Polymeric glutamate sugar ester as well as biological fermentation preparation method and application thereof

Abstract

The invention relates to polymerized glutamate sugar ester as well as a biological fermentation preparation method and application thereof, and belongs to the technical field of biological fermentation. According to the scheme, amino acid and functional oligosaccharide are taken as substrates, and efficient directional assembly of high-molecular polymerized glutamate sugar ester is realized by designing an integrated cell factory biosynthesis technology. The invention provides a brand-new polyglutamic acid sugar ester compound and a biological fermentation method of the polyglutamic acid sugar ester, the biological fermentation method is suitable for industrial continuous production, and the prepared polyglutamic acid sugar ester is narrower in molecular weight distribution and better in product uniformity.

Description

Technical Field

[0001] The present application relates to a polymerized glutamic acid sugar ester and a biological fermentation preparation method and application thereof, belonging to the field of biological fermentation technology. Background Art

[0002] Polyglutamic acid (γ-PGA) is a natural anionic polymer formed by the polymerization of glutamic acid monomers through γ-amide bonds. It exhibits excellent biodegradability, biocompatibility, and exceptional water and moisture retention. In agriculture, γ-PGA, due to its unique molecular structure, exhibits remarkable fertilizer efficiency enhancement, soil improvement, and stress resistance and growth promotion capabilities. The numerous free carboxyl groups on its molecular chain can chelate trace elements such as calcium and magnesium in the soil, preventing fertilizer solidification and improving fertilizer utilization. Its strong water-holding capacity effectively inhibits soil moisture evaporation, contributing to drought resistance and soil moisture conservation. γ-PGA also promotes crop root development and enhances crop resistance to abiotic stresses such as drought and salinity.

[0003] Seaweed, particularly green and brown algae, is rich in a variety of bioactive polysaccharides and oligosaccharides. For example, rhamnosyl oligosaccharides (ROS) from green algae and alginate oligosaccharides (AOS) from brown algae have been shown to exhibit diverse bioactivities, including promoting plant growth, stimulating plant immune responses, enhancing plant stress resistance, and improving crop quality. These seaweed oligosaccharides act as signaling molecules, regulating the expression of relevant genes in plants and inducing disease resistance. They also serve as natural organic nutrients.

[0004] Ester-linked bioactive molecules play a crucial role in regulating plant growth. Brassinolide, a well-known sterol ester, is currently one of the most active and widely used plant growth regulators, significantly enhancing crop photosynthesis, yield, and stress resistance. Similarly, diethylaminoethyl ester (DA-6), a carboxylic acid ester, also exhibits high plant growth regulating activity. Furthermore, sugar ester biosurfactants such as rhamnolipids have also been found to promote plant growth. These studies suggest that linking active groups via ester bonds can not only produce synergistic effects but may also impart novel functionalities to the molecules, arguing that their structural characteristics determine their functional diversity.

[0005] Traditional polyamino acids and carbohydrates are prepared by chemical catalytic esterification, which has the following defects: 1) many by-products (such as sulfonation by-products ≥15% in concentrated sulfuric acid catalytic reactions) and low purity of the target product (<85%); 2) high reaction energy consumption (requiring high temperature >80°C or high pressure conditions); 3) difficulty in controlling the product molecular weight distribution (polydispersity index PDI>1.5); 4) chemical catalyst residues make post-processing complicated.

[0006] Therefore, it is necessary to provide a preparation process method that can overcome the process defects of the traditional chemical catalytic esterification method, while making the molecular weight distribution of the product narrower and the product uniformity better.

[0007] It should be noted that the above background technology is only used to illustrate the technical route of the present application solution and does not necessarily constitute the prior art. Summary of the Invention

[0008] In order to solve the above problems, a polymerized glutamate sugar ester and its bio-fermentation preparation method and application are provided. The present application specifically provides an efficient preparation method of polymerized glutamate sugar ester based on enzyme-catalyzed self-assembly technology. By adopting the "integrated cell factory" biosynthesis technology, the precise assembly of glutamate and functional oligosaccharides under mild conditions (below 45°C, pH 5.0-7.0) is achieved. The cost is reduced by 50%-60% compared with the chemical catalytic esterification method, and the molecular weight distribution of the prepared polymerized glutamate sugar ester is narrower and the product uniformity is better.

[0009] In this application, the structures of polyglutamic acid and functional oligosaccharides are fused, the carboxyl group of glutamic acid reacts with the hydroxyl group of functional oligosaccharides to form an ester bond, and then a new multifunctional biopolymer with a network macromolecular structure is formed through intermolecular polymerization and cross-linking, thereby realizing the functional integration and enhancement of functional oligosaccharides and polyglutamic acid.

[0010] The prepared polyglutamic acid sugar ester is a glycoesterified derivative of poly-γ-glutamic acid (γ-PGA). Its structure is further stabilized through glycoesterification, further enhancing its biological activities, including growth promotion, stress resistance, water and fertilizer conservation in crops, and anti-inflammatory, immune regulation, and intestinal microecological regulation in humans. It has significant application potential in green agriculture (such as water-retaining and drought-resistant agents), sustained-release drug carriers (such as anti-tumor drug delivery systems), and functional food additives (such as prebiotic enhancers).

[0011] The present application provides a bio-fermentation preparation method of polymerized glutamic acid sugar ester, which comprises the steps of using engineered bacteria to perform bio-fermentation using glutamic acid or its salt and functional oligosaccharides as substrates; The engineered bacteria express acyltransferase, glycosyltransferase, pgsB 、 pgsC and pgsA ; The functional oligosaccharide is selected from one or more oligosaccharides or derivatives thereof obtained from animal, plant or microorganism oligosaccharides, or oligosaccharides or derivatives thereof obtained by degradation of animal, plant or microorganism polysaccharides.

[0012] Optionally, the functional oligosaccharide is selected from one or more seaweed oligosaccharides; Optionally, the seaweed oligosaccharide is selected from one or more of green algae oligosaccharide, brown algae oligosaccharide, and red algae oligosaccharide; Optionally, the seaweed oligosaccharide is selected from one or more of rhamnose oligosaccharide, alginate oligosaccharide, fucoidan oligosaccharide, carrageenan oligosaccharide and agar oligosaccharide; Optionally, the rhamnosaccharide is derived from the enzymatic or chemical hydrolysis product of green algae polysaccharide; Optionally, the alginate oligosaccharide is derived from the enzymatic or chemical hydrolysis product of alginate in brown algae; Optionally, the rhamnose oligosaccharide is an oligosaccharide compound with α-L-rhamnose monomers as the main constituent units, linearly connected by 1,3 and / or 1,4 glycosidic bonds; Optionally, the alginate oligosaccharide is a linear oligosaccharide formed by random or block copolymerization of β-D-mannuronic acid and α-L-guluronic acid through 1,4 glycosidic bonds.

[0013] Optionally, the engineered bacteria are controlled to express pgsB 、 pgsC and pgsA Synthesizing polyglutamic acid or its salt from glutamic acid or its salt; The engineered bacteria are controlled to express acyltransferase and glycosyltransferase, and the polymerized glutamate sugar ester is synthesized by using polyglutamic acid or its salt and functional oligosaccharide.

[0014] Optionally, the engineered bacteria activates the expression of the acyltransferase and glycosyltransferase by adding regulatory components; Optionally, the regulating component is xylose.

[0015] Optionally, the expression of the engineered bacteria acyltransferase and glycosyltransferase is regulated by a xylose-inducible xylA promoter.

[0016] Optionally, the acyltransferase and glycosyltransferase share a common promoter, and a linker is included between the acyltransferase and the glycosyltransferase; Optionally, the pgsB The promoter is P43; Optionally, the pgsA The promoter is xylA; Optionally, the pgsC The promoter is veg.

[0017] Optionally, the pgsB Originated from Bacillus subtilis ; Optionally, the pgsC Originated from Bacillus licheniformis ; Optionally, the pgsA Originated from Bacillus amyloliquefaciens .

[0018] Optional, for Bacillus licheniformis ATCC 9945.

[0019] Optional, for Bacillus amyloliquefaciens FZB42.

[0020] Optional, for Bacillus subtilis 168.

[0021] Optionally, the acyltransferase includes one or more of RhlA, NtASAT1, NtASAT2, and AtSAT1; Optionally, the glycosyltransferase includes one or more of RhlB, WasF, UrdGT2, UrdGT4, GtfC, and GtfD; Optionally, the acyltransferase is RhlA; Optionally, the glycosyltransferase is RhlB.

[0022] Optionally, the RhlA and RhlB are derived from Pseudomonas aeruginosa .

[0023] Optionally, the NtASAT1 or NtASAT2 is derived from tobacco Nicotiana tabacum ; Optionally, the AtSAT1 is derived from Arabidopsis thaliana Arabidopsis thaliana ; Optionally, the WsaF is derived from Geobacillus stearothermophilus Geobacillus stearothermophilus ; Optionally, the UrdGT2 or UrdGT4 is derived from Streptomyces fradiae ; Optionally, the GtfC or GtfD is derived from Streptomyces spp ; Optionally, the promoter of the acyltransferase is a xylose-inducible promoter; Optionally, the promoter of the acyltransferase is the xylose-inducible promoter xylA; Optionally, the promoter of the glycosyltransferase is a xylose-inducible promoter; Optionally, the promoter of the glycosyltransferase is the xylose-inducible promoter xylA; Optionally, the acyltransferase and glycosyltransferase share a common promoter, and a linker is contained between the acyltransferase and glycosyltransferase genes.

[0024] Optionally, the linker encodes a peptide chain of (Gly4Ser)3.

[0025] Optionally, the engineered bacteria contains an expression vector, and the expression vector contains the structure of a xylose-inducible promoter xylA-RhlA-linker-RhlB.

[0026] Optionally, the engineered bacteria contains an expression vector containing a xylose-inducible promoter xylA-RhlB - Structure of linker-RhlA.

[0027] Optionally, the engineered bacteria are one or more of Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens.

[0028] This application uses Bacillus subtilis as a base strain and realizes the synthesis of polyglutamic acid sugar esters through the design of two modules. The first module is the synthesis of polyglutamic acid (γ-PGA). The synthesis of γ-PGA is formed by the polymerization of glutamic acid or its salts catalyzed by the γ-PGA synthase complex (PgsBCA). PgsB and PgsC together form the catalytic site, while PgsA transports γ-PGA outside the cell membrane. This is achieved by integrating γ-PGA synthase complexes from different sources into the genome and by adding promoters of different strengths. pgsB 、 pgsC and pgsA The second module is to catalyze the esterification reaction between polyglutamic acid and functional oligosaccharides. It mainly involves heterologous expression of acyltransferase and glycosyltransferase. Pseudomonas aeruginosa RhlA (acyltransferase) and RhlB (glycosyltransferase) are fused through a flexible linker to shorten the substrate transfer distance. The exogenous acyltransferase and glycosyltransferase are expressed by plasmids to couple the two modules, ultimately achieving the esterification of glutamate and functional oligosaccharides into polymerized glutamate sugar esters.

[0029] It should be noted that the purpose of selecting Bacillus subtilis is that it itself expresses the polyglutamate (γ-PGA) synthase complex. Overexpression can further increase the content of the polyglutamate (γ-PGA) synthase complex, which is beneficial to production. Those skilled in the art can directly use Bacillus subtilis or other engineered bacteria that express the polyglutamate (γ-PGA) synthase complex or engineered bacteria that do not express the polyglutamate (γ-PGA) synthase complex to overexpress the polyglutamate (γ-PGA) synthase complex to achieve the same effect. Therefore, the selection of engineered bacteria can be adjusted and selected by those skilled in the art, and setting and selecting the culture conditions according to different engineered bacteria is also a basic operation.

[0030] The present application provides a bio-fermentation preparation method of polyglutamic acid sugar ester, which comprises the steps of using engineered bacteria to perform bio-fermentation using polyglutamic acid or its salt and functional oligosaccharides as substrates; The engineered bacteria express acyltransferase and glycosyltransferase; The functional oligosaccharide is selected from one or more oligosaccharides or derivatives thereof obtained from the degradation of animal, plant or microbial polysaccharides; Optionally, the acyltransferase and glycosyltransferase share a common promoter, and a linker is contained between the acyltransferase and the glycosyltransferase.

[0031] Optionally, the functional oligosaccharide is selected from one or more seaweed oligosaccharides; Optionally, the seaweed oligosaccharide is selected from one or more of green algae oligosaccharide, brown algae oligosaccharide, and red algae oligosaccharide; Optionally, the seaweed oligosaccharide is selected from one or more of rhamnose oligosaccharide, alginate oligosaccharide, fucoidan oligosaccharide, carrageenan oligosaccharide and agar oligosaccharide; Optionally, the rhamnosaccharide is derived from the enzymatic or chemical hydrolysis product of green algae polysaccharide; Optionally, the alginate oligosaccharide is derived from the enzymatic or chemical hydrolysis product of alginate in brown algae; Optionally, the rhamnose oligosaccharide is an oligosaccharide compound with α-L-rhamnose monomers as the main constituent units, linearly connected by 1,3 and / or 1,4 glycosidic bonds; Optionally, the alginate oligosaccharide is a linear oligosaccharide formed by random or block copolymerization of β-D-mannuronic acid and α-L-guluronic acid through 1,4 glycosidic bonds.

[0032] The present application provides a polymerized glutamate sugar ester having the following general structure:

[0033] R is a substituent derived from functional oligosaccharides, wherein the functional oligosaccharides are selected from one or more oligosaccharides or derivatives thereof obtained from the degradation of animal, plant, or microbial polysaccharides; The molecular weight of the polymerized glutamate sugar ester is 10-5000 kDa, and the molecular weight distribution of the polymerized glutamate sugar ester is ≤1.5.

[0034] Optionally, the molecular weight distribution of the polymerized glutamate sugar ester is ≤1.4; The molecular weight distribution of the polymerized glutamate sugar ester is ≤1.3; The molecular weight distribution of the polymerized glutamate sugar ester is ≤1.2; The molecular weight distribution of the polymerized glutamate sugar ester is ≤1.1; Optionally, the functional oligosaccharide is selected from one or more seaweed oligosaccharides; Optionally, the seaweed oligosaccharide is selected from one or more of green algae oligosaccharide, brown algae oligosaccharide, and red algae oligosaccharide; Optionally, the seaweed oligosaccharide is selected from one or more of rhamnose oligosaccharide, alginate oligosaccharide, fucoidan oligosaccharide, carrageenan oligosaccharide and agar oligosaccharide; Optionally, the rhamnosaccharide is derived from the enzymatic or chemical hydrolysis product of green algae polysaccharide; for example, refer to the method disclosed in the applicant's self-developed process CN201610691257.5 patent.

[0035] Optionally, the alginate oligosaccharide is derived from the enzymatic or chemical hydrolysis product of alginate in brown algae; Optionally, the rhamnose oligosaccharide is an oligosaccharide compound with α-L-rhamnose monomers as the main constituent units, linearly connected by 1,3 and / or 1,4 glycosidic bonds; Optionally, the alginate oligosaccharide is a linear oligosaccharide formed by random or block copolymerization of β-D-mannuronic acid and α-L-guluronic acid through 1,4 glycosidic bonds.

[0036] Optionally, the functional oligosaccharide is obtained by sulfation modification, phosphorylation modification, oxidation modification, esterification modification, amino modification, carboxymethylation modification, acetylation modification, cross-linking, graft copolymerization or chelation with metal ions.

[0037] The present application provides the polymerized glutamate sugar ester prepared by the above method or the application of the above polymerized glutamate sugar ester in agriculture, medicine and food.

[0038] Optional applications in functional agricultural inputs, drug sustained-release carriers, and food additives; Optionally, the invention can be used in fertilizer synergists, biostimulants, water-retaining and drought-resistant agents, anti-tumor drug delivery systems and prebiotic synergists.

[0039] The beneficial effects of this application include but are not limited to: 1. The polyglutamic acid sugar esters provided herein are glycoesterified derivatives of poly-γ-glutamic acid (γ-PGA). Glycoesterification makes their structure more stable, further enhancing their biological activities, including growth promotion, stress resistance, and water and fertilizer conservation in crops, and anti-inflammatory, immune regulation, and intestinal microecological regulation in humans. Furthermore, the polyglutamic acid sugar esters prepared through bioengineering fermentation have a narrower molecular weight distribution and better product uniformity.

[0040] 2. By adopting the "integrated cell factory" biosynthesis technology, precise assembly of glutamic acid and functional oligosaccharides can be achieved under mild conditions (below 45°C, pH 5.0-7.0). The cost is 50%-60% lower than the chemical catalytic esterification method, and the final dosage form is flexible, and both liquid and solid dosage forms can be achieved.

[0041] 3. This application overcomes the industrial bottlenecks faced by traditional esterification technologies, such as low product purity, high energy consumption, and process discontinuity. The bio-fermentation preparation process for polymerized glutamic acid sugar esters provided in this application is a targeted, efficient, and low-energy integrated continuous preparation technology, offering significant technical advantages over traditional chemical catalytic esterification methods.

[0042] 4. This application utilizes a one-pot process with dynamic regulation, utilizing a step-by-step coupling of biofermentation and bioenzymatic catalysis to achieve efficient synthesis of polyglutamic acid and polyglutamic acid esters. This modularization of the reaction compartments facilitates control of the reaction process. Furthermore, the full-stage biosynthesis approach results in mild reaction conditions, minimal production equipment requirements, lower costs, no environmental pollution, and simpler and more efficient product separation. The high specificity and selectivity of the enzyme contribute to higher product quality and purity.

[0043] 5. The polymerized glutamic acid sugar esters provided in this application have significant application potential in green agriculture (e.g., fertilizer synergists, biostimulants, water-retaining and drought-resistant agents), drug sustained-release carriers (e.g., anti-tumor drug delivery systems), and functional food additives (e.g., prebiotic synergists), and possess significant economic value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 Schematic diagram of the structure of the polymerized glutamic acid sugar ester involved in this application; Figure 2 This is a schematic diagram of a specific structure of the polymerized glutamic acid sugar ester involved in this application; Figure 3 The biosynthetic pathway of the polymeric glutamate sugar ester involved in this application; Figure 4 This is a liquid chromatography chart of the molecular weight of the product of the polymerized glutamic acid sugar ester chemical process involved in Example 1 of the present application; Figure 5 This is a liquid chromatography diagram of the molecular weight of the products of the biosynthetic route of polymerized glutamate sugar esters involved in Example 3 of the present application; Figure 6 This is a liquid chromatography diagram of the molecular weight of the products of the biosynthetic route of polymerized glutamate sugar esters involved in Example 4 of the present application; Figure 7 This is an infrared spectrum of the product of the biosynthesis route of polymerized glutamate sugar esters involved in Example 3 of the present application; Figure 8 This is an infrared spectrum of the product of the biosynthesis route of polymerized glutamate sugar esters involved in Example 4 of the present application; Figure 9 This is a graph showing the yield of polymerized rhamnosyl glutamate by shake flask fermentation of different engineered strains involved in Example 4 of this application; Figure 10 The polymerized rhamnosyl glutamate powder involved in Example 6 of the present application is in solution at different dilution ratios; Figure 11 The stability of the polymerized rhamnosyl glutamate stock solution involved in Example 6 of the present application at different temperatures; Figure 12 The stability of the polymerized rhamnosyl glutamate involved in Example 6 of the present application in acidic (pH = 1-6) and alkaline (pH = 7-12) solutions; Figure 13 The water retention effect of polyglutamic acid fucoidan in Example 7 of this application (A is water, B is polyglutamic acid fucoidan); Figure 14 This is the chelating effect of poly-rhamnosyl glutamate and poly-fucoidoglutamate on calcium ions involved in Example 8 of the present application (A is pure water, B is poly-rhamnosyl glutamate, and C is poly-fucoidoglutamate). DETAILED DESCRIPTION

[0045] The present application is described in detail below with reference to examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and reagents in the examples of the present application are purchased through commercial channels.

[0046] The polymerized glutamate sugar ester provided in the present application has a long chain of polyglutamic acid as the main skeleton, and functional oligosaccharides with biological activity are grafted onto it as side chains through stable ester bonds. The fertilizer efficiency-enhancing and water-retention functions of γ-PGA are organically combined with the immune stimulation and growth-promoting functions of seaweed oligosaccharides through ester bonds. It is expected to achieve multiple goals of fertilizer efficiency-enhancing, water retention and drought resistance, immune stimulation and growth promotion at the same time, and is expected to serve as a multifunctional and efficient new agricultural biomaterial.

[0047] From a chemical perspective, each glutamic acid unit in the polyglutamic acid molecular chain contains a free carboxyl group (-COOH), while the seaweed oligosaccharide molecules are rich in hydroxyl groups (-OH). The two have the theoretical basis for esterification reaction to form a covalent ester bond (-COO-).

[0048] However, achieving this reaction and precisely controlling the product structure presents significant technical challenges. Conventional methods that theoretically enable this type of esterification reaction primarily rely on chemical synthesis. This typically requires the use of concentrated sulfuric acid as a catalyst, performed under high temperature and high pressure; or in an organic solvent system, using condensing agents such as dicyclohexylcarbodiimide (DCC) and N,N'-carbonyldiimidazole (CDI) and catalysts such as 4-dimethylaminopyridine (DMAP), performed under high temperature and anhydrous conditions.

[0049] These methods have many inherent defects: 1) Harsh reaction conditions: high temperature and strong acid may cause degradation of heat-labile sugar molecules and γ-PGA chains; 2) Use of toxic reagents: condensation agents such as DCC and their by-products are toxic and may remain in the product, limiting their application in green agriculture; 3) Poor selectivity and heterogeneous product structure: It is difficult to control the esterification site in the reaction, which may lead to cross-linking, excessively wide molecular weight distribution (large PDI), low degree of substitution and other problems, and large differences between product batches; 4) Complicated post-processing and high cost: Multiple purifications are required to remove chemical reagents, which increases process complexity and production costs; 5) Environmentally unfriendly: The use and handling of organic solvents and chemical reagents violates the principles of green chemistry and sustainable development.

[0050] To this end, the present application specifically develops a green, efficient, and structure-controllable preparation process for polymerized glutamate sugar esters, which is crucial for promoting the application of such high-performance biomaterials in agriculture and other fields. It can overcome the above-mentioned defects of existing chemical synthesis methods and provide a biosynthesis method based on synthetic biology and fermentation engineering. This technology has disruptive advantages such as mild conditions, simple processes, and environmental friendliness, providing a new polymer material and technical solution for this field.

[0051] The main technical ideas of the present application are described below using green algae-derived rhamnosyl sulfate oligosaccharide as an active side chain polymerized rhamnosyl glutamate and brown algae-derived alginate oligosaccharide as an active side chain as examples.

[0052] It should be noted that the functional oligosaccharides used as side chains may also be natural plant or animal or microbial oligosaccharides, oligosaccharides obtained by degradation of natural plant or animal or microbial polysaccharides, enzymatically / chemically synthesized oligosaccharides or their derivatives.

[0053] Example 1 Preparation of polyglutamic acid rhamnosyl ester by acid catalysis Raw materials used: poly-γ-glutamic acid (molecular weight 80 kDa, degree of polymerization n=650), rhamnosaccharide (extracted from seaweed, DP=5, purity ≥95%).

[0054] The synthesis steps are as follows: 1) Substrate premixing: γ-PGA (10 g) + rhamnose oligosaccharide (8 g) + purified water (200 ml) are magnetically stirred (500 rpm, 50°C, 2 h) until completely dissolved to obtain a premixed solution; 2) Acid-catalyzed reaction: The premixed solution is added to a round-bottom flask, and concentrated sulfuric acid (6 ml, accounting for 3% of the system volume) is added. The temperature is maintained at 70°C in a water bath and mechanically stirred (300 rpm, 8 h); 3) Neutralization and purification: The reaction solution is cooled to 25°C, and 0.5 M NaOH is slowly added dropwise to pH = 7. The temperature is maintained at <30°C in an ice bath. The product is purified and concentrated using an ultrafiltration membrane (molecular weight cutoff 10 kDa) to obtain the polyglutamic acid rhamnosyl ester product.

[0055] The results and data analysis are as follows: 1) Conversion rate determination (HPLC) Mobile phase: acetonitrile-water (70:30, v / v); flow rate: 1.0 ml / min; detection wavelength: 210 nm.

[0056] The results showed that the conversion rate was 82.5% (±1.2%, n=3).

[0057] 2) Product purity (HPLC area normalization method) The results are as follows Figure 4 As shown, the retention time of the main peak (polyglutamic acid rhamnosyl ester) is 14.2 min, the peak area accounts for 64.3%, and the purity is 64.3%. It is speculated that the secondary peak is the polyglutamic acid β-ester isomer.

[0058] 3) Molecular weight distribution (GPC) Mobile phase: 0.1 M Na2SO4 solution; flow rate: 1.0 ml / min; detection wavelength: 210 nm; calibration with PS standard.

[0059] The results showed: Mw=85300 Da, Mn=52650 Da, PDI=1.62.

[0060] 4) Byproduct analysis (GC-MS / HPLC-MS) Table 1 By-product analysis results

[0061] In summary, traditional chemical catalysis can be used to synthesize polyglutamic acid rhamnosyl esters. However, this method has the following drawbacks: 1) uncontrollable side reactions, such as acid hydrolysis and cleavage of polyglutamic acid and oligosaccharide chains, hydrolysis and release of the sulfate group of rhamnose sulfate, and acidic dehydration of rhamnose to produce furfural; 2) high energy consumption and high pollution; heating the ton-scale reaction system to 70°C consumes a large amount of energy, and the COD of the reaction wastewater after neutralization is high, requiring two-stage reverse osmosis treatment, further increasing costs; 3) poor product uniformity (PDI = 1.62); Given that the quality and preparation process of products prepared by traditional chemical catalysis methods need to be optimized, this application proposes an updated process for preparing polymerized glutamate glycolipids based on a microbial fermentation method, which iterates the above-mentioned traditional chemical catalysis process.

[0062] Example 2 Construction of an engineered strain for producing polyglutamic acid sugar esters The construction of the engineered strain for producing polyglutamate sugar esters is mainly divided into two modules. First, using glutamate or its salts as the substrate, the polyglutamate (γ-PGA) synthase complex is overexpressed in Bacillus subtilis to catalyze the polymerization of glutamate into γ-PGA. When γ-PGA is polymerized to a certain extent, the expression of the second module, namely acyltransferase and glycosyltransferase, is activated, and the production and secretion of polyglutamate sugar esters is achieved by adding functional oligosaccharides.

[0063] The steps for engineering strain construction and key enzyme screening are as follows: 1) Origin of the first module γ-PGA synthase complex Table 2 Sources of γ-PGA synthase complex

[0064] 2) Design of promoter strength gradient in the first module Table 3 Promoter strength and regulatory characteristics

[0065] Table 4 Design of different promoter strength combinations in the first module

[0066] 3) First module genome integration (CRISPR-Cas9 system) 3.1) Integration site design: Target: amyE Gene loci (non-essential regions that do not affect growth); Donor plasmid: containing expression cassette (e.g. promoter combination 1: P43- pgsA -xylA- pgsB- veg- pgsC ), screening was performed for the different donor plasmids constructed in Table 4; 3.2) Electroporation and screening: Prepare Bacillus subtilis competent cells (pretreated with 0.1 M CaCl2), and then co-electroporate the following: donor plasmid to be screened (10 μg) and CRISPR plasmid pJOE8999 (5 μg, containing Cas9 and sgRNA) under the following electroporation conditions: 2.5 kV, 5 ms, 4°C; resuscitation: SMM liquid medium, shaken at 37°C for 1 h; PCR verification and screening.

[0067] Integration of expression cassettes with different promoter combinations into the Bacillus subtilis genome amyE Six engineered strains B-1, B-2, B-3, B-4, B-5, and B-6 were obtained from the site (corresponding to promoter combinations 1 to 6 in Table 4 ).

[0068] 4) The second module heterologously expresses acyltransferase and glycosyltransferase Will come from Pseudomonas aeruginosa The RhlA (acyltransferase) and RhlB (glycosyltransferase) genes were connected by a DNA sequence encoding a (Gly4Ser)3 flexible linker to form a fusion gene. rhlA -linker- rhlB The fusion gene was cloned into the downstream of the xylose-inducible promoter xylA of the Bacillus subtilis expression vector pAX01 to construct the recombinant plasmid pAX01- rhlAB pAX01- rhlAB The plasmids were respectively introduced into the six bacterial strains constructed in 3) to obtain the polyglutamic acid sugar ester production engineering strains BS-1, BS-2, BS-3, BS-4, BS-5, and BS-6 assembled with two modules.

[0069] Example 4 Preparation of Polymeric Rhamnosyl Glutamate 1) Screening and verification of high-yield polyglutamic acid ester production strains Table 5 Screening and verification of high-yield polymerized rhamnosyl glutamate strains

[0070] The results are as follows Figure 9 As shown, it can be seen that the engineered strain BS-6 has the highest yield of poly-rhamnosyl glutamate, reaching 25.8 g / L, so the obtained engineered strain BS-6 was used for subsequent process optimization.

[0071] 2) Fermentation and induced expression The engineered strain BS-6 was inoculated into LB medium containing 5 μg / mL erythromycin and activated at 37°C for 12 hours. The inoculum size was 8% and transferred to a 5 L fermentor (working volume 3 L). The initial medium composition was: 60 g / L sodium glutamate, 30 g / L glucose, 5 g / L yeast extract, and 0.1 mM MnCl2. The control parameters were: temperature 37°C, pH 7.0, dissolved oxygen >30% (stirring speed 300 rpm). The key induction operation (performed simultaneously at 36 hours of fermentation) was: adding sterile xylose to a final concentration of 5 g / L (activating the xylA promoter, turning on RhlA (acyltransferase) and RhlB (glycosyltransferase) and pgsAgene expression); the fermentation temperature was adjusted to 30°C; the stirring rate was reduced to 160 rpm to reduce shear stress; and the expression was induced for 8 hours.

[0072] 3) Intracellular enzyme-catalyzed glycoesterification reaction After induction, sterile rhamnosaccharide solution (200 g / L, dissolved in 50 mM Tris-HCl buffer, pH 7.5) was added at a flow rate of 5 g / L / h. The reaction conditions were maintained at 30°C, pH 7.0, and a stirring rate of 160 rpm. Samples were taken every 2 hours during the addition to monitor substrate consumption.

[0073] 4) Self-assembly of biosynthetic products 12 hours after the addition, the viscosity of the fermentation broth increased significantly (>5000 cP), indicating that the self-assembly of the polymerized rhamnosyl glutamic acid ester was complete. The reaction was terminated by centrifugation (8000 rpm, 10 minutes) to collect the supernatant, and the product was detected by HPLC. The results are as follows: Figure 5 As shown in Figure 2, the weight average molecular weight of the product is 12.82 kDa and PDI=1.09. Figure 7 As shown, at 1725 cm -1 There is a characteristic absorption peak of ester carbonyl (OC=O), indicating that the product is successfully esterified.

[0074] Example 5 Preparation of polyglutamic acid fucoidan This example is basically the same as Example 4, except that the sterile rhamnosaccharide solution is replaced by a sterile alginate oligosaccharide solution (prepared by enzymatic hydrolysis and purification of alginate extracted from kelp).

[0075] HPLC detection of the product showed the following results: Figure 6 As shown in Figure 2, the weight average molecular weight of the product is 12.75 kDa and PDI = 1.10. Figure 8 As shown, at 1745 cm -1 There is a characteristic absorption peak of ester carbonyl (OC=O), indicating that the product is successfully esterified.

[0076] Example 6 Performance Test of Polymeric Rhamnosyl Glutamate like Figure 10 As shown, the poly(rhamnosyl glutamate) powder exhibited good solubility at various dilution ratios (20-2000 times), with stable solutions and no observed insoluble matter. This property demonstrates that poly(rhamnosyl glutamate) can flexibly adapt to and meet the needs of various application scenarios, including aerial spraying, foliar spraying, flushing, and drip irrigation, demonstrating its broad application potential and practicality.

[0077] like Figure 11As shown, poly rhamnose glutamate can ensure the stability of product indicators under both high and low temperature conditions, meeting the demand for product stability in the production of new fertilizers. Through a large amount of test data, it was found that the loss rate of poly rhamnose glutamate in fertilizer production is stably controlled within 10%.

[0078] like Figure 12 The following figure shows the solution state of poly(rhamnosyl glutamate) under acidic and alkaline conditions. It can be seen that poly(rhamnosyl glutamate) is stable in the pH range of 1 to 12, with clear solutions and no flocculation or precipitation. This property indicates that poly(rhamnosyl glutamate) can be combined with fertilizers and pesticides of different acidity and alkalinity, and has a wide range of applications.

[0079] Example 7 Water Retention Performance Test of Polyglutamic Acid Fucoidan Materials: soil sample, polyglutamic acid fucoidan (2-fold dilution).

[0080] Methods: 1) Place 30 g of soil in a beaker and add 10 ml of polyglutamic acid fucoidan diluent. For the control group, add an equal amount of water. 2) Allow the soil to stand until it is fully soaked, then turn it upside down on filter paper and observe the size of the water diffusion zone and the soil quality.

[0081] like Figure 13 As shown, in the group with polyglutamic acid fucoidan ester, water diffused slowly, the water halo area was small, and the soil was loose; in the control group with pure water, water diffused quickly, the water halo area was large, and the soil was hard.

[0082] Example 8 Testing of Calcium Chelating Performance of Poly-rhamnosyl Glutamate and Poly-fucosyl Glutamate Materials: 7% calcium chloride solution, 7% ammonium sulfate solution, 5% polyglutamic acid rhamnosyl ester aqueous solution, 5% polyglutamic acid fucoidan ester aqueous solution.

[0083] Methods: 1) Add 50 ml of calcium chloride solution to three beakers, add 0.5 ml of polyglutamic acid rhamnosyl ester aqueous solution and 0.5 ml of polyglutamic acid fucoidan aqueous solution to two of them, and stir evenly; 2) Add 50 ml of ammonium sulfate solution and let it stand for observation; 3) Add water to the calcium solution as a control and observe in the same way.

[0084] like Figure 14 As shown, after 1 minute, a large amount of calcium sulfate precipitation was generated in the clear water (without the addition of polyglutamic acid rhamnosyl esters and polyglutamic acid fucoidan esters), and the two polyglutamic acid sugar ester groups were clear and stable.

[0085] Example 9 pH buffering effect test of polymerized rhamnosyl glutamate Materials: 5 g / L potassium sulfate solution (pH 3.6), 60 g / L urea solution (pH 9.2), 5% poly-rhamnosyl glutamate aqueous solution, and water.

[0086] Methods and results: Adjust the acidic solution: 1) Measure the initial pH value of potassium sulfate solution to 3.6; 2) Add 0.5 ml of polyglutamic acid rhamnosyl ester aqueous solution, stir evenly, and measure the pH value to 4.61.

[0087] Adjust the alkaline solution: 1) Measure the initial pH value of the urea solution to 9.2; 2) Add 0.5 ml of the aqueous solution of polyglutamic acid rhamnosyl ester, stir evenly, and measure the pH value to 7.11.

[0088] The buffering effect is shown in Table 6.

[0089] Table 6 Buffering effect test results

[0090] According to the results in Table 6, the polymerized rhamnosyl glutamate provided in the present application has a pH buffering effect.

[0091] This application scheme iterates the process of preparing polymerized rhamnosyl glutamate esters by traditional chemical catalysis. The prepared polymerized rhamnosyl glutamate ester products have a narrower molecular weight distribution and higher uniformity, achieving higher quality polymerized rhamnosyl glutamate ester product updates and iterations, which is of great significance for improving the market competitiveness of polymerized rhamnosyl glutamate ester products and has important industrial application value.

[0092] The foregoing is merely an embodiment of the present application, and the scope of protection of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, various modifications and variations can be made to the present application. Any modifications, equivalent substitutions, improvements, etc. made within the technical ideas and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A bio-fermentation method for preparing polymerized glutamic acid sugar esters, characterized in that: The preparation method comprises the steps of using engineered bacteria to carry out biological fermentation with glutamic acid or its salt and functional oligosaccharides as substrates; The engineered bacteria express acyltransferase, glycosyltransferase, pgsB 、 pgsC and pgsA ; The functional oligosaccharide is selected from one or more oligosaccharides or derivatives thereof obtained from animal, plant or microorganism oligosaccharides, or oligosaccharides or derivatives thereof obtained by degradation of animal, plant or microorganism polysaccharides.

2. The bio-fermentation preparation method of polymerized glutamic acid sugar ester according to claim 1, characterized in that: The functional oligosaccharide is selected from one or more of green algae oligosaccharides, brown algae oligosaccharides, and red algae oligosaccharides.

3. The bio-fermentation preparation method of polymerized glutamic acid sugar ester according to claim 1, characterized in that: Control the engineering bacteria to express first pgsB 、 pgsC and pgsA Synthesizing polyglutamic acid or its salt from glutamic acid or its salt; The engineered bacteria are controlled to express acyltransferase and glycosyltransferase, and the polymerized glutamate sugar ester is synthesized by using polyglutamic acid or its salt and functional oligosaccharide.

4. The bio-fermentation preparation method of polymerized glutamic acid sugar ester according to claim 1, characterized in that: The acyltransferase and glycosyltransferase share a promoter, and a linker is included between the acyltransferase and the glycosyltransferase.

5. A bio-fermentation method for preparing polymerized glutamic acid sugar esters, characterized in that: The preparation method comprises the steps of using engineered bacteria to carry out biofermentation using polyglutamic acid or its salt and functional oligosaccharides as substrates; The engineered bacteria express acyltransferase and glycosyltransferase; The functional oligosaccharide is selected from one or more oligosaccharides or derivatives thereof obtained from animal, plant or microorganism oligosaccharides, or oligosaccharides or derivatives thereof obtained by degradation of animal, plant or microorganism polysaccharides.

6. The bio-fermentation preparation method of polymerized glutamic acid sugar ester according to claim 5, characterized in that: The functional oligosaccharide is selected from one or more of green algae oligosaccharides, brown algae oligosaccharides, and red algae oligosaccharides.

7. A polymerized glutamic acid sugar ester, characterized in that: The polymerized glutamic acid sugar ester has the following general structure: R is a substituent derived from functional oligosaccharides, wherein the functional oligosaccharides are selected from one or more oligosaccharides or derivatives thereof obtained from the degradation of animal, plant, or microbial polysaccharides; The molecular weight of the polymerized glutamate sugar ester is 10-5000 kDa, and the molecular weight distribution of the polymerized glutamate sugar ester is ≤1.

5.

8. The polymerized sugar glutamate ester according to claim 7, characterized in that The functional oligosaccharide is selected from one or more of green algae oligosaccharides, brown algae oligosaccharides, and red algae oligosaccharides.

9. The polymerized sugar glutamate ester according to claim 8, characterized in that The functional oligosaccharide is obtained by sulfation modification, phosphorylation modification, oxidation modification, esterification modification, amination modification, carboxymethylation modification, acetylation modification, cross-linking, graft copolymerization or chelation with metal ions.

10. Use of the polymerized glutamate sugar ester prepared by the method according to any one of claims 1 to 6 or the polymerized glutamate sugar ester according to any one of claims 7 to 9 in agriculture, medicine and food.

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