A method for converting straw into amylose

Abstract

The present application relates to the technical field of biological catalysis, and discloses a method for converting straw into amylose, which comprises the following steps: pretreating straw by using a pretreatment system composed of choline chloride / ethylene glycol and NaHCO3 to remove lignin, then washing and drying the pretreated straw as a substrate, then constructing a multi-enzyme synergistic catalytic system, which comprises an engineered bacterium whole-cell catalyst obtained by using ice nucleation protein to fix and display CBP1942 and PGP on the surface of E. coli, and a cellulase preparation with low beta-glucosidase activity, then adding one or more of molybdate, vanadate and pyrophosphate into the catalytic system, and finally converting the substrate into amylose under suitable reaction conditions in the catalytic system. The method effectively improves the lignin removal rate, enhances the multi-enzyme synergistic effect, and inhibits the degradation of phosphorylated intermediates, so that the yield of amylose is increased by about 5 times.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, and in particular to a method for converting straw into amylose. Background Technology

[0002] With the increasing severity of the global energy crisis and environmental problems, developing renewable resources to replace traditional fossil resources has become an important development direction in the field of biomanufacturing. Traditional chemical production processes are highly dependent on non-renewable resources such as oil and coal, which not only consume huge amounts of resources but also cause serious carbon emissions and environmental pollution. Lignocellulose is the most abundant renewable organic carbon source in nature, widely found in crop straw (such as corn straw, wheat straw, and rice straw), and is mainly composed of cellulose, hemicellulose, and lignin. However, due to the encapsulation effect of lignin and the complex cell wall structure, natural lignocellulose has strong resistance to degradation, limiting its high-value conversion and utilization. Therefore, developing efficient pretreatment and biocatalysis technologies to achieve efficient utilization of straw resources is one of the current research priorities.

[0003] Amylose, an important functional polysaccharide, has wide applications in food, medicine, and materials. Traditional amylose production primarily relies on the extraction and separation of starch from agricultural crops, a process limited by agricultural planting cycles, land resources, and climate conditions, making it difficult to meet the demands of future sustainable development. In contrast, the artificial synthesis of amylose through biocatalysis holds promise for transforming food production from traditional agriculture to industrialized biomanufacturing, thereby improving production controllability and stability. Enzymatically synthesized amylose possesses significant advantages such as uniform degree of polymerization and controllable structure, allowing for further applications in the preparation of high-performance materials. For example, it can be used as a chromatographic packing material for the efficient separation and purification of chiral compounds, holding significant value in the pharmaceutical industry.

[0004] Currently, the synthesis of amylose from lignocellulose still faces many challenges, mainly including: low substrate conversion efficiency, with lignin in natural straw hindering effective contact between enzymes and cellulose substrates; insufficient synergistic efficiency of multi-enzyme systems, leading to easy diffusion and loss of intermediate products during cascade reactions; and the susceptibility of phosphorylation intermediates in the reaction system to degradation by phosphatases, causing carbon flow to deviate from the target product synthesis pathway, severely limiting the overall yield improvement. Therefore, it is urgent to develop new technologies to improve the conversion efficiency of straw to amylose. Summary of the Invention

[0005] The purpose of this invention is to provide a method for converting straw into amylose, which solves the problems of low substrate conversion efficiency, insufficient multi-enzyme synergy, and easy degradation of phosphorylated intermediates in the existing technology when converting straw into amylose.

[0006] To achieve the above objectives, the present invention provides a method for converting straw into amylose, comprising the following steps: Straw was pretreated using a pretreatment system consisting of choline chloride / ethylene glycol and NaHCO3 to remove lignin. The pretreated straw was washed and dried before being used as a substrate; A multi-enzyme synergistic catalytic system was constructed, comprising an engineered bacterial whole-cell catalyst obtained by immobilizing cellobiose phosphorylase CBP1942 and starch phosphorylase PGP on the surface of Escherichia coli using ice nucleoprotein, and a cellulase preparation with low β-glucosidase activity. One or more of molybdate, vanadate, and pyrophosphate are added to the catalytic system; Under suitable reaction conditions, the substrate is converted into amylose in the catalytic system.

[0007] The straw is pretreated using a pretreatment system composed of choline chloride / ethylene glycol and NaHCO3 to remove lignin. Specifically, this includes: The pretreatment conditions are: temperature 120–160℃, NaHCO3 addition amount 1–5wt%, solid-liquid ratio 1:10–1:30, and reaction time 1–5h.

[0008] The straw is pretreated using a pretreatment system composed of choline chloride / ethylene glycol and NaHCO3 to remove lignin. Specifically, this includes: The pretreatment conditions were: temperature 140℃, NaHCO3 addition amount 2wt%, solid-liquid ratio 1:20, and reaction time 3h.

[0009] Specifically, the construction of a multi-enzyme synergistic catalytic system includes: The cellulase preparation with low β-glucosidase activity is used in combination with the whole-cell catalyst of the engineered bacteria to achieve cascade transformation of the substrate.

[0010] Specifically, the addition of one or more of molybdate, vanadate, and pyrophosphate to the catalytic system includes: The molybdate, vanadate, and pyrophosphate are selected from one or more of the following compounds: (NH4)2MoO4, Na2MoO4, Na3VO4, and pyrophosphate.

[0011] This invention discloses a method for converting straw into amylose. First, the straw is pretreated using a pretreatment system composed of choline chloride / ethylene glycol and NaHCO3 to remove lignin. Then, the pretreated straw is washed and dried to serve as a substrate. Next, a multi-enzyme synergistic catalytic system is constructed. This system includes an engineered bacterial whole-cell catalyst obtained by immobilizing cellobiose phosphorylase CBP1942 and starch phosphorylase PGP on the surface of *E. coli* using ice nucleoprotein, and a cellulase preparation with low β-glucosidase activity. Then, one or more of molybdate, vanadate, and pyrophosphate are added to the catalytic system. Finally, under suitable reaction conditions, the substrate is converted into amylose in the catalytic system. This method effectively improves the lignin removal rate (up to 81.08%), enhances the synergistic effect of multiple enzymes, and inhibits the degradation of phosphorylation intermediates, thereby increasing the amylose yield by approximately 5 times. It has advantages such as a wide range of substrate sources, high conversion efficiency, and green sustainability. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0013] Figure 1 This is a Western blotting analysis of the free enzyme CBP / PGP and the recombinant enzyme INP-CBP / INP-PGP of the present invention.

[0014] Figure 2 This is a graph showing the effect of adding miglitol on the yield of cellulase produced by Trichoderma reesei fermentation.

[0015] Figure 3 This is a flowchart of the steps in the method for converting straw into amylose according to the present invention. Detailed Implementation

[0016] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0017] Please refer to Figures 1 to 3 ,in, Figure 1 This is a Western blot analysis of the free enzyme CBP / PGP and the recombinant enzyme INP-CBP / INP-PGP of the present invention, where M is the protein molecular weight marker, lane 1 is the supernatant of the free enzyme CBP, lanes 2 and 3 are the outer membrane components of the recombinant enzyme INP-CBP, lanes 4 and 5 are the outer membrane components of the recombinant enzyme INP-PGP, and lane 6 is the supernatant of the free enzyme PGP. Miglitol can effectively inhibit β-glucosidase. Figure 2 This is a graph showing the effect of adding miglitol on the yield of cellulase produced by Trichoderma reesei fermentation. Figure 3 This is a flowchart of the steps in the method for converting straw into amylose according to the present invention.

[0018] This invention provides a method for converting straw into amylose, comprising the following steps: S101: Straw is pretreated using a pretreatment system consisting of choline chloride / ethylene glycol and NaHCO3 to remove lignin; S102: The pretreated straw is washed and dried before being used as a substrate; S103: Construct a multi-enzyme synergistic catalytic system, the catalytic system comprising an engineered bacterial whole-cell catalyst obtained by immobilizing cellobiose phosphorylase CBP1942 and starch phosphorylase PGP on the surface of Escherichia coli using ice nucleoprotein, and a cellulase preparation with low β-glucosidase activity. S104: One or more of molybdate, vanadate, and pyrophosphate are added to the catalytic system; S105: Under suitable reaction conditions, the substrate is converted into amylose in the catalytic system.

[0019] Specifically, the pretreatment method for corn stalks: This embodiment provides a corn stalk pretreatment method based on a weak base-assisted deep eutectic solvent (DES), and the specific steps are as follows: (1) Raw material processing: Crush the corn stalks and pass them through a 40-60 mesh sieve. Take the portion that passes through the sieve and dry it to a constant weight before using it.

[0020] (2) Preparation of deep eutectic solvent (DES): Choline chloride (ChCl) was vacuum dried at 80°C and 0.08 MPa for 6 hours to remove moisture, and then cooled to room temperature. Choline chloride was mixed with ethylene glycol (EG) at a molar ratio of 1:2 and stirred in an oil bath at 60°C (200 rpm) for 1 hour until a homogeneous and transparent liquid was formed. The resulting DES was stored in a vacuum desiccator for 24 hours for later use.

[0021] (3) Weak base-assisted DES pretreatment: Weigh 2g (dry basis) of corn stalks and mix them with 40g of DES at a solid-liquid ratio of 1:20 (w / w). Add a weak base additive, NaHCO3, to the system at an amount equal to 2wt% of the mass of DES. Place the mixture in an oil bath and react under the following conditions: temperature: 140℃, time: 3h, stirring speed: 200rpm, pressure: atmospheric pressure.

[0022] (4) Solid-liquid separation and washing: After the reaction was completed and cooled to room temperature, the solid was separated by vacuum filtration using a G3 glass frit funnel. The resulting solid was washed sequentially as follows: with 200 mL of 50% (v / v) ethanol aqueous solution, and then repeatedly washed with 80°C deionized water until the pH of the filtrate was neutral.

[0023] (5) Drying and storage: The washed solids were dried to constant weight at 80°C and stored in a desiccator for later use.

[0024] (6) Pretreatment effect Under the above conditions, as shown in Table 1, the lignin removal rate in corn straw is about 81%, and the cellulose content is increased to about 50%, which significantly improves the accessibility of the substrate to subsequent enzymatic hydrolysis and biotransformation.

[0025] Method for constructing surface-display engineered bacteria: This embodiment provides a method for constructing multi-enzyme catalytic strains based on the Escherichia coli surface display system, which can be used to achieve efficient cascade transformation of substrates.

[0026] (1) Gene design and synthesis: Enzyme genes from different sources were selected, including: Cellobiose phosphorylase (EC2.4.1.20); α-glucan phosphorylase (GenBank:D00520.1); The above genes were codon-optimized to fit the E. coli expression system, and flexible linker peptides (G4S) were introduced at the 5' or 3' end of the genes to ensure the structural stability of the fusion protein.

[0027] (2) Construction of surface display carrier: An outer membrane protein of *E. coli* (such as Ice Nucleation Protein, INP) was selected as the anchoring protein to construct a fusion expression vector with the following structure: promoter—anchoring protein—linking peptide—target enzyme—terminator. A strong promoter (T7) was selected, and the anchoring protein facilitated the surface display of the target enzyme. Plasmid construction was performed using the Gibson Assembly method.

[0028] (3) Construction of engineered bacteria: The recombinant plasmid was transformed into competent *Escherichia coli* W3110(DE3) cells, plated on LB agar plates containing antibiotics (e.g., 50 μg / mL kanamycin), and incubated at 37°C for 12–16 h. Positive clones were screened. The correct insertion of the target gene was verified by colony PCR and sequencing.

[0029] (4) Induced expression and surface display: Positive strains were inoculated into LB liquid medium (containing antibiotics) and cultured at 37°C until OD600≈0.6–0.8. IPTG was added to induce expression (final concentration 0.1–0.5 mM), and cultured at 20°C for another 12–16 h.

[0030] (5) Surface display verification: like Figure 1 Membrane proteins were extracted and Western blotting was performed to verify whether the target enzymes were successfully displayed on the cell surface. The results showed that the two enzymes were displayed on the E. coli membrane surface.

[0031] Enzymatic synthesis system of amylose: Using 1% corn stalks as a substrate, BL21-INPN-CBP1942 and BL21-INPN-PGP bacterial suspensions were taken at a 1:1 volume ratio, centrifuged at 12000 rpm for 3 min, and the precipitate was collected. Cellulase with 30 FPU / g low β-glucosidase and 75 μmol / L maltodextrin were added as primers. The system was brought to a final volume of 30 mL using 20 mmol / L phosphate buffer (pH 7). 20 g / L sodium molybdate, 20 g / L sodium vanadate, or 20 g / L pyrophosphate were added to the reaction mixture. Thorough mixing was ensured to guarantee sufficient contact between the bacterial cells and corn stalks. The reaction system was incubated at 55℃ for 72 h, and samples were taken. The enzymes were inactivated by boiling in water for 5 min. Subsequently, the mixture was centrifuged at 12000 rpm for 5 min, and the supernatant was transferred to another clean EP tube. After appropriate dilution, amylose yield was determined using an amylose assay kit. The reaction system with the addition of 20 g / L sodium molybdate, 20 g / L sodium vanadate, or 20 g / L pyrophosphate can produce 0.83 g / L amylose, which is five times higher than the yield without the addition.

[0032] Table 1 compares the cellulose, hemicellulose, and lignin content in corn straw before and after pretreatment.

[0033] By using a deep eutectic solvent system and alkali synergistic pretreatment, the lignin removal rate is significantly improved, and the cellulose availability is enhanced. Surface display technology enables the spatial localization of key enzymes, improving cascade reaction efficiency and reducing intermediate product diffusion losses. By introducing phosphatase inhibitors, the degradation of phosphorylation intermediates is effectively inhibited, improving the target product generation efficiency. Experiments show that this invention can increase amylose yield by approximately 5 times. Using agricultural waste as raw material reduces dependence on food and fossil resources, resulting in good environmental and economic benefits. The obtained amylose has a uniform degree of polymerization distribution and can be used in high-value-added applications, such as chiral separation materials.

[0034] The above-disclosed embodiments are merely one or more preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that implementing all or part of the above embodiments and making equivalent changes in accordance with the claims of this application still fall within the scope of this application.

Claims

1. A method for converting straw into amylose, characterized in that, Includes the following steps: Straw was pretreated using a pretreatment system consisting of choline chloride / ethylene glycol and NaHCO3 to remove lignin. The pretreated straw was washed and dried before being used as a substrate; A multi-enzyme synergistic catalytic system was constructed, comprising an engineered bacterial whole-cell catalyst obtained by immobilizing cellobiose phosphorylase CBP1942 and starch phosphorylase PGP on the surface of Escherichia coli using ice nucleoprotein, and a cellulase preparation with low β-glucosidase activity. One or more of molybdate, vanadate, and pyrophosphate are added to the catalytic system; Under suitable reaction conditions, the substrate is converted into amylose in the catalytic system.

2. The method for converting straw into amylose as described in claim 1, characterized in that, The straw was pretreated using a pretreatment system consisting of choline chloride / ethylene glycol and NaHCO3 to remove lignin. Specifically, this included: The pretreatment conditions are: temperature 120–160℃, NaHCO3 addition amount 1–5wt%, solid-liquid ratio 1:10–1:30, and reaction time 1–5h.

3. The method for converting straw into amylose as described in claim 1, characterized in that, The straw was pretreated using a pretreatment system consisting of choline chloride / ethylene glycol and NaHCO3 to remove lignin. Specifically, this included: The pretreatment conditions were: temperature 140℃, NaHCO3 addition amount 2wt%, solid-liquid ratio 1:20, and reaction time 3h.

4. The method for converting straw into amylose as described in claim 1, characterized in that, The construction of a multi-enzyme synergistic catalytic system specifically includes: The cellulase preparation with low β-glucosidase activity is used in combination with the whole-cell catalyst of the engineered bacteria to achieve cascade transformation of the substrate.

5. The method for converting straw into amylose as described in claim 1, characterized in that, The catalytic system is supplemented with one or more of molybdate, vanadate, and pyrophosphate, specifically including: The molybdate, vanadate, and pyrophosphate are selected from one or more of the following compounds: (NH4)2MoO4, Na2MoO4, Na3VO4, and pyrophosphate.

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