阅读说明：本技术 一种淀粉转化制备昆布二糖的方法 (Method for preparing laminaribiose by starch conversion ) 是由 游淳 孙尚尚 于 2018-08-08 设计创作，主要内容包括：本发明公布了一种构建体外多酶分子机器,通过多酶级联催化来制备昆布二糖的方法,属于昆布二糖的酶催化制备领域。本发明所公开的昆布二糖的制备方法包括将淀粉中的葡萄糖单元分别通过淀粉磷酸化酶和葡萄糖苷酶转化生成葡萄糖-1-磷酸和葡萄糖,然后葡萄糖-1-磷酸与葡萄糖通过昆布二糖磷酸化酶合成昆布二糖。过程中通过添加促使淀粉完全磷解的其他辅助酶,例如异淀粉酶,可以进一步提高淀粉的利用率以及昆布二糖的最终浓度。该技术方法具有底物廉价易得,生产成本低,产品得率高,分离纯化简单等优势,可实现昆布二糖的规模化生产。(The invention discloses a method for preparing laminaribiose by constructing an in-vitro multienzyme molecular machine and through multienzyme cascade catalysis, belonging to the field of enzymatic preparation of laminaribiose. The preparation method of the laminaribiose disclosed by the invention comprises the steps of converting glucose units in starch into glucose-1-phosphate and glucose by starch phosphorylase and glucosidase respectively, and then synthesizing the laminaribiose by the laminaribiose phosphorylase through the glucose-1-phosphate and the glucose. The utilization of starch and the final concentration of laminaribiose can be further improved by adding other auxiliary enzymes, such as isoamylase, which promote complete starch phospholysis during the process. The method has the advantages of cheap and easily available substrate, low production cost, high product yield, simple separation and purification, and the like, and can realize the large-scale production of laminaribiose.)

1. A method for preparing laminaribiose by enzyme catalysis is characterized in that starch is taken as a substrate, and starch phosphorylase, glucosidase and laminaribiose phosphorylase are added for multi-enzyme catalytic reaction.

2. The method of claim 1, wherein the substrate is isoamylase treated starch.

3. The method of claim 2, wherein the substrate is treated with isoamylase in a system comprising a buffer and metal ions.

4. The method of claim 2 or 3, wherein the isoamylase treatment is performed at 10-99 ℃ for 0.5-72 hours, more preferably at 30-95 ℃ for 1-48 hours, even more preferably at 50-90 ℃ for 1-12 hours, and most preferably at 85 ℃ for 3 hours.

5. The method of any one of claims 1 to 4, wherein a buffer, phosphate, metal ions are also added to the multi-enzyme catalytic reaction.

6. The method according to any of claims 1 to 5, wherein the reaction temperature of the multi-enzyme catalyzed reaction is 10 to 95 ℃, more preferably 20 to 80 ℃, more preferably 30 to 60 ℃, and most preferably 50 ℃.

7. The method according to any of claims 1 to 6, wherein 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, most preferably 12 hours.

8. The method according to any one of claims 1 to 7, wherein the starch phosphorylase, the glucosidase and the laminaribiose phosphorylase are added in a ratio of 1 to 3:1:1 to 3 in the multi-enzyme catalytic reaction.

9. The process according to any one of claims 1 to 8, wherein the starch phosphorylase, the glucosidase and the laminaribiose phosphorylase are added in a ratio of 2:1:3 in the multi-enzyme catalyzed reaction.

10. A method according to any one of claims 1 to 9, wherein the starch phosphorylase is derived from Thermotoga maritima (Thermotoga maritima), Clostridium thermocellum (Clostridium thermocellum), thermus thermophilus (thermmushilus), or has an amino acid sequence which is at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the starch phosphorylase from said source; preferably, the starch phosphorylase is derived from Thermotoga maritima (Thermotoga maritima), 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 a starch phosphorylase derived from Thermotoga maritima (Thermotoga maritima);

preferably, the laminaribiose phosphorylase is derived from Paenibacillus sp, euglena gracilis, Acholeplasma laevis or has an amino acid sequence which is at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the laminaribiose phosphorylase from the above sources; preferably, the laminaribiose phosphorylase is derived from Paenibacillus sp or has an amino acid sequence at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to a laminaribiose phosphorylase derived from Paenibacillus sp.

Preferably, the glucosidase enzyme is derived from Aspergillus niger (Aspergillus niger), beet (Betavulgaris), Paecilomyces lilacinus (Paecilomyces lilacinus), or has an amino acid sequence which is at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to the glucosidase enzyme of the above-mentioned source; preferably, the glucosidase is derived from Paecilomyces lilacinus (Paecilomyces lilacinus) 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 a glucosidase derived from Paecilomyces lilacinus (Paecilomyces lilacinus).

Preferably, the isoamylase is derived from Sulfolobus (Sulfolobus tokodaii), arabidopsis thaliana (arabidopsis thaliana), Flavobacterium sp, or has an amino acid sequence which is at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to that of the isoamylase of the above source; preferably, the isoamylase is derived from Sulfolobus (Sulfolobus tokodaii) or has an amino acid sequence which is at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to an isoamylase derived from Sulfolobus (Sulfolobus tokodaii).

Technical Field

The invention belongs to the field of biological manufacturing, and particularly relates to a method for preparing laminaribiose by using starch as a raw material through an enzymatic method.

Background

Laminaribiose is an oligosaccharide linked by β -1,3 glycosidic bonds, is mainly used in the agricultural field, can be used for promoting seed germination, and can also be used as a natural preservative.

At present, laminaribiose is mainly produced by hydrolyzing polysaccharides such as pine needles or laminarin with traditional dilute acid. The process has low yield and high cost, and can result in high laminaribiose price. Laminaribiose can also be prepared by a chemical process, i.e. laminaribiose is obtained by O-glycosylation from the Koenigs-Knorr process with halo sugar groups as glycosyl donors. However, the final product is not easily purified and the final yield is less than 10%.

With the development of industrial enzyme biotechnology, there have been scientists who have attempted enzymatic synthesis of laminaribiose. Japanese scientists have utilized 3 enzymes (sucrose phosphorylase, glucose isomerase and laminaribiose phosphorylase) to produce laminaribiose from sucrose as a substrate, however, laminaribiose yield is only about 50%, resulting in higher cost for subsequent product separation.

Therefore, it is highly desirable to develop a method for preparing laminarin with low cost, low pollution and high yield.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for preparing laminaribiose, which takes starch as a substrate and produces the laminaribiose through in vitro multi-enzyme catalytic reaction catalysis.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the invention provides a method for preparing laminaribiose by using an enzyme catalysis reaction, which is characterized in that starch is used as a substrate, and starch phosphorylase (α -glucan phosphorylase, EC 2.4.1.1, α GP), glucosidase (α -glucan lyase, EC 3.2.1.20, AG) and laminaribiose phosphorylase (EC 2.4.1.31, LBP) are added to carry out a multi-enzyme catalysis reaction.

Preferably, the starch is a mixture of any one or more of soluble starch, soluble amylose, soluble amylopectin, amylodextrin, maltodextrin, maltopolysaccharide and maltose in any proportion.

Preferably, the concentration of starch in the multi-enzyme catalyzed reaction is 1-200g/L, more preferably 5-50g/L, more preferably 8-20g/L, and most preferably 10 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 50 ℃.

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 12 hours.

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

It will be understood by those skilled in the art that various buffers can be used in the present invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer such as sodium citrate buffer, etc., preferably, the buffer is HEPES buffer. Preferably, the pH of the buffer is 5.0-8.0, more preferably 6.0-7.5, and most preferably 6.5. Preferably, the concentration of the buffer in the reaction system is 10 to 500mM, more preferably 20 to 150mM, still more preferably 50 to 120mM, and most preferably 100 mM.

It will be appreciated by those skilled in the art that various phosphates may be used in the present invention, such as potassium phosphate, sodium phosphate, and the like, preferably the phosphate is potassium phosphate. Preferably, the concentration of phosphate in the reaction system is 1 to 50mM, more preferably 2 to 30mM, still more preferably 5 to 15mM, and most preferably 20 mM.

It will be appreciated by those skilled in the art that various metal ions can be used in the present invention, such as zinc ions, magnesium ions, manganese ions, etc., preferably, the metal ions are zinc ions. Preferably, the concentration of zinc ion in the reaction system is 1 to 20mM, more preferably 2 to 15mM, still more preferably 3 to 10mM, and most preferably 5 mM.

In a preferred embodiment, starch treated with isoamylase (isoamylase, EC 3.2.1.68, IA) is used as a substrate, and starch phosphorylase (α -glucan phosphorylase, EC 2.4.1.1, α GP), glucosidase (α -glucanase, EC 3.2.1.20, AG) and laminaribiose phosphorylase (EC 2.4.1.31, LBP) are added to perform a multi-enzyme catalytic reaction.

Preferably, the isoamylase treatment is carried out at 10 to 99 ℃ for 0.5 to 72 hours, more preferably at 30 to 95 ℃ for 1 to 48 hours, still more preferably at 50 to 90 ℃ for 1 to 12 hours, and most preferably at 85 ℃ for 3 hours.

Preferably, when starch is treated with isoamylase, the concentration of the starch is 1 to 500g/L, more preferably 10 to 300g/L, even more preferably 50 to 250g/L, and most preferably 200 g/L; the amount of isoamylase to be used is 0.1 to 20U/mL, more preferably 0.5 to 10U/mL, still more preferably 1 to 8U/mL, and most preferably 5U/mL.

Preferably, the isoamylase treats the starch in a system containing a buffer, metal ions.

It will be appreciated by those skilled in the art that various buffers can be used in the present invention, such as sodium acetate buffer, HEPES buffer, citrate buffer such as sodium citrate buffer, and the like, preferably, the buffer is sodium acetate buffer. Preferably, the pH of the buffer is from 4.0 to 8.0, more preferably from 4.5 to 6.5, most preferably 5.5. Preferably, the concentration of the buffer in the reaction system is 1 to 50mM, more preferably 2 to 20mM, still more preferably 3 to 10mM, and most preferably 5 mM.

It will be appreciated by those skilled in the art that various metal ions can be used in the present invention, such as zinc ions, magnesium ions, manganese ions, etc., preferably, the metal ions are exemplified by zinc. Preferably, the concentration of zinc ion in the reaction system is 0.01 to 10mM, more preferably 0.1 to 5mM, still more preferably 0.2 to 1mM, and most preferably 0.5 mM.

In the present invention, the starch phosphorylase, glucosidase and laminaribiose phosphorylase may be added in any ratio in the multi-enzyme catalytic reaction.

Preferably, the ratio of the starch phosphorylase, the glucosidase and the laminaribiose phosphorylase added is 1-3: 1: 1-3.

Further preferably, the ratio of starch phosphorylase, glucosidase and laminaribiose phosphorylase added is 2:1: 3.

Preferably, the amount of starch phosphorylase used in the multi-enzyme catalyzed reaction is 0.1-50U/mL, more preferably 0.5-10U/mL, still more preferably 1-5U/mL, and most preferably 2U/mL.

Preferably, the amount of glucosidase used in the multi-enzyme catalyzed reaction is 0.1-50U/mL, more preferably 0.5-10U/mL, more preferably 1-5U/mL, and most preferably 1U/mL.

Preferably, the laminaribiose phosphorylase is used in an amount of 0.1-50U/mL, more preferably 0.5-10U/mL, even more preferably 1-5U/mL, and most preferably 3U/mL in the multi-enzyme catalyzed reaction.

In the present invention, starch phosphorylase, laminaribiose phosphorylase, isoamylase and glucosidase of various sources can be used. For example, the starch phosphorylase may be derived from Thermotoga maritima (Thermotoga maritima), Clostridium thermocellum (Clostridium thermocellum), Thermus thermophilus (Thermus thermophilus), etc., preferably, the starch phosphorylase is derived from Thermotoga maritima; the laminaribiose phosphorylase may be derived from paenibacillus (paenibacillus sp.), Euglena Gracilis (Euglena Gracilis), Acholeplasma (acholeplasia laiwii), etc., preferably, the laminaribiose phosphorylase is derived from paenibacillus; the isoamylase may be derived from sulfolobus (sulfolobutokodaii), Arabidopsis thaliana (Arabidopsis thaliana), Flavobacterium sp, etc., preferably, the isoamylase is derived from sulfolobus; the glucosidase may be derived from Aspergillus niger (Aspergillus niger), beet (Betavulgaris), Paecilomyces lilacinus (Paecilomyces lilacinus), etc., and preferably, the glucosidase is derived from Paecilomyces lilacinus. The present invention can also use starch phosphorylase, laminaribiose phosphorylase, isoamylase and glucosidase having an amino acid sequence at least 60%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% identical to each of the enzymes derived as described above.

The invention takes starch as a substrate, adds starch phosphorylase, glucosidase and laminaribiose phosphorylase to prepare a three-enzyme reaction system, and the enzyme catalysis approach comprises the following steps: converting glucose units in the starch into glucose-1-phosphate and glucose by using starch phosphorylase and glucosidase respectively; converting glucose and glucose-1-phosphate to laminaribiose by laminaribiose phosphorylase.

Because the starch is a mixture consisting of amylose and amylopectin with different chain lengths, glucose units of the amylose are connected with α -1,4 glycosidic bonds, the amylopectin is connected with a main chain of the starch through α -1,6 glycosidic bonds, and the starch phosphorylase cannot decompose α -1,6 glycosidic bonds, in order to improve the yield of glucose-1-phosphoric acid, debranching enzyme-isoamylase which can decompose α -1,6 glycosidic bonds in the starch is added into a reaction system, thereby improving the utilization rate of the starch.

As the inorganic phosphorus is circulated in the reaction process, the reaction can be started by only adding a small amount of phosphate buffer solution and can be continuously carried out, so that the use of phosphate does not cause environmental pressure in the actual production.

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

in the invention, starch is used as a raw material in a multi-enzyme catalytic reaction, the laminaribiose is converted by in vitro multi-enzyme catalysis, and the enzyme capable of promoting the hydrolysis of the starch is added through process optimization, so that the conversion efficiency is obviously improved, the yield is high, and the separation cost of the laminaribiose is greatly reduced. The method has the advantages of simplicity, high utilization rate of raw materials, high yield of laminaribiose, low separation cost, environmental friendliness and the like, and can realize the large-scale production of the laminaribiose.

Drawings

FIG. 1 is a schematic diagram of the in vitro multi-enzyme catalytic pathway for starch conversion to laminaribiose, wherein IA is isoamylase, α GP is starch phosphorylase, AG is glucosidase, and LBP is laminaribiose phosphorylase.

FIG. 2 shows SDS-PAGE detection of 4 key enzymes, wherein M is Marker, IA and α GP were purified by heat treatment, and AG and LBP were purified by Ni-NTA column.

FIG. 3 is a graph showing the synthesis of laminaribiose from starch at 10g/L under in vitro multi-enzyme catalysis under initial conditions, wherein FIG. 3A is a graph showing the reaction progress of the synthesis of laminaribiose from starch under in vitro multi-enzyme catalysis under initial conditions; FIG. 3B shows the HPLC analysis results of synthesizing laminaribiose from starch under in vitro multi-enzyme catalysis under the initial conditions.

FIG. 4 is a graph of laminaribiose synthesis under in vitro multi-enzyme catalysis of 10g/L IA treated starch under initial conditions.

FIG. 5 is a graph showing the reaction progress of synthesizing laminaribiose by multi-enzyme catalysis of 10g/L IA treated starch in vitro under optimal conditions.

FIG. 6 is a graph showing the reaction progress of synthesizing laminaribiose from starch treated with high concentration of IA in vitro multi-enzyme catalysis under optimal conditions.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

The following materials were used in the examples of the present invention

Soluble starch, product of ACROS company, product number: 424490020, respectively;

pET20b vector, Novagen, Madison, WI;

coli expression strain BL21(DE3), Invitrogen, Carlsbad, CA;

all enzymes of the present invention can be purchased from Sigma, and all enzymes can be obtained by prokaryotic expression according to genetic engineering methods.