be concluded that the other fragment is a GlcN at the non-reducing end. I.e., the sequence is DA.

FIG. 3 is a first-order mass spectrum of GlcN-GlcN of complex-sequence chitobiose.