CN113005160A

CN113005160A - Method for preparing cellobiose by starch conversion

Info

Publication number: CN113005160A
Application number: CN201911327630.9A
Authority: CN
Inventors: 游淳; 孙尚尚
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2021-06-22
Anticipated expiration: 2039-12-20
Also published as: CN113005160B

Abstract

The invention discloses a method for constructing an in vitro multi-enzyme molecular machine and preparing cellobiose through multi-enzyme cascade catalysis, belonging to the field of enzymatic preparation of cellobiose. The preparation method of cellobiose disclosed by the invention comprises the steps of hydrolyzing branched chains in starch by isoamylase, converting glucose units in debranched chain starch by starch phosphorylase and glucosidase to generate glucose-1-phosphate and glucose, and synthesizing cellobiose by the cellobiose phosphorylase through the glucose-1-phosphate and the glucose. By optimizing the process, the utilization rate of starch and the yield of cellobiose can be further improved. The technical method has the advantages of cheap and easily obtained substrate, low production cost, high product yield, simple separation and purification and the like, and can realize the large-scale production of cellobiose.

Description

Method for preparing cellobiose by starch conversion

Technical Field

The invention belongs to the field of biological manufacturing, and particularly relates to a method for preparing cellobiose by using starch as a raw material through in vitro multi-enzyme catalysis.

Background

Cellobiose is oligosaccharide connected by beta-1, 4 glycosidic bonds, has the sweetness of 30 percent of that of cane sugar and almost zero calorie, is mainly used as a functional sweetener in the field of food, and is soluble dietary fiber which can be used as prebiotics to promote the reproduction of beneficial flora in intestinal tracts.

Currently, cellobiose production is mainly based on hydrolysis of high purity cellulose with dilute acids, enzymes or microorganisms. The acidolysis of cellulose products is not single, resulting in low cellobiose yield, high separation cost and easy environmental stress. The difficulty of the enzymolysis and microbial degradation processes lies in that the cellulose has poor solubility, some pretreatment processes are needed, the products are mixed, the yield is low, and the product cellobiose can inhibit the activity of the cellulase. The defects of the traditional modes lead the price of the cellobiose to be high, and limit the application prospect of the health food additive.

With the development of industrial enzyme biotechnology, there have been scientists who have begun attempting to enzymatically synthesize cellobiose. Japanese scientists have attempted to produce cellobiose from starch as a substrate using 3 enzymes (glucan phosphorylase, glucoamylase and cellobiose phosphorylase), but the yield of cellobiose is only about 40% at the highest, resulting in higher cost for subsequent product isolation.

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

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for preparing cellobiose, which takes cheap starch as a substrate and produces cellobiose through catalysis of an in vitro multi-enzyme reaction system.

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

the invention provides a method for preparing cellobiose by using enzyme catalytic reaction, which is characterized in that starch is used as a substrate, isoamylase (isoamyylase, EC 3.2.1.68, IA), starch phosphorylase (alpha-glucan phosphorylase, EC 2.4.1.1, alpha GP), glucosidase (alpha-glucosidases, EC 3.2.1.20, alpha G) and cellobiose phosphorylase (cellobiose phosphorylase, EC 2.4.1.20, CBP) are added to construct a multi-enzyme catalytic reaction system, and the enzyme catalytic reaction is carried out. In the invention, starch is used as a substrate, starch is catalyzed by isoamylase to obtain debranched chain starch, the debranched chain starch is catalyzed by starch phosphorylase and glucosidase to be respectively converted into glucose-1-phosphate and glucose, and the glucose-1-phosphate and the glucose are catalyzed by cellobiose phosphorylase to generate cellobiose.

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 enzyme-catalyzed reaction system is 1 to 200g/L, more preferably 5 to 50g/L, even more preferably 8 to 20g/L, and most preferably 10 g/L.

In a preferred embodiment, when the starch contains α -1,6 glycosidic linkages (e.g., soluble starch, soluble amylopectin, amylodextrin, maltodextrin, maltopolysaccharide), isoamylase (isoamylase, EC 3.2.1.68, IA) is added to the reaction system; more preferably, isoamylase is added to the reaction system, and after a reaction period, starch phosphorylase, glucosidase and cellobiose phosphorylase are added.

Preferably, isoamylase is added into the reaction system first, and the reaction is carried out at 10-99 ℃ for 0.5-72 hours, further preferably at 30-95 ℃ for 1-48 hours, more preferably at 50-90 ℃ for 1-12 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.

Preferably, the reaction system is added with starch phosphorylase, glucosidase and cellobiose phosphorylase, and the reaction is continued at 10-95 ℃ for 0.5-150 hours, more preferably at 20-80 ℃ for 1-60 hours, even more preferably at 30-60 ℃ for 6-48 hours, and most preferably at 50 ℃ for 12 hours

Preferably, buffer solution, phosphate and metal ions are also added into the reaction system.

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 the invention, the starch phosphorylase, the glucosidase and the cellobiose phosphorylase added in the enzyme catalysis reaction system can be in any proportion.

Preferably, the ratio of the added starch phosphorylase, the added glucosidase and the added cellobiose phosphorylase is 1.5-3: 0.5: 1-3.

Further preferably, the ratio of starch phosphorylase, glucosidase and cellobiose phosphorylase added is 2.5:0.5: 1.2.

Preferably, the amount of the starch phosphorylase to be used in the reaction system is 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, still more preferably 1 to 5U/mL, and most preferably 2.5U/mL.

Preferably, the amount of the glucosidase to be used in the reaction system is 0.1 to 50U/mL, more preferably 0.2 to 10U/mL, still more preferably 0.3 to 5U/mL, and most preferably 0.5U/mL.

Preferably, the cellobiose phosphorylase is used in an amount of 0.1 to 50U/mL, more preferably 0.5 to 10U/mL, still more preferably 1 to 5U/mL, and most preferably 1.2U/mL in the reaction system.

In the present invention, starch phosphorylase, cellobiose phosphorylase, isoamylase and glucosidase of various origins 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 cellobiose phosphorylase may be derived from Clostridium thermocellum (Paenibacillus sp.), Euglena Gracilis (Euglena Gracilis), Acholeplasma (Acholeplastia laidii), etc., preferably, the cellobiose phosphorylase is derived from Clostridium thermocellum; the isoamylase may be derived from Sulfolobus (Sulfolobus tokodaii), 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 (Beta vulgaris), Paecilomyces lilacinus (Paecilomyces lilacinus), etc., and preferably, the glucosidase is derived from Paecilomyces lilacinus. The present invention may also use starch phosphorylase, cellobiose 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, and adds isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase to prepare a four-enzyme reaction system, wherein the enzyme catalysis approach comprises the following steps: hydrolyzing amylopectin in starch by isoamylase, and converting glucose units in debranched amylose into glucose-1-phosphate and glucose by starch phosphorylase and glucosidase; glucose and glucose-1-phosphate are converted to cellobiose by cellobiose phosphorylase.

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

Drawings

FIG. 1 is a schematic diagram of an in vitro multi-enzyme catalytic pathway for starch conversion to cellobiose; wherein: IA is isoamylase, alpha GP is starch phosphorylase, alpha G is glucosidase and CBP is cellobiose phosphorylase.

FIG. 2 shows SDS-PAGE detection of 4 key enzymes; wherein: m is Marker, IA and α GP are purified by heat treatment, α G and CBP are purified by Ni-NTA column.

FIG. 3 is a graph of cellobiose synthesis under in vitro multi-enzyme catalysis of 10g/L IA treated starch under initial conditions.

FIG. 4 is a graph showing the reaction progress of synthesizing cellobiose by multi-enzyme catalysis of 10g/L IA-treated starch in vitro under optimal conditions.

FIG. 5 is a graph showing the reaction progress of synthesizing cellobiose from starch treated with high concentration of IA under 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.

Example 1 in vitro Multi-enzyme catalysis of starch conversion to Cellobiose

The catalytic pathway for the conversion of starch to cellobiose by an in vitro multi-enzyme catalytic system is shown in FIG. 1. The key enzyme and key steps involved in the method comprise: (1) isoamylase (IA, EC 3.2.1.68) for hydrolyzing amylopectin of starch to obtain debranched amylopectin; (2) starch phosphorylase (α GP, EC 2.4.1.1) for the release of glucose-1-phosphate from debranched amylose; (3) a glucosidase (α G, EC 3.2.1.20) for releasing glucose from debranched amylose; (4) cellobiose phosphorylase (CBP, EC 2.4.1.20) for catalyzing the production of cellobiose from glucose-1-phosphate and glucose.

In this example, isoamylase was derived from Sulfolobus (Sulfolobus tokodaii) and the gene thereof is numbered ST0928 on KEGG, and the genomic DNA of this strain was purchased from DSMZ, german collection of strains. The starch phosphorylase is derived from Thermotoga maritima (Thermotoga maritima), and the number of the gene on KEGG is TM 1168; the glucosidase is derived from Paecilomyces lilacinus (Paecilomyces lilacinus), and the number of the gene on KEGG is QAQ 81244; cellobiose phosphorylase is derived from Clostridium thermocellum (Clostridium thermocellum) and has the gene numbered Cth 0275 on KEGG. These genomic DNAs are all available from the ATCC's official website (www.atcc.org). These four genes were obtained by PCR from the corresponding genomic DNA using F1/R1, F2/R2, F3/R3 and F4/R4, respectively, wherein F1: GTTTAACTTTAAGAAGGAGATATAGTGCTGGAGAAACTTCCCGAG, R1: GTGGTGGTGGTGGTGCTCGAGTCAGAGAACCTTCTTCCAGAC, F2: CATCATCATCATCATCACAGCAGCGGCTTGAAAAAAACATGGTGGAAAGAAG, R2: GTGGTGGTGGTGGTGGTGCTCGAGTTCTTTCCAGATGTATACGCGCGCC, F3: GTTTAACTTTAAGAAGGAGATATACCATGGGTCAGAAAGGCTGGAAATTTC, R3: CAGTGGTGGTGGTGGTGGTGCTCGAGACTAATATTACGGCCCAGGGTCAC, F4: GTTTAACTTTAAGAAGGAGATATAATGGTTTTTTCACACAAGGATAGACC, R4: GTGGTGGTGGTGGTGGTGCTCGAGCTAATATTCAATCCTCCTATATACC, and cloned into pET20b vector (Novagen, Madison, Wis.) by the method of Simple Cloning (You C, Zhang XZ, Zhang Y-HP.2012.Simple Cloning via direct transformation of PCR products (DNA Multimer) to Escherichia coli and Bacillus subtilis. Appl. environ. Microbiol.78(5):1593-5.) to obtain corresponding expression vectors pET20b-StIA, pET20b-Tm α GP, pET20b-Pl α G and pET20 b-PsCBP. Then, these four plasmids were transformed into escherichia coli expression bacteria BL21(DE3) (Invitrogen, Carlsbad, CA), and protein expression and purification were performed, and the results of protein purification are shown in fig. 2.

The reaction system contained 5mM sodium acetate buffer (pH 5.5), 0.5mM divalent zinc ion, 5U/mL isoamylase, and 200g/L starch, and the reaction was catalyzed at 85 ℃ for 3 hours to obtain IA-treated starch.

Then, the reaction system contained 100mM HEPES buffer (pH 6.5), 5mM divalent zinc ion, 20mM potassium phosphate (pH 6.5), 1U/mL starch phosphorylase, 1U/mL glucosidase, 2U/mL cellobiose phosphorylase, and 10g/L IA-treated starch, and catalyzed at 50 ℃ for 12 hours.

And detecting the concentration of the cellobiose by using high performance liquid chromatography. 94.5. mu.L of the reaction sample was taken, and 5.5. mu.L of 10% sulfuric acid was added to terminate the reaction. Centrifuging to take the supernatant, and detecting the area and height of the cellobiose peak by HPLC to calculate the concentration of the cellobiose.

The final cellobiose concentration at the end of the reaction (FIG. 3) was 18mM, calculated from the slope of the standard curve, and the conversion to starch (10g/L, about 55.5mM glucose equivalents, 2 molecules glucose equivalents to 1 molecule cellobiose) was 64.8%.

Example 2 increasing the yield of Cellobiose by optimizing the enzyme ratio

Isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase were prepared as in example 1, and IA-treated starch was prepared as in example 1.

After gradual optimization, the optimal potassium phosphate concentration is determined to be 20mM, and the optimal enzyme adding amount is 2.5U/mL of starch phosphorylase, 0.5U/mL of glucosidase and 1.2U/mL of cellobiose phosphorylase. For cellobiose production, the reaction system contained 100mM HEPES buffer (pH 6.5), 5mM divalent zinc ion, 20mM potassium phosphate (pH 6.5), 2.5U/mL starch phosphorylase, 0.5U/mL glucosidase, 1.2U/mL cellobiose phosphorylase, and 10g/L IA-treated starch, and the reaction was catalyzed at 50 ℃ for 12 hours, and cellobiose was detected as in example 1.

It was determined that after the reaction, the final cellobiose concentration (FIG. 4) was 22.4mM and the conversion to starch (10g/L, about 55.5mM dextrose equivalent) was 80.7%, with a significant increase in conversion and a significant decrease in glucose concentration compared to the initial conditions.

EXAMPLE 3 production of cellobiose from high starch concentrations

For cellobiose production, 100mM HEPES buffer (pH 6.5), 5mM divalent zinc ion, 20mM potassium phosphate (pH 6.5), 12.5U/mL starch phosphorylase, 2.5U/mL glucosidase, 6U/mL cellobiose phosphorylase, and 50g/L IA-treated starch were reacted at 50 ℃ for 24 hours, and cellobiose was detected as in example 1.

After the reaction was completed, the final concentration of cellobiose (FIG. 5) was 101mM (34.5g/L), and the conversion rate was not significantly decreased as compared with 10g/L of starch.

Then, the reaction system contained 100mM HEPES buffer (pH 6.5), 5mM divalent zinc ion, 10mM potassium phosphate (pH 6.5), 25U/mL starch phosphorylase, 5U/mL glucosidase, 12U/mL cellobiose phosphorylase, and 100g/L IA-treated starch, and catalyzed at 50 ℃ for 24 hours, and cellobiose was detected as in example 1.

After the reaction was complete, the final cellobiose concentration (FIG. 5) was 219mM (69g/L), and the conversion was not significantly reduced compared to 10g/L starch.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing cellobiose by enzyme catalysis is characterized in that starch is taken as a substrate, isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase are added to establish a multi-enzyme catalytic reaction system, and enzyme catalytic reaction is carried out.

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, and metal ions are further added to the enzymatic reaction system.

6. The method according to any of claims 1 to 5, wherein the reaction temperature of the enzyme-catalyzed reaction system 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 enzyme-catalyzed reaction system is 0.5 to 150 hours, more preferably 1 to 60 hours, even more preferably 6 to 48 hours, and most preferably 12 hours.

8. The method according to any one of claims 1 to 7, wherein the starch phosphorylase, the glucosidase and the cellobiose phosphorylase are added in a ratio of 1.5 to 3:0.5:1 to 3 in the enzyme-catalyzed reaction system.

9. The method according to any one of claims 1 to 8, wherein the starch phosphorylase, the glucosidase and the cellobiose phosphorylase are added to the enzyme-catalyzed reaction system at a ratio of 2.5:0.5: 1.2.

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 (Thermus thermophilus), 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 cellobiose phosphorylase is derived from Clostridium thermocellum (Clostridium thermocellum), Euglena Gracilis (Euglena Gracilis), Cholesterol-free bacterium (Acholeplasia laidlani), 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 cellobiose phosphorylase from the above sources; preferably, the cellobiose phosphorylase is derived from Clostridium thermocellum (Clostridium thermocellum) 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 the cellobiose phosphorylase derived from Clostridium thermocellum (Clostridium thermocellum); preferably, the glucosidase enzyme is derived from Aspergillus niger (Aspergillus niger), beet (Beta vulgaris), 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 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).