阅读说明：本技术 一种α-葡聚糖寡糖的制备方法 (Preparation method of alpha-glucan oligosaccharide ) 是由 马江锋 姜岷 张振龙 方艳 吴昊 董维亮 于 2021-08-16 设计创作，主要内容包括：本发明属于寡糖的制备技术领域,具体涉及一种酶法制备α-葡聚糖寡糖的方法。将从细丽毛壳菌中提取的α-D-右旋糖苷酶基因SEQ ID NO.1导入到毕赤酵母GS115中进行表达,然后用分离得到的右旋糖苷酶将低分子量右旋糖苷进行降解,通过控制酶的添加量和反应时间来控制合成α-葡聚糖寡糖,然后通过膜过滤、冷冻干燥得到分子量1kDa左右的α-葡聚糖寡糖。传统酸降解法具有耗能率高、氯化物残留高且设备易腐蚀等问题。该方法反应可控、工艺简单和原料利用率高等优点,且该方法产物均一,不产生还原性单糖。(The invention belongs to the technical field of oligosaccharide preparation, and particularly relates to a method for preparing alpha-glucan oligosaccharide by an enzyme method. The alpha-D-dextranase gene SEQ ID NO.1 extracted from chaetomium elegans is introduced into pichia pastoris GS115 for expression, then the dextran enzyme obtained by separation is used for degrading the low molecular weight dextran, the synthesis of alpha-glucan oligosaccharide is controlled by controlling the addition amount and the reaction time of the enzyme, and then the alpha-glucan oligosaccharide with the molecular weight of about 1kDa is obtained by membrane filtration and freeze drying. The traditional acid degradation method has the problems of high energy consumption rate, high chloride residue, easy corrosion of equipment and the like. The method has the advantages of controllable reaction, simple process, high utilization rate of raw materials and the like, and the product of the method is uniform and does not produce reducing monosaccharide.)

1. A method for preparing alpha-glucan oligosaccharide by an enzyme method is characterized by comprising the following steps: the method comprises the following steps:

(1) integrating the alpha-D-dextranase gene as shown in SEQ ID NO.1 to a yeast protein expression plasmid vector to obtain a recombinant plasmid vector containing a target gene;

(2) preparation ofE.coliDh5 α competence;

(3) introducing the plasmid containing the alpha-D-dextranase gene obtained in the step (1) intoE.coliDh5 α;

(4) subjecting the product obtained in step (3)E.coliDh5 alpha, and then cultured fromE.coli Extracting plasmids from Dh5 alpha;

(5) linearizing the plasmids extracted in the step (4);

(6) preparing pichia pastoris GS115 competence;

(7) introducing the linearized plasmid obtained in the step (5) into pichia pastoris GS 115;

(8) fermenting and culturing the pichia pastoris GS115 containing the alpha-D-dextranase gene obtained in the step (7), and centrifuging to obtain a crude enzyme solution of dextranase;

(9) mixing the crude enzyme solution obtained in the step (8) with a dextran solution with low molecular weight according to a proportion for reaction;

(10) performing membrane filtration on the reaction solution in the step (9) to obtain a solution of alpha-glucan oligosaccharide;

(11) and (4) carrying out rotary evaporation and concentration on the solution of the alpha-glucan oligosaccharide obtained in the step (10), and freeze-drying to obtain the alpha-glucan oligosaccharide with the molecular weight of about 1 kDa.

2. The enzymatic method of producing α -glucan oligosaccharides according to claim 1, characterized in that: the alpha-D-dextranase gene in the step (1) is derived from chaetomium elegansChaetomium gracileCGMCC3.3783, the yeast is Pichia pastoris GS115, and the yeast protein expression plasmid vector is pPIC9 k.

3. The enzymatic method of producing α -glucan oligosaccharides according to claim 1, characterized in that: the method described in step (4)E.coli The culture medium used for the Dh5 alpha expansion culture is LB culture medium.

4. The enzymatic method of producing α -glucan oligosaccharides according to claim 1, characterized in that: the restriction endonuclease used for the linearization described in step (5) was ScaI.

5. The enzymatic method of producing α -glucan oligosaccharides according to claim 1, characterized in that: the introduction method in the step (7) is an electrical transformation method of pichia pastoris, the electric shock condition is 2000V, 200 omega and 25 muF, and the discharge time is 4.9 ms.

6. The enzymatic method of producing α -glucan oligosaccharides according to claim 1, characterized in that: the strain activation culture medium used in the fermentation culture in the step (8) is a yeast extract peptone glucose culture medium, YPD culture medium for short, the seed culture medium is BMGY culture medium, and the fermentation culture medium is BMMY culture medium.

7. The enzymatic method of producing α -glucan oligosaccharides according to claim 1, characterized in that: the centrifugation condition in the step (8) is 8000r and 10 min.

8. The enzymatic method of producing α -glucan oligosaccharides according to claim 1, characterized in that: the addition amount of the enzyme in the step (9) is 5U/ml, the mass concentration of the glucan is 10%, and the molecular weight of the low-molecular-weight dextran is about 8 kDa.

9. The enzymatic method of producing α -glucan oligosaccharides according to claim 1, characterized in that: the reaction time in the step (10) is 6 h.

10. The enzymatic method of producing α -glucan oligosaccharides according to claim 1, characterized in that: the filter membrane used in the membrane filtration in the step (10) is a filter membrane with the pore size capable of intercepting 1k of molecular weight.

Technical Field

The invention belongs to the technical field of oligosaccharide preparation, and particularly relates to a preparation method for preparing alpha-glucan oligosaccharide by an enzyme method.

Background

Oligosaccharides are low-polymerization degree saccharides which are polymerized from 2 to 9 monosaccharides and are linked to form a linear chain or a linear chain through glycosidic bonds, and are also called oligosaccharides, and the molecular formula of the oligosaccharides can be generally expressed as (C6H10O5) n (n is 2-9), and the molecular weight of the oligosaccharides is about 300-. The unique structure of the oligosaccharide makes the oligosaccharide have obvious difference with monosaccharide and polysaccharide in the aspects of physicochemical properties, physiological functions and the like. The oligosaccharide and the derivatives thereof are important substances with biological activity, can promote the growth of bifidobacterium, have the characteristics and functions of low calorie, tooth decay resistance, tumor resistance, diabetes prevention and the like, and recently research shows that the oligosaccharide also has the effects of anticoagulation, oxidation resistance, immune system regulation and the like.

Oligosaccharides are ubiquitous in plants in various ways, most of the plants contain more oligosaccharides and similar substances in fruits and seeds, and monosaccharides constituting the oligosaccharides mainly comprise xylose, galactose, glucose, arabinose, fructose and the like. There are thousands of oligosaccharides that have been identified, and they can be classified into homo-oligosaccharides and hetero-oligosaccharides according to the difference in monosaccharides that constitute the oligosaccharides, the homo-oligosaccharides being oligosaccharides polymerized from the same monosaccharide, and the hetero-oligosaccharides being oligosaccharides polymerized from two or more different types of monosaccharides. The alpha-glucan oligosaccharide is homooligosaccharide formed by polymerizing 2-9 glucose monomers, has various biological activities of the oligosaccharide, and is widely applied to the fields of food, medicine, cosmetics and the like.

At present, the industrial preparation method of the alpha-glucan oligosaccharide mainly degrades glucan with high molecular weight into oligosaccharide with lower molecular weight by acid or ultrasonic wave, and the glucanase is used for reducing the viscosity of the polysaccharide in the sugar industry, and no relevant literature research is found for preparing the glucan oligosaccharide. Compared with an acid method and an ultrasonic degradation method, the method for producing glucan oligosaccharide by enzyme catalysis has the advantages of mild enzyme catalysis reaction, easier process control, lower equipment requirement and less energy consumption. And the alpha-glucanase has certain selectivity when degrading the glucan with large molecular weight, and when the molecular weight is less than a certain amount, the glucan oligosaccharide can not be degraded any more, so that the molecular weight of the glucan oligosaccharide product prepared by the enzyme method is more uniform. The method provides experience for realizing large-scale preparation of glucan oligosaccharide by an industrial enzyme method.

Disclosure of Invention

The purpose of the invention is: provides a method for preparing alpha-glucan oligosaccharide by an enzyme method. The method has the advantages of simple and controllable process, high preparation efficiency of the alpha-glucan oligosaccharide, small influence on the structure and biological activity of the alpha-glucan oligosaccharide, realization of separation and mass preparation of oligosaccharide mixtures, and possibility of enzyme preparation for industrial production of the alpha-glucan oligosaccharide.

The preparation method of the alpha-glucan oligosaccharide comprises the following steps:

(1) integrating the alpha-D-dextranase gene as shown in SEQ ID NO.1 to a yeast protein expression plasmid vector to obtain a recombinant plasmid vector containing a target gene;

(2) preparation ofE.coli Dh5 α competence;

(3) introducing the plasmid containing the alpha-D-dextranase gene obtained in the step (1) intoE.coli Dh5 α;

(4) subjecting the product obtained in step (3)E.coliDh5 alpha, and then cultured fromE.coliExtracting plasmids from Dh5 alpha;

(5) linearizing the plasmids extracted in the step (4);

(6) preparing pichia pastoris GS115 competence;

(7) introducing the linearized plasmid obtained in the step (5) into pichia pastoris GS 115;

(8) fermenting and culturing the pichia pastoris GS115 containing the alpha-D-dextranase gene obtained in the step (7), and centrifuging to obtain a crude enzyme solution of dextranase;

(9) mixing the crude enzyme solution obtained in the step (8) with a dextran solution with low molecular weight according to a certain proportion for reaction;

(10) performing membrane filtration on the reaction liquid reacted for a certain time in the step (9) to obtain a solution of alpha-glucan oligosaccharide;

(11) and (3) carrying out rotary evaporation concentration and freeze drying on the solution of the alpha-glucan oligosaccharide obtained in the step (10) to obtain the alpha-glucan oligosaccharide with the molecular weight of about 1 k.

Wherein:

the alpha-D-dextranase gene in the step (1) is derived from Chaetomium gracile (with the number of CGMCC 3.3783).

The yeast in the step (1) is pichia pastoris GS115, and the yeast protein expression plasmid vector is pPIC9 k.

The culture medium used for the E.coli Dh5 alpha expansion culture in the step (4) is LB culture medium. The LB culture medium formula is: 10g/L of tryptone and 5g/L, NaCl10g/L of yeast powder.

The enzyme used for the linearization described in step (5) is abbreviated as ScaI.

The introduction method in the step (7) is an electrical transformation method of pichia pastoris, the electric shock condition is 2000V, 200 omega and 25 muF, and the discharge time is 4.9 ms.

The strain activation culture medium used in the fermentation culture in the step (8) is a yeast extract peptone glucose culture medium, YPD culture medium for short, the seed culture medium is BMGY culture medium, and the fermentation culture medium is BMMY culture medium.

The molecular weight of the low molecular weight dextran in the step (9) is about 8kDa, and the dextran is prepared in the early stage of a laboratory.

The filter membrane used in the membrane filtration in the step (10) is a filter membrane with the pore size capable of intercepting 1k of molecular weight.

Compared with the prior art, the invention has the following beneficial effects:

the preparation method for preparing the alpha-glucan oligosaccharide by the enzyme method has the advantages of simple and controllable process, high preparation efficiency, small influence on the structure and biological activity of the alpha-glucan oligosaccharide, no generation of reducing monosaccharide and realization of efficient separation of the alpha-glucan oligosaccharide.

Drawings

FIG. 1 is a high performance liquid chromatogram of a low molecular weight glucan substrate and glucan oligosaccharide product of the invention.

FIG. 2 is a Fourier infrared spectrum of the product of the invention, alpha-glucan oligosaccharides.

FIG. 3 is the NMR spectrum of the product of the present invention, alpha-glucan oligosaccharide.

FIG. 4 is a carbon spectrum of the product of the present invention, alpha-glucan oligosaccharide.

Detailed Description

The present invention is further described below with reference to examples.

Example 1

The method for constructing the dextran enzyme-producing engineering bacteria described in this embodiment 1 includes the following steps:

constructing a recombinant plasmid expression vector containing a target gene;

amplifying and linearizing the recombinant plasmid expression vector;

preparing pichia pastoris GS115 competence;

introducing the linearized recombinant plasmid vector into pichia pastoris GS 115;

the target gene in the step (1) is derived from an alpha-D-dextranase gene of Chaetomium gracile (with the number of CGMCC 3.3783), and is shown as SEQ ID NO. 1.

The yeast protein expression plasmid vector in the step (1) is pPIC9 k. The alpha-D-dextranase gene was inserted between the SnaB I and EcoRI cleavage sites of pPIC9k and was synthesized by Kinsley.

The recombinant plasmid expression vector amplification method in the step (2) is to introduce the recombinant plasmid into Escherichia coli Dh5 alpha and amplify the recombinant plasmid expression vector by culturing Dh5 alpha. Preparing an LB culture medium: 10g/L of tryptone and 5g/L, NaCl10 g/10 g/L of yeast powder. Dh5 α transformation experiments: mu.l of the recombinant plasmid vector was added to a centrifuge tube containing Dh 5. alpha. competent cells. After ice-cooling for 20min, water bath at 42 ℃ is carried out for 90s, and ice-cooling is carried out for 5 min. Adding 800 μ lLB culture medium, shaking at 37 deg.C and 60r/min, and warm bathing for 45 min. 150 ampicillin-resistant LB plates were plated and incubated overnight at 37 ℃.

The enzyme used for linearization of the recombinant plasmid expression vector in step (2) is ScaI. And (3) plasmid extraction: after overnight culture of E.coli Dh5 α containing the recombinant plasmid expression vector, plasmids were extracted according to the plasmid miniprep kit. Linearization experiment: the enzyme digestion reaction system comprises 2000ng of recombinant plasmid vector, ScaI 2. mu.l, 10 × L5. mu.l and ddH2O, the total volume is 50. mu.l, and the reaction is carried out for 3h at 37 ℃. After the reaction is finished, whether the linearization is complete is detected through gel electrophoresis, and a small amount of agarose gel recovery kit is used for recovering the linearized expression vector fragments.

The preparation method of pichia pastoris GS115 competence in the step (3) is the preparation of competent cells by an electrical transformation method. Preparation of YPD Medium: 22g/L glucose, 10g/L yeast powder, 20g/L tryptone, 115 ℃ and 20min for sterilization for later use. Preparing pichia pastoris competent cells: 50 mul of Pichia pastoris original strain is inoculated into a 5mL YPD test tube, and the strain grows overnight at 30 ℃ and 200 rpm; then taking 150 mul, transferring to a 250mL PYD shake flask, culturing at 30 ℃ and 200rpm until the OD600 is 1.3-1.5, transferring the bacterial liquid in the shake flask to a 100mL centrifuge tube, centrifuging at 4 ℃ and 5000rpm for 10min, pouring off the supernatant, and then re-suspending the cells by using 50mL precooled sterile double distilled water; centrifugation as above, after removal of the supernatant, the cells were resuspended in 25 ml of pre-cooled sterile double distilled water; centrifugation as above, decanting the supernatant and resuspending the cells with 4ml of pre-cooled 1M sorbitol; centrifugation as above, decanting the supernatant and resuspending the cells with 100. mu.l of pre-cooled 1M sorbitol; the prepared competent cells were dispensed into 1.5ml EP tubes per 60. mu.l.

The transformation method in the step (4) is an electric transformation method. Pichia pastoris GS115 electrotransformation experiment: mixing 60 mul of yeast competent cells with 10-15 mul of linearized fragments with the concentration of 200ng/ml, and transferring into a precooled 0.2cm3 electric transfer cup; placing on ice for 5min, and then clicking, wherein the electric shock condition is 2000V, 200 omega and 25 muF, and the discharge time is 4.9 ms; immediately adding 1ml of precooled 1M sorbitol into an electric rotating cup after electric shock, and transferring the content into a sterilized centrifuge tube; standing for 2h at 30 ℃; and taking 100 mul of a geneticin G418YPD plate, and picking a single colony for verification in 3-4 days.

Example 2 fermentation experiment of engineering bacteria for producing dextranase

Preparing a seed culture medium BMGY: 10g/L of yeast powder, 20g/L of tryptone, K2HPO42.23g/L, KH2PO4g/L, 10ml/L, YNB 134g/L of glycerol, 4 x 10-5g/L of biotin and 10g/L of histidine.

Preparing a fermentation medium BMMY: 10g/L yeast powder, 20g/L tryptone, K2 HPO42.23g/L, KH2PO4g/L, YNB 134g/L biotin 4 x 10-5g/L histidine 10g/L and 2% methanol.

Defining enzyme activity: the amount of 1. mu. mol glucose produced by degradation of the substrate per ml of enzyme solution in 1 min.

And (3) enzyme activity detection experiment, namely taking acetic acid-sodium acetate buffer solution with pH of 5 as a solvent, and preparing dextran TB7 into sugar solution with mass fraction of 3%. 100 μ l of the crude enzyme solution and 900 μ l of the prepared sugar solution were mixed in a 2ml centrifuge tube and reacted in a 55 ℃ water bath for 15 min. After the reaction, 100. mu.l of the reaction mixture was put into a 5ml centrifuge tube, 300. mu.l of water and 300. mu.l of DNS solution were added thereto, boiled for 5min, and diluted to 4ml with water. Reading is detected by an ultraviolet spectrophotometer at the wavelength of 540nm, and the enzyme activity is calculated by a standard curve.

Fermentation experiment: inoculating 50ml of recombinant Pichia pastoris engineering bacteria stored at-80 ℃ into a 5ml YPD test tube; after culturing for 16h, inoculating 500 μ l of the mixture to 50ml of seed culture medium BMGY for culturing; when the OD600 of the seed liquid is 4-5, transferring the bacterial liquid into a sterilized 100ml centrifugal tube, and centrifuging at 4 ℃ and 6000rpm for 10 min; pouring out the supernatant, re-suspending the thallus with partial BMMY culture medium, transferring to 100ml fermentation culture medium, fermenting at 30 deg.C and 200rpm, detecting thallus concentration and enzyme activity every day, and stopping fermentation when the enzyme activity increase amplitude is not obvious; centrifuging at 4 deg.C and 8000rpm for 10min, and collecting supernatant as crude enzyme solution for catalysis.

Example 3

The method for preparing alpha-glucan oligosaccharide described in example 3 consists of the following steps:

(1) mixing the crude enzyme solution obtained by centrifuging the fermentation liquor and dextran TB7 concentrated solution with molecular weight of about 8kDa prepared in the early stage of a laboratory in a reaction container for reaction.

(2) After the reaction, the reaction solution was taken out and separated and purified.

(3) Concentration and crystallization.

The reaction system in the reactor in the step (1) is 450ml of crude enzyme solution with the mass fraction of 10% dextran TB 7500 ml, the mass fraction of 250ml of acetic acid-sodium acetate buffer solution and the enzyme activity of 13U/ml, and the total volume is 1.2L. The enzyme activity concentration of the whole catalytic system is 5U/L, the pH value is adjusted to 5.0, and the reaction is carried out for 4 hours at the temperature of 55 ℃.

The separation and purification experiment in the step (2) is as follows: the reaction solution was taken out of the reaction vessel and boiled for 10min to denature the proteins in the reaction solution, and the residual cells, denatured proteins and some other solid insoluble matter in the reaction solution were removed by filtration through a microfiltration membrane. Removing some protein molecules and glucan with large molecular weight by using an ultrafiltration membrane capable of intercepting 5kDa through membrane pores, and collecting the permeate containing the alpha-glucan oligosaccharide.

The concentration operation described in step (3) is to evaporate most of the water by a rotary evaporator to concentrate the α -glucan oligosaccharide solution. Freezing and crystallizing the concentrated alpha-glucan oligosaccharide solution, putting the solution into a freezing vacuum drier for 2 days, and grinding the solution into powder. The total yield of oligosaccharide was calculated to be 65.65%.

Sequence listing

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