Enzyme for cutting off maltodextrin branched chain as well as construction and application of enzyme

Abstract

The invention relates to gene cloning and expression preparation of an enzyme for cutting off a maltodextrin branched chain and application of the enzyme in multi-enzyme cascade catalysis of starch. The DNA sequence of the maltodextrin branched chain excision enzyme is shown as SEQ ID NO.1, and the amino acid sequence of the maltodextrin branched chain excision enzyme is shown as SEQ ID NO.2. The enzyme for cutting off the maltodextrin branch chain can be subjected to mass soluble expression in escherichia coli and pichia pastoris, is good in thermal stability and high in maltodextrin debranching activity, and can cooperate with multi-enzyme cascade catalysis to improve the product yield and the starch utilization rate.

Description

Technical Field

[0001] This invention relates to an enzyme for removing the side chains of maltodextrin, its construction and application, and belongs to the fields of genetic engineering and enzyme engineering. Background Technology

[0002] Amylose is a linear glucan composed of α-1,4-glycosidic bonds, while amylopectin consists of branches on amylose linked by α-1,6-glycosidic bonds. Natural starch is usually amylopectin. In the starch processing industry, it is usually necessary to remove the branches with α-1,6-glycosidic bonds to form amylose for further utilization. Starch debranching enzymes mainly include pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68), which can be used to remove α-1,6-glycosidic bonds from pullulan, amylopectin, or maltodextrin. For example, in the malt syrup preparation process, starch is liquefied under the catalysis of amylase to produce maltodextrin with a lower degree of polymerization. Subsequently, starch debranching enzymes and saccharifying enzymes synergistically hydrolyze maltodextrin at 60-65℃, realizing the conversion of substrate into malt syrup. Furthermore, in the multi-enzyme cascade catalysis, α-glucan phosphorylase (αGP, EC 2.4.1.1) sequentially cleaves glucose units from the non-reducing end of maltodextrin in the presence of inorganic phosphorus, generating glucose-1-phosphate (G1P). Subsequent enzyme cascade catalysis allows G1P to be converted into high-value products such as glucosamine, fructose, and cellobiose. In this enzyme-catalyzed reaction, the α-1,6-glycosidic bond in the substrate hinders the utilization of maltodextrin by αGP, requiring the co-catalysis of starch debranching enzymes to improve substrate utilization.

[0003] Compared to pullulanase, which has a strong hydrolytic ability towards pullulanose, isoamylase exhibits higher debranching activity towards amylopectin and glycogen. Enzymes reported to have debranching activity against maltodextrin or amylopectin typically react at temperatures below the starch saccharification temperature (60-65℃). Literature reports the high thermal stability of *Cephalotaxus fortunei* (a type of fungus). Sulfolobus tokodaii The source of isoamylase has lower specific enzyme activity and poor heterologous expression (Kun Cheng, Fei Zhang, Fangfang Sun, Hongge Chen, YH Percival Zhang. Doubling power output of starch biobattery treated by the most thermostable isoamylase from an archaeon). Sulfolobus tokodaii (Scientific Reports. 2015. 5: 13184). Therefore, discovering enzymes that can efficiently cleave starch or maltodextrin branches at 60-70℃ is of great significance for the starch deep processing industry and multi-enzyme cascade catalysis. Summary of the Invention

[0004] This invention relates to an enzyme that removes branched chains from maltodextrin, its construction and application, and provides a gene for an enzyme with activity that removes branched chains from maltodextrin and the protein it encodes.

[0005] Another object of the present invention is to provide the application of this enzyme.

[0006] This invention is achieved through the following technical solution:

[0007] A gene for a thermostable enzyme that cleaves the branched chains of maltodextrin, the DNA sequence of which is shown in SEQ ID NO.1, is 2157 bp in length. This gene was obtained from the KEGG (Kyoto Encyclopedia of Genes and Genomes) database from Bacillus thermophilus (Bacillus). Geobacillus kaustophilus It was obtained by screening the genome and its code is: GK2827.

[0008] The DNA sequence encodes a heat-resistant enzyme that cleaves the branches of maltodextrin, and its amino acid sequence is shown in SEQ ID NO.2, comprising 718 amino acids and a molecular weight of 80.4 kDa.

[0009] The method for heterologous expression and preparation of the heat-resistant enzyme that cleaves maltodextrin branches in *E. coli* includes the following steps: using the Novizan Seamless Cloning Kit (catalog number C115-01), the encoding gene is constructed between the Nde I and Xho I restriction sites of the pET28a vector to obtain the recombinant expression plasmid pET28a-GK2827. The recombinant plasmid is then transformed into *E. coli*. E. coli BL21(DE3) was induced to express by IPTG, cells were harvested, sonicated, heat-treated at 70°C, and centrifuged at 12,000 rpm for 20 minutes to obtain crude enzyme solution. The His-tagged enzyme that cleaves the maltodextrin branch was purified from the crude enzyme solution using a nickel column.

[0010] The method for heterologous expression and preparation of the heat-resistant enzyme that cleaves maltodextrin branches in Pichia pastoris includes the following steps: Using the Novizan Seamless Cloning Kit (catalog number C115-01), the encoding gene is constructed between the EcoRI and SalI restriction sites of the pPICZαA vector to obtain the recombinant expression plasmid pPICZαA-GK2827. The recombinant plasmid is transformed into Pichia pastoris GS115, and expression is induced with methanol. The cell culture medium is harvested to obtain a crude enzyme solution. The His-tagged enzyme that cleaves maltodextrin branches is purified from the crude enzyme solution using a nickel column.

[0011] The optimal reaction conditions for the heat-resistant enzyme that cleaves the branched chains of maltodextrin are: catalytic reaction at 70°C in 100 mM HEPES buffer (pH 7.0).

[0012] The heat-resistant enzyme that cleaves the branches of maltodextrin exhibits the following specific activities under optimal reaction conditions: 848 U / mg for hydrolyzing maltodextrin with a DE value (dextrose equivalent) of 10 (purchased from Dongxiao Biotechnology Co., Ltd.); 76.4 U / mg for hydrolyzing maltodextrin with a DE value of 4-7 (purchased from Sigma-Aldrich, catalog number 419672); 26.7 U / mg for hydrolyzing pullulanose (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number 419672); 2.4 U / mg for hydrolyzing soluble starch (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number S104452); and 0.8 U / mg for hydrolyzing corn amylopectin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number A120984).

[0013] The heat-resistant enzyme that cleaves the maltodextrin branch chain has a half-life of 46.6 h at 65 °C, a half-life of 5 h at 75 °C, and a half-life of 0.26 h at 85 °C.

[0014] The heat-resistant enzyme that cleaves the maltodextrin branches can be used in the multi-enzyme cascade catalysis of starch synthesis into cellobiose. Without the addition of the maltodextrin-cleaving enzyme, the multi-enzyme reaction conditions are: 100 mM HEPES buffer (pH 7.0), 100 g / L maltodextrin with a DE value of 10, 15 U / mL α-glucan phosphorylase (αGP), 50 U / mL cellobiose phosphorylase (CBP, EC 2.4.1.20), and 1.5 U / mL α-glucosidase (αG, EC 3.2.1.20). The reaction is carried out at 60°C for 12 h, producing 33 g / L cellobiose and 19.6 g / L glucose, with a starch utilization rate of 52.6%. The heat-resistant enzyme that cleaves maltodextrin branches was added to a multi-enzyme reaction to construct the following system: 100 mM HEPES buffer (pH 7.0), 100 g / L maltodextrin with a DE value of 10, 15 U / mL α-glucan phosphorylase (αGP), 50 U / mL cellobiose phosphorylase (CBP, EC 2.4.1.20), 1.5 U / mL α-glucosidase (αG, EC 3.2.1.20), and 0.02 g / L maltodextrin branch-cleaving enzyme. After reacting at 60°C for 12 h, 54 g / L cellobiose and 26.6 g / L glucose were produced, with a starch utilization rate of 80.6%. Using this maltodextrin branch-cleaving enzyme can significantly improve the enzymatic hydrolysis efficiency of starch.

[0015] The maltodextrin-cleaving enzymes screened in this invention can be expressed in large quantities in soluble form in *E. coli* and also efficiently secreted in *Pichia pastoris*, exhibiting good thermostability. The maltodextrin-cleaving enzymes described in this invention demonstrate highly efficient hydrolytic activity against maltodextrins with DE values ​​of 10 and 4-7, significantly improving substrate utilization in the multi-enzyme cascade catalysis of starch to cellobiose. The maltodextrin-cleaving enzymes described in this invention can perform maltodextrin debranching at 60-65℃, consistent with starch saccharification conditions, allowing for one-pot multi-enzyme reactions and greatly reducing the process difficulty and production cost of maltodextrin debranching. Attached Figure Description

[0016] Figure 1 Gel image of heterologous expression of the maltodextrin-cleaving enzyme in *E. coli*. M: Protein standard of standard molecular weight; T: *E. coli* BL21(DE3) cell lysate expressing the maltodextrin-cleaving enzyme; S: Supernatant of cell lysate after centrifugation at 12,000 rpm for 20 minutes; H: Supernatant of cell lysate after heat treatment at 70°C for 20 minutes and centrifugation at 12,000 rpm for 20 minutes; Ni: Maltodextrin-cleaving enzyme purified from crude enzyme solution using a nickel column.

[0017] Figure 2 Substrate selectivity of enzymes that cleave the maltodextrin branch chain.

[0018] Figure 3 Enzymes that cleave maltodextrin branches catalyze the synthesis of cellobiose from starch in a cascade of enzymes. (A) Catalyzing the production of cellobiose and glucose from starch without the addition of enzymes that cleave maltodextrin branches. (B) Catalyzing the production of cellobiose and glucose from starch with the addition of 0.02 g / L of enzymes that cleave maltodextrin branches. Dashed lines represent the amount of glucose, and solid lines represent the amount of cellobiose. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0020] Example 1: Preparation of an enzyme to cleave the branched chains of maltodextrin

[0021] The gene for the heat-resistant enzyme that cleaves the branched chains of maltodextrin described in this invention has a DNA sequence as shown in SEQ ID NO.1, with a full length of 2157 bp. This gene was obtained from the KEGG (Kyoto Encyclopedia of Genes and Genomes) database from Bacillus thermophilus (…). Geobacillus kaustophilus The DNA sequence was obtained by screening the genome of GK2827. The DNA sequence encodes a heat-resistant enzyme that cleaves the branched chains of maltodextrin, and its amino acid sequence is shown in SEQ ID NO. 2, comprising 718 amino acids and a molecular weight of 80.4 kDa.

[0022] The method for heterologous expression and preparation of the heat-resistant enzyme that cleaves maltodextrin branches in Escherichia coli includes the following steps:

[0023] (1) Constructing the vector: According to the instructions of the Novizan Seamless Cloning Kit (Catalog No. C115-01), the gene and vector linear fragments were amplified by conventional PCR using the genome of Bacillus thermophilus and the pET28a vector as templates. The recombinant product was obtained by recombination reaction. The recombinant product was transformed into Escherichia coli TOP10, single clones were picked, plasmids were extracted by shaking culture, and the recombinant vector pET28a-GK2827 was obtained by sequencing verification. The gene of the enzyme that cuts the branch chain of maltodextrin was inserted between the Nde I and Xho I restriction sites of the pET28a vector.

[0024] (2) Transform Escherichia coli with the recombinant plasmid E. coli BL21(DE3) was induced to express by IPTG, cells were harvested, sonicated, heat-treated at 70°C, and centrifuged at 12,000 rpm for 20 minutes to obtain crude enzyme solution. The His-tagged enzyme that cleaves the maltodextrin branch was purified from the crude enzyme solution using a nickel column.

[0025] The method for heterologous expression and preparation of the heat-resistant enzyme that cleaves maltodextrin branches in Pichia pastoris includes the following steps:

[0026] (1) Constructing the vector: According to the instructions of the Novizan Seamless Cloning Kit (Catalog No. C115-01), the gene and vector linear fragments of Bacillus thermophilus genome and pPICZαA vector were amplified by conventional PCR technology. The recombinant product was obtained by recombination reaction. The recombinant product was transformed into Pichia pastoris GS115. Single clones were picked, and plasmids were extracted by shaking culture. Sequencing verification yielded the recombinant vector pPICZαA-GK2827. The gene of the enzyme that cuts the branch chain of maltodextrin was inserted between the EcoRI and SalI restriction sites of the pPICZαA vector.

[0027] (2) The recombinant plasmid was transformed into Pichia pastoris GS115, and expression was induced with methanol. The cell culture medium was harvested to obtain crude enzyme solution. The His-tagged enzyme that cleaves the maltodextrin branch was purified from the crude enzyme solution using a nickel column.

[0028] Experimental results are as follows Figure 1 As shown, the enzyme that removes the maltodextrin branch chain is expressed in large quantities in Escherichia coli, with an expression level close to 50%. After heat treatment, the purity of the enzyme that removes the maltodextrin branch chain in the crude enzyme solution reaches more than 80%.

[0029] Example 2 Determination of optimal reaction conditions for enzymes that cleave maltodextrin branches

[0030] The enzyme activity of the prepared heat-resistant enzyme that cleaves the branched chains of maltodextrin was determined according to the method published in the literature (Y.-H. Percival Zhang and Lee R. Lynd, Determination of the number-average degree of polymerization of cellodextrins and cellulose with application to enzymatic hydrolysis. Biomacromolecules, 2005, 6, 1510-1515).

[0031] Optimal reaction temperature: Maltodextrin (purchased from Dongxiao Biotechnology Co., Ltd.) with 100 mM HEPES (pH 7.0) and a DE value (dextrose equivalent) of 10 at 10 g / L was used. The reaction was carried out at 40~80℃ for 5 min, and the reduced end produced was measured to calculate the specific enzyme activity.

[0032] Optimal pH for reaction: 100 mM citrate buffer (pH 5.0~6.5), 100 mM phosphate buffer (pH 5.5~7.0), and 100 mM HEPES buffer (pH 6.5~7.5) were used respectively. Maltodextrin with a DE value of 10 was added and reacted at 70℃ for 5 min. The reduced end produced was measured and the specific enzyme activity was calculated.

[0033] The specific enzyme activity unit (U / mg) is defined as the amount of enzyme required to catalyze the production of 1 micromolar of the reducing end in 1 minute under the corresponding conditions.

[0034] Experimental results show that the optimal reaction conditions for the heat-resistant enzyme that cleaves the maltodextrin branch chain are: catalytic reaction in 100 mM HEPES buffer (pH 7.0) at 70°C.

[0035] Example 3: Determination of the thermal stability of enzymes that cleave maltodextrin branches

[0036] The heat-resistant enzyme that cleaves the maltodextrin branch chain was diluted to 0.1 g / L with 100 mM HEPES (pH 7.0) and incubated in water baths at 65℃, 75℃, and 85℃. Samples were taken at different time points, and the specific enzyme activity of the enzyme solutions was measured at 70℃ using 100 mM HEPES buffer (pH 7.0).

[0037] Residual enzyme activity calculation method: The residual enzyme activity is calculated by dividing the specific enzyme activity of the enzyme solution sampled at each time point by the specific enzyme activity of the enzyme solution at 0 h, and then multiplying by 100%. The residual enzyme activity of the enzyme solution with an incubation time of 0 h is set to 100%.

[0038] Enzyme half-life: The incubation time at which the residual enzyme activity reaches 50% is the enzyme's half-life.

[0039] Experimental results show that the heat-resistant enzyme that cleaves the maltodextrin branch chain has a half-life of 46.6 h at 65 °C, a half-life of 5 h at 75 °C, and a half-life of 0.26 h at 85 °C.

[0040] Example 4: Determination of substrate selectivity of enzymes that cleave maltodextrin branches

[0041] The substrate selectivity of the heat-resistant enzyme that cleaves the maltodextrin branch chain was determined using the following experimental steps:

[0042] Using maltodextrin with a DE value of 10 (purchased from Dongxiao Biotechnology Co., Ltd.), maltodextrin with a DE value of 4-7 (purchased from Sigma-Aldrich, catalog number 419672), pullulan (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number 419672), soluble starch (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number S104452), and corn amylopectin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number A120984) as substrates, the specific enzyme activities of the enzymes to various substrates were determined in 100 mM HEPES buffer (pH 7.0) at 70℃.

[0043] Experimental results are as follows Figure 2 As shown, the heat-resistant enzyme that cleaves the branches of maltodextrin has the following specific enzyme activity under optimal reaction conditions: 848 U / mg for hydrolyzing maltodextrin with a DE value of 10, 76.4 U / mg for hydrolyzing maltodextrin with a DE value of 4-7, 26.7 U / mg for hydrolyzing pullulanose, 2.4 U / mg for hydrolyzing soluble starch, and 0.8 U / mg for hydrolyzing corn amylopectin.

[0044] Example 5: Enzyme-assisted multi-enzyme cascade catalysis of starch synthesis into cellobiose by cleaving maltodextrin branches.

[0045] This embodiment utilizes a multi-enzyme cascade catalysis to synthesize cellobiose from maltodextrin with a DE value of 10. Firstly, three enzymes were recombinantly expressed: derived from... Thermococcus kodakarensis aGP (KEGG code TK1406), αG (KEGG code GK0615) from Bacillus thermophilus, and CBP (KEGG code Cthe_0275) from Clostridium thermophilum.

[0046] High-performance liquid chromatography (HPLC) was used to quantitatively analyze cellobiose and glucose. The chromatographic column used was a Bio-Rad HPX-87H, the mobile phase was 5 mM sulfuric acid aqueous solution, the flow rate was 0.5 mL / min, the column temperature was 55℃, and a differential refractive index detector was used.

[0047] A reaction mixture containing 100 mM HEPES buffer (pH 7.0), 100 g / L maltodextrin with a DE value of 10, 15 U / mL α-glucan phosphorylase (αGP), 50 U / mL cellobiose phosphorylase (CBP), and 1.5 U / mL α-glucosidase (αG) was reacted at 60 °C. HPLC analysis showed that after 12 h of reaction, 33 g / L cellobiose and 19.6 g / L glucose were produced, with a starch utilization rate of 52.6%. Figure 3 A).

[0048] A reaction mixture containing 100 mM HEPES buffer (pH 7.0), 100 g / L maltodextrin with a DE value of 10, 15 U / mL α-glucan phosphorylase (αGP), 50 U / mL cellobiose phosphorylase (CBP), 1.5 U / mL α-glucosidase (αG), and 0.02 g / L maltodextrin-cleaving enzyme was reacted at 60 °C. HPLC analysis showed that after 12 h of reaction, 54 g / L cellobiose and 26.6 g / L glucose were produced, with a starch utilization rate of 80.6%. Figure 3 B).

[0049] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An enzyme which removes the branched chains of maltodextrins, characterized in that: The enzyme for removing branched chains of maltodextrin is shown in the amino acid sequence of SEQ ID NO.

2.

2. The enzyme for cleaving maltodextrin branches as described in claim 1, characterized in that, Preparation by heterologous expression in E. coli BL21 (DE3) or Pichia pastoris GS115.

3. The debranching enzyme of claims 1-2, characterized in that, The temperature for catalyzing the reaction is 30-85°C, preferably 40-80°C, more preferably 60-80°C, and most preferably 60-70°C. Preferably, the pH for catalyzing the reaction is 4.5-8.0, more preferably 6.0-7.5, and most preferably 6.5-7.

0.

4. The debranching enzyme of claims 1-3, characterized in that, The enzyme for removing branched chains of maltodextrin is shown in the amino acid sequence of SEQ ID NO.

2.

5. A method of catalyzing the synthesis of cellobiose from starch by a concerted multi-enzyme cascade of enzymes that cleave the maltodextrin side-chains, characterized in that, The enzyme for removing branched chains of maltodextrin is shown in the amino acid sequence of SEQ ID NO.

2.

6. The enzyme for removing branched chains of maltodextrin according to any one of claims 1-5, comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to SEQ ID NO.

2.

7. Preparation and use of the enzyme for removing branched chains of maltodextrin according to any one of claims 1-6 in hydrolyzing α-1,6-glucosidic bonds of starch.

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