Polymerized glutamic acid sugar ester, and a method for preparing the same by biofermentation and use thereof

Abstract

The application relates to a polymeric glutamic acid sugar ester and a biological fermentation preparation method and application thereof, and belongs to the technical field of biological fermentation. According to the application, amino acids and functional oligosaccharides are used as substrates, and a 'one-body cell factory' biosynthesis technology is designed to realize efficient directional assembly of high-molecular polymeric glutamic acid sugar esters. The application provides a brand-new polymeric glutamic acid sugar ester compound, and provides a biological fermentation method of the polymeric glutamic acid sugar ester, which is suitable for industrialized continuous production, and the prepared polymeric glutamic acid sugar ester has a narrower molecular weight distribution and better product uniformity.

Description

Technical Field

[0001] This application relates to a polymeric glutamic acid glycoester, its preparation method by bio-fermentation, and its application, belonging to the field of bio-fermentation technology. Background Technology

[0002] Polyglutamic acid (γ-PGA) is a natural anionic polymer formed by the polymerization of glutamic acid monomers through γ-amide bonds. It possesses excellent biodegradability, biocompatibility, and superior water retention capacity. In agriculture, γ-PGA, due to its unique molecular structure, exhibits remarkable fertilizer enhancement, soil improvement, and stress resistance and growth promotion functions. The numerous free carboxyl groups on its molecular chain can chelate micronutrients 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, playing a role in drought resistance and moisture conservation. Simultaneously, γ-PGA can promote crop root development and enhance crop resistance to abiotic stresses such as drought and salinity.

[0003] Seaweed, especially green and brown algae, is rich in various bioactive polysaccharides and oligosaccharides. For example, rhamnosaccharide sulfate (ROS) from green algae and alginate oligosaccharide (AOS) from brown algae have been shown to have various bioactivities, such as promoting plant growth, stimulating plant immune responses, enhancing plant stress resistance, and improving crop quality. These seaweed oligosaccharides, as signaling molecules, can regulate the expression of related genes in plants, induce disease resistance, and can also serve as natural organic nutrients.

[0004] Ester-linked bioactive molecules play a crucial role in plant growth regulation. Brassinolide (a sterol ester), a well-known plant growth regulator, is one of the most active and widely used, significantly enhancing crop photosynthesis, increasing yield, and improving stress resistance. Similarly, aminoethyl ester (DA-6), a carboxylic acid ester, also exhibits high plant growth-regulating activity. Furthermore, glycolipid biosurfactants such as rhamnolipids have been found to promote plant growth. These studies demonstrate that linking active groups via ester bonds can produce synergistic effects and may even endow molecules with entirely new functions; the structural characteristics of these molecules determine their functional diversity.

[0005] Traditional polyamino acid and sugar esterification preparation methods mostly employ chemical catalytic esterification, which has the following drawbacks: 1) numerous byproducts (e.g., sulfonation byproducts ≥15% in concentrated sulfuric acid catalysis), resulting in low purity of the target product (<85%); 2) high energy consumption (requiring high temperature >80℃ or high pressure conditions); 3) difficulty in controlling the molecular weight distribution of the product (polydispersity index PDI >1.5); 4) chemical catalyst residues leading to complex post-processing.

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

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

[0008] To address the aforementioned issues, this application provides a method for preparing and applying a polymeric glutamate glycoester via bio-fermentation. Specifically, it provides an efficient method for preparing a polymeric glutamate glycoester based on enzyme-catalyzed self-assembly technology. By employing an "integrated cell factory" biosynthesis technology, the precise assembly of glutamate and functional oligosaccharides is achieved under mild conditions (below 45°C, pH 5.0~7.0). The cost is reduced by 50%~60% compared to chemical catalytic esterification, and the prepared polymeric glutamate glycoester exhibits a narrower molecular weight distribution and better product uniformity.

[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 ester bonds, and then forms a novel multifunctional biopolymer with a network macromolecular structure through intermolecular polymerization and cross-linking, thereby realizing the functional integration and enhancement of functional oligosaccharides and polyglutamic acid.

[0010] The prepared polyglutamic acid glycoester is a glycoesterified derivative of poly-γ-glutamic acid (γ-PGA). Glycoesterification modification enhances its structural stability, further strengthening its bioactivity in crops, including growth promotion, stress resistance, water and fertilizer retention, as well as its anti-inflammatory, immunomodulatory, and gut microbiota regulation effects in humans. It shows significant application potential in green agriculture (e.g., 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).

[0011] This application provides a method for the bio-fermentation preparation of polyglutamic acid sugar esters, the method comprising the step of using engineered bacteria to carry out bio-fermentation with glutamic acid or its salt and functional oligosaccharides as substrates;

[0012] The engineered bacteria express acyltransferases and glycosyltransferases. pgsB , pgsC and pgsA ;

[0013] The functional oligosaccharide is selected from one or more of the following: plant, animal, or microbial oligosaccharides or their derivatives, or oligosaccharides or their derivatives obtained from the degradation of plant, animal, or microbial polysaccharides.

[0014] Optionally, the functional oligosaccharide is selected from one or more of seaweed oligosaccharides;

[0015] Optionally, the seaweed oligosaccharide is selected from one or more of green algae oligosaccharides, brown algae oligosaccharides, and red algae oligosaccharides;

[0016] Optionally, the seaweed oligosaccharide is selected from one or more of rhamnosaccharide, alginate oligosaccharide, fucoidan oligosaccharide, carrageenan oligosaccharide, and agar oligosaccharide;

[0017] Optionally, the rhamnooligosaccharide is derived from the enzymatic or chemical hydrolysis products of green algae polysaccharides;

[0018] Optionally, the alginate oligosaccharide is derived from the enzymatic or chemical hydrolysis products of alginate in brown algae;

[0019] Optionally, the rhamnooligosaccharide is an oligosaccharide compound formed by linear linkage of α-L-rhamnosaccharide monomers through 1,3 and / or 1,4 glycosidic bonds as the main constituent units;

[0020] 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.

[0021] Optionally, the engineered bacteria can be controlled to first express... pgsB , pgsC and pgsA Polyglutamic acid or its salts are synthesized from glutamic acid or its salts;

[0022] After controlling the engineered bacteria to express acyltransferase and glycosyltransferase, the polyglutamic acid glycoester is synthesized by combining polyglutamic acid or its salt with functional oligosaccharides.

[0023] Optionally, the engineered bacteria can activate the expression of the acyltransferase and glycosyltransferase by adding regulatory components;

[0024] Optionally, the regulating ingredient is xylose.

[0025] Optionally, the expression of the engineered bacterial acyltransferase and glycosyltransferase is regulated by the xylose-inducible xylA promoter.

[0026] Optionally, the acyltransferase and glycosyltransferase share a single promoter, and a linker is present between the acyltransferase and glycosyltransferase.

[0027] Optionally, the pgsB The promoter is P43;

[0028] Optionally, the pgsA The promoter is xylA;

[0029] Optionally, the pgsC The promoter is veg.

[0030] Optionally, the pgsBSource Bacillus subtilis ;

[0031] Optionally, the pgsC Source Bacillus licheniformis ;

[0032] Optionally, the pgsA Source Bacillus amyloliquefaciens .

[0033] Optional, for Bacillus licheniformis ATCC 9945.

[0034] Optional, for Bacillus amyloliquefaciens FZB42.

[0035] Optional, for Bacillus subtilis 168.

[0036] Optionally, the acyltransferase includes one or more of RhlA, NtASAT1, NtASAT2, and AtSAT1;

[0037] Optionally, the glycosyltransferase includes one or more of RhlB, WasF, UrdGT2, UrdGT4, GtfC, and GtfD;

[0038] Optionally, the acyltransferase is RhlA;

[0039] Optionally, the glycosyltransferase is RhlB.

[0040] Optionally, RhlA and RhlB are derived from... Pseudomonas aeruginosa .

[0041] Optionally, the NtASAT1 or NtASAT2 is derived from tobacco. Nicotiana tabacum ;

[0042] Optionally, the AtSAT1 is derived from Arabidopsis thaliana. Arabidopsis thaliana ;

[0043] Optionally, the WsaF is derived from *Bacillus stearothermophilus*. Geobacillus stearothermophilus ;

[0044] Optionally, the UrdGT2 or UrdGT4 is derived from... Streptomyces fradiae ;

[0045] Optionally, the GtfC or GtfD is derived from... Streptomyces spp ;

[0046] Optionally, the promoter of the acyltransferase is a xylose-inducible promoter;

[0047] Optionally, the promoter of the acyltransferase is the xylose-inducible promoter xylA;

[0048] Optionally, the promoter of the glycosyltransferase is a xylose-inducible promoter;

[0049] Optionally, the promoter of the glycosyltransferase is the xylose-inducible promoter xylA;

[0050] Optionally, the acyltransferase and glycosyltransferase share a single promoter, and a linker is included between the acyltransferase and glycosyltransferase genes.

[0051] Optionally, the linker encodes a peptide chain of (Gly4Ser)3.

[0052] Optionally, the engineered bacteria contains an expression vector containing the structure of the xylose-inducible promoter xylA-RhlA-linker-RhlB.

[0053] Optionally, the engineered bacteria contains an expression vector containing the xylose-inducible promoter xylA-RhlB. - The structure of linker-RhlA.

[0054] Optionally, the engineered bacteria are one or more of Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens.

[0055] This application uses Bacillus subtilis as the chassis strain and achieves the synthesis of polyglutamic acid esters through a two-module design. The first module is the synthesis of polyglutamic acid (γ-PGA), which 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 to the extracellular space. This is achieved by integrating γ-PGA synthase complexes from different sources into the genome and by adding promoters of varying strengths. pgsB , pgsC and pgsA The second module catalyzes the esterification reaction of polyglutamic acid with functional oligosaccharides at different expression levels. This mainly involves the heterologous expression of acyltransferases and glycosyltransferases, which... Pseudomonas aeruginosa RhlA (acyltransferase) and RhlB (glycosyltransferase) are fused through a flexible linker to shorten the substrate delivery distance. By expressing exogenous acyltransferase and glycosyltransferase through plasmids, the two modules are coupled, ultimately achieving the esterification of glutamate with functional oligosaccharides into polyglutamate glycoesters.

[0056] It should be noted that the purpose of selecting Bacillus subtilis is to enable it to express polyglutamate (γ-PGA) synthase complex. Overexpression can further increase the content of polyglutamate (γ-PGA) synthase complex, which is beneficial for production. Those skilled in the art can directly use Bacillus subtilis or other engineered bacteria that express polyglutamate (γ-PGA) synthase complex or engineered bacteria that do not express polyglutamate (γ-PGA) synthase complex to overexpress polyglutamate (γ-PGA) synthase complex, and 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 culture conditions according to different engineered bacteria is also a basic operation.

[0057] This application provides a method for the bio-fermentation preparation of polyglutamic acid glycoesters, the method comprising the step of using engineered bacteria to carry out bio-fermentation with polyglutamic acid or its salt and functional oligosaccharides as substrates;

[0058] The engineered bacteria express acyltransferases and glycosyltransferases;

[0059] The functional oligosaccharide is selected from one or more of the following: plant, microbial oligosaccharides or their derivatives, or oligosaccharides or their derivatives obtained by degradation of plant, microbial, or animal polysaccharides.

[0060] Optionally, the acyltransferase and glycosyltransferase share a single promoter, and a linker is present between the acyltransferase and glycosyltransferase.

[0061] Optionally, the functional oligosaccharide is selected from one or more of seaweed oligosaccharides;

[0062] Optionally, the seaweed oligosaccharide is selected from one or more of green algae oligosaccharides, brown algae oligosaccharides, and red algae oligosaccharides;

[0063] Optionally, the seaweed oligosaccharide is selected from one or more of rhamnosaccharide, alginate oligosaccharide, fucoidan oligosaccharide, carrageenan oligosaccharide, and agar oligosaccharide;

[0064] Optionally, the rhamnooligosaccharide is derived from the enzymatic or chemical hydrolysis products of green algae polysaccharides;

[0065] Optionally, the alginate oligosaccharide is derived from the enzymatic or chemical hydrolysis products of alginate in brown algae;

[0066] Optionally, the rhamnooligosaccharide is an oligosaccharide compound formed by linear linkage of α-L-rhamnosaccharide monomers through 1,3 and / or 1,4 glycosidic bonds as the main constituent units;

[0067] 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.

[0068] This application provides a polymeric glutamic acid glycoester, which has the following general formula:

[0069]

[0070] R is a substituent derived from a functional oligosaccharide, wherein the functional oligosaccharide is selected from one or more of plant, animal, or microbial oligosaccharides or their derivatives, or oligosaccharides or their derivatives obtained from the degradation of plant, animal, or microbial polysaccharides.

[0071] The polymeric glutamate ester has a molecular weight of 10~5000 kDa and a molecular weight distribution of ≤1.5.

[0072] Optionally, the molecular weight distribution of the polymeric glutamate ester is ≤1.4;

[0073] The molecular weight distribution of the polymeric glutamic acid glycoester is ≤1.3;

[0074] The molecular weight distribution of the polymeric glutamic acid glycoester is ≤1.2;

[0075] The molecular weight distribution of the polymeric glutamate ester is ≤1.1;

[0076] Optionally, the functional oligosaccharide is selected from one or more of seaweed oligosaccharides;

[0077] Optionally, the seaweed oligosaccharide is selected from one or more of green algae oligosaccharides, brown algae oligosaccharides, and red algae oligosaccharides;

[0078] Optionally, the seaweed oligosaccharide is selected from one or more of rhamnosaccharide, alginate oligosaccharide, fucoidan oligosaccharide, carrageenan oligosaccharide, and agar oligosaccharide;

[0079] Optionally, the rhamnooligosaccharide is derived from the enzymatic or chemical hydrolysis products of green algae polysaccharides; for example, refer to the method disclosed in the applicant's self-developed process patent CN201610691257.5.

[0080] Optionally, the alginate oligosaccharide is derived from the enzymatic or chemical hydrolysis products of alginate in brown algae;

[0081] Optionally, the rhamnooligosaccharide is an oligosaccharide compound formed by linear linkage of α-L-rhamnosaccharide monomers through 1,3 and / or 1,4 glycosidic bonds as the main constituent units;

[0082] 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.

[0083] Optionally, the functional oligosaccharide is obtained by sulfation modification, phosphorylation modification, oxidation modification, esterification modification, amino modification, carboxymethylation modification, acetylation modification, crosslinking, graft copolymerization, or chelation with metal ions.

[0084] This application provides information on the application of the polyglutamic acid ester prepared by the above method or the above-mentioned polyglutamic acid ester in agriculture, medicine, and food.

[0085] Optionally, applications in functional agricultural inputs, drug sustained-release carriers, and food additives;

[0086] Optionally, applications in fertilizer synergists, biostimulants, water-retaining and drought-resistant agents, antitumor drug delivery systems, and prebiotic synergists.

[0087] The beneficial effects of this application include, but are not limited to:

[0088] 1. The polyglutamic acid sugar ester provided in this application is a sugar esterification derivative of poly-γ-glutamic acid (γ-PGA). The structure is more stable through sugar esterification modification, and its biological activities such as promoting crop growth, resisting stress, retaining water and fertilizer, anti-inflammatory, immune regulation and intestinal microecological regulation in humans are further enhanced. Moreover, the polyglutamic acid sugar ester prepared by bioengineering fermentation has a narrower molecular weight distribution and better product uniformity.

[0089] 2. By adopting the "integrated cell factory" biosynthesis technology, the precise assembly of glutamic acid and functional oligosaccharides can be achieved under mild conditions (below 45℃, pH 5.0~7.0), reducing costs by 50%~60% compared to chemical catalytic esterification. The final dosage form is flexible, and both liquid and solid dosage forms can be achieved.

[0090] 3. Breaking through the industrialization bottlenecks faced by traditional esterification technology, such as low product purity, high energy consumption, and discontinuous process, the bio-fermentation preparation process of polyglutamic acid sugar ester provided in this application, as a directional, efficient, and low-energy integrated continuous preparation technology, has significant technical advantages compared with traditional chemical catalytic esterification methods.

[0091] 4. The proposed method adopts a one-pot dynamic control approach, utilizing a stepwise coupling of bio-fermentation and bio-enzyme to achieve efficient synthesis of polyglutamic acid and polyglutamic acid sugar esters. This approach regionalizes and modularizes the reaction, making it easier to control the reaction process. Furthermore, the entire process utilizes biosynthesis, resulting in mild reaction conditions, low equipment requirements, lower costs, no environmental pollution, and simpler and more efficient product separation. Due to the high specificity and selectivity of the enzymes, the product quality and purity are also higher.

[0092] 5. The polyglutamic acid ester provided in this application has significant application potential in green agriculture (such as fertilizer synergists, biostimulants, water-retaining and drought-resistant agents), drug sustained-release carriers (such as anti-tumor drug delivery systems), and functional food additives (such as prebiotic synergists), and has important economic value and application prospects. Attached Figure Description

[0093] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0094] Figure 1 This is a schematic diagram of the structure of the polymeric glutamic acid glycoester involved in this application;

[0095] Figure 2 This is a schematic diagram of a specific structure of the polymeric glutamic acid glycoester involved in this application;

[0096] Figure 3 This application pertains to the biosynthetic pathway of the polyglutamic acid glycoester involved in the present application.

[0097] Figure 4 This is a liquid chromatogram of the molecular weight of the product of the chemical route of polymeric glutamic acid ester involved in Example 1 of this application;

[0098] Figure 5 This is a liquid chromatogram of the molecular weight of the product of the polymeric glutamic acid glycoester biosynthetic route involved in Example 3 of this application;

[0099] Figure 6 This is a liquid chromatogram of the molecular weight of the product of the polymeric glutamic acid glycoester biosynthetic route involved in Example 4 of this application;

[0100] Figure 7 The infrared spectrum of the product of the biosynthetic route of polymeric glutamic acid glycoester involved in Example 3 of this application;

[0101] Figure 8 The infrared spectrum of the product of the biosynthetic route of polymeric glutamic acid ester involved in Example 4 of this application;

[0102] Figure 9 This is a graph showing the yield results of shake-flask fermentation of polyglutamic acid rhamnosyl ester by different engineered strains involved in Example 4 of this application;

[0103] Figure 10 Examples 6 of this application show the solution states of the polyglutamic acid rhamnosyl ester powder at different dilution ratios.

[0104] Figure 11 The stability of the polyglutamic acid rhamnosyl ester stock solution involved in Example 6 of this application at different temperatures;

[0105] Figure 12 The stability of the polymeric rhamnosyl glutamate ester involved in Example 6 of this application in acidic (pH=1-6) and alkaline (pH=7-12) solutions;

[0106] Figure 13 The results show the water retention effect of polyglutamic acid fucoidan ester involved in Example 7 of this application (A is water, B is polyglutamic acid fucoidan ester).

[0107] Figure 14 The chelating effect of polyglutamic acid rhamnosyl ester and polyglutamic acid fucoidan on calcium ions in Example 8 of this application is shown (A is water, B is polyglutamic acid rhamnosyl ester, and C is polyglutamic acid fucoidan). Detailed Implementation

[0108] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0109] The polyglutamic acid glycoester provided in this application uses the long chain of polyglutamic acid as the main backbone, and attaches bioactive functional oligosaccharides as side links through stable ester bonds. The fertilizer-enhancing and water-retaining functions of γ-PGA are organically combined with the immune-stimulating and growth-promoting functions of seaweed oligosaccharides through ester bonds. It is expected to achieve multiple goals of fertilizer enhancement, water retention and drought resistance, immune stimulation and growth promotion at the same time, and is expected to serve as a multifunctional and highly efficient new agricultural biomaterial.

[0110] From a chemical perspective, each glutamic acid unit in the polyglutamic acid molecular chain contains a free carboxyl group (-COOH), while seaweed oligosaccharide molecules are rich in hydroxyl groups (-OH). The two have the theoretical basis to undergo esterification to form covalent ester bonds (-COO-).

[0111] However, achieving this reaction and precisely controlling the structure of the product presents a significant technical challenge. The conventional method for theoretically realizing such esterification reactions is primarily chemical synthesis. This method typically requires the use of concentrated sulfuric acid as a catalyst under high temperature and pressure; or it can be carried out in an organic solvent system using condensing agents such as dicyclohexylcarbodiimide (DCC), N,N'-carbonyldiimidazole (CDI), and catalysts such as 4-dimethylaminopyridine (DMAP) under high temperature and anhydrous conditions.

[0112] These methods have several inherent drawbacks: 1) Harsh reaction conditions: High temperatures and strong acids may degrade thermally unstable sugar molecules and γ-PGA chains; 2) Use of toxic reagents: Condensing agents such as DCC and their byproducts 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 sites in the reaction, which may lead to problems such as cross-linking, excessively wide molecular weight distribution (high PDI), and low degree of substitution, resulting in large batch-to-batch differences in the product; 4) Cumbersome post-processing and high cost: Multiple purifications are required to remove chemical reagents, increasing the complexity of the process 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.

[0113] Therefore, this application develops a green, efficient, and structurally controllable process for preparing polymeric glutamic acid 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 process, and environmental friendliness, providing a brand-new polymer material and technical solution for this field.

[0114] The main technical concept of this application will be explained below using polyrhamnosaccharide glutamic acid with rhamnosaccharide sulfate from green algae as the active side chain and polyfucoidoester glutamic acid with alginate oligosaccharide from brown algae as the active side chain as examples.

[0115] It should be noted that the functional oligosaccharides used as side chains can also be natural plant or animal oligosaccharides, oligosaccharides obtained by degradation of natural plant or animal polysaccharides, enzymatically / chemically synthesized oligosaccharides, or their derivatives.

[0116] Example 1: Preparation of polyglutamic acid rhamnosine ester by acid catalysis

[0117] Raw materials used: poly-γ-glutamic acid (molecular weight 80 kDa, degree of polymerization n=650), rhamnosaccharide (seaweed extract, DP=5, purity ≥95%).

[0118] The synthesis steps are as follows: 1) Substrate premixing: γ-PGA (10 g) + rhamnooligosaccharide (8 g) + purified water (200 ml) were magnetically stirred (500 rpm, 50℃, 2 h) until completely dissolved to obtain a premixed solution; 2) Acid-catalyzed reaction: The premixed solution was added to a round-bottom flask, and concentrated sulfuric acid (6 ml, accounting for 3% of the system volume) was added. The mixture was kept at a constant temperature of 70℃ in a water bath and mechanically stirred (300 rpm, 8 h); 3) Neutralization and purification: The reaction solution was cooled to 25℃, and 0.5M NaOH was slowly added dropwise until pH=7. The temperature was maintained at <30℃ in an ice bath. The mixture was purified and concentrated by ultrafiltration membrane separation (molecular weight cutoff 10 kDa) to obtain the polyglutamic acid rhamnosaccharide ester product.

[0119] The results and data analysis are as follows:

[0120] 1) Conversion rate determination (HPLC)

[0121] Mobile phase: acetonitrile-water (70:30, v / v); flow rate: 1.0 ml / min; detection wavelength: 210 nm.

[0122] The results showed a conversion rate of 82.5% (±1.2%, n=3).

[0123] 2) Product purity (HPLC area normalization method)

[0124] The results are as follows Figure 4 As shown, the main peak (polyglutamic acid rhamnosyl ester) has a retention time of 14.2 min, a peak area of ​​64.3%, and a purity of 64.3%. It is speculated that the secondary peak is the β-esterified isomer of polyglutamic acid.

[0125] 3) Molecular weight distribution (GPC)

[0126] Mobile phase: 0.1 M Na2SO4 solution; flow rate: 1.0 ml / min; detection wavelength: 210 nm; calibration with PS standard.

[0127] The results showed that Mw = 85300 Da, Mn = 52650 Da, and PDI = 1.62.

[0128] 4) Byproduct analysis (GC-MS / HPLC-MS)

[0129] Table 1. Byproduct analysis results

[0130]

[0131] In summary, traditional chemical catalysis can be used to synthesize polyglutamic acid rhamnose ester. However, this method has the following drawbacks: 1) Uncontrollable side reactions, such as hydrolysis and breakage of polyglutamic acid and oligosaccharide chains, hydrolysis and shedding of the sulfate groups of rhamnose sulfate, and acidic dehydration of rhamnose to produce furfural; 2) High energy consumption and high pollution, as heating the ton-scale reaction system to 70°C requires a large amount of energy, and the reaction wastewater has a high COD after neutralization, requiring two-stage reverse osmosis treatment, further increasing costs; 3) Poor product uniformity (PDI=1.62).

[0132] Given that the quality of products prepared by traditional chemical catalysis methods and the preparation process need to be optimized, this application proposes an updated process for preparing polyglutamic acid glycolipids based on microbial fermentation, which iterates over the aforementioned traditional chemical catalysis process.

[0133] Example 2 Construction of engineered strains for the production of polyglutamic acid esters

[0134] The construction of engineered strains for producing polyglutamic acid glycoesters mainly consists of two modules. First, using glutamic acid or its salts as substrates, the polyglutamic acid (γ-PGA) synthase complex is overexpressed in Bacillus subtilis to catalyze the polymerization of glutamic acid into γ-PGA. When γ-PGA has polymerized to a certain extent, the expression of the second module, namely acyltransferase and glycosyltransferase, is initiated. The production and secretion of polyglutamic acid glycoesters are achieved through the addition of functional oligosaccharides.

[0135] The steps for constructing engineered strains and screening key enzymes are as follows:

[0136] 1) Source of the γ-PGA synthase complex in the first module

[0137] Table 2 Sources of γ-PGA synthase complex

[0138]

[0139] 2) First module promoter strength gradient design

[0140] Table 3 Promoter strength and regulation characteristics

[0141]

[0142] Table 4 Design of different promoter strength combinations in Module 1

[0143]

[0144] 3) First module: Genome integration (CRISPR-Cas9 system)

[0145] 3.1) Integration site design:

[0146] Target: amyEGene loci (non-essential regions that do not affect growth);

[0147] Donor plasmid: containing expression cassette (e.g., promoter combination 1: P43-) pgsA -xylA- pgsB- veg- pgsC ), and screen for different donor plasmids constructed in Table 4;

[0148] 3.2) Electroconversion and Screening:

[0149] Prepare competent Bacillus subtilis cells (pretreated with 0.1 M CaCl2) and then co-electropoize them: donor plasmid to be screened (10 μg), CRISPR plasmid pJOE8999 (5 μg, containing Cas9 and sgRNA), electroporation conditions: 2.5 kV, 5 ms, 4℃; recovery: SMM liquid medium, 37℃ shaker for 1 h; PCR verification and screening.

[0150] Expression cassettes with different promoter combinations were integrated 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).

[0151] 4) The second module heterologously expresses acyltransferases and glycosyltransferases

[0152] will come from Pseudomonas aeruginosa The RhlA (acyltransferase) and RhlB (glycosyltransferase) genes are linked by a DNA sequence encoding a (Gly4Ser)3 flexible linker to form a fusion gene. rhlA -linker- rhlB The fusion gene was cloned downstream of the xylA promoter in the Bacillus subtilis expression vector pAX01 to construct the recombinant plasmid pAX01- rhlAB ; pAX01- is converted via electrical conversion (2.5 kV, 5 ms) rhlAB The plasmids were introduced into the six strains constructed in step 3) to obtain engineered strains for the production of polyglutamic acid esters, namely BS-1, BS-2, BS-3, BS-4, BS-5, and BS-6, which were equipped with two modules.

[0153] Example 4 Preparation of Polymeric Rhamnosyl Glutamate

[0154] 1) Screening and validation of engineered strains for high-yield production of polyglutamic acid esters

[0155] Table 5 Screening and validation of strains producing high-polymerized rhamnosine esters.

[0156]

[0157] The results are as follows Figure 9 As shown, the engineered strain BS-6 produced the highest yield of polyglutamic acid rhamnosyl ester, reaching 25.8 g / L. Therefore, the obtained engineered strain BS-6 was used for subsequent process optimization.

[0158] 2) Fermentation and Induced Expression

[0159] 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 was then transferred to a 5 L fermenter (working volume 3 L) at an 8% inoculation rate. The initial culture medium composition was: 60 g / L sodium glutamate, 30 g / L glucose, 5 g / L yeast extract, and 0.1 mM MnCl2. Control parameters were: temperature 37°C, pH 7.0, dissolved oxygen >30% (stirring speed 300 rpm). Key induction procedures (performed simultaneously at 36 hours of fermentation): Sterile xylose was added to a final concentration of 5 g / L (to activate the xylA promoter, initiating RhlA (acyltransferase) and RhlB (glycosyltransferase) and... pgsA Gene expression was induced; the fermentation temperature was adjusted to 30℃; the stirring rate was reduced to 160 rpm to reduce shear force; and expression was induced for 8 hours.

[0160] 3) Intracellular enzymes catalyze glycoesterification reactions

[0161] After induction, a 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 stirring speed of 160 rpm. Samples were taken every 2 hours during the addition process to monitor substrate consumption.

[0162] 4) Self-assembly of biosynthetic products

[0163] Twelve hours after the addition of feedstock, the viscosity of the fermentation broth increased significantly (>5000 cP), indicating that the self-assembly of polyglutamic acid rhamnosyl ester was complete. The reaction was terminated by centrifugation (8000 rpm, 10 minutes), collection of the supernatant, and HPLC analysis of the product. The results are as follows: Figure 5 As shown, the product has a weight-average molecular weight of 12.82 kDa and a PDI of 1.09. Infrared detection results are as follows... Figure 7 As shown, at 1725 cm -1 The presence of a characteristic absorption peak for the carbonyl group (OC=O) indicates that the product has been successfully esterified.

[0164] Example 5: Preparation of polyglutamic acid fucoidan ester

[0165] This embodiment is basically the same as embodiment 4, except that the flow-added sterile rhamnosaccharide solution is replaced with the flow-added sterile alginate oligosaccharide solution (prepared by enzymatic hydrolysis and purification of alginate extracted from kelp).

[0166] The product was detected by HPLC, and the results are as follows: Figure 6 As shown, the product has a weight-average molecular weight of 12.75 kDa and a PDI of 1.10. Infrared detection results are as follows... Figure 8 As shown, at 1745 cm -1 The presence of a characteristic absorption peak for the carbonyl group (OC=O) indicates that the product has been successfully esterified.

[0167] Example 6 Performance Testing of Polyglutamic Acid Rhamnose Ester Product

[0168] like Figure 10 As shown, polyglutamic acid rhamnosyl ester powder exhibited good solubility at different dilution ratios (20-2000 times), with stable solutions and no observed insoluble matter. This characteristic indicates that, in practical applications, polyglutamic acid rhamnosyl ester can flexibly adapt to and meet the needs of various application scenarios such as aerial spraying, foliar spraying, fertigation, and drip irrigation, demonstrating its broad application potential and practicality.

[0169] like Figure 11 As shown, polyglutamic acid rhamnosyl ester can maintain stable product indicators under both high and low temperature conditions, meeting the requirements for product stability in the production of new fertilizers. Through a large amount of test data, it was found that the loss rate of polyglutamic acid rhamnosyl ester in fertilizer production is stably controlled within 10%.

[0170] like Figure 12 As shown, the solution states of polyrhamnosyl glutamate under acidic and alkaline conditions reveal that it remains stable within a pH range of 1–12, producing clear and transparent solutions without flocculation or precipitation. This characteristic indicates that polyrhamnosyl glutamate can be compounded with fertilizers / pesticides of varying acidity and alkalinity, making it suitable for a wide range of applications.

[0171] Example 7: Water Retention Performance Test of Polyglutamic Acid Fucoidan Ester

[0172] Materials: Soil sample, polyglutamic acid fucoidan ester (2x dilution).

[0173] Methods: 1) Place 30 g of soil in a beaker and add 10 ml of polyglutamic acid fucoidan dilution. Add an equal amount of water to the control group. 2) Let stand until the soil is thoroughly soaked, then invert the beaker onto filter paper and observe the size of the water diffusion circle and the soil condition.

[0174] like Figure 13As shown, in the group with added polyglutamic acid fucoidan, water diffusion was slow, the water halo area was small, and the soil was loose; in the control group with clear water, water diffusion was fast, the water halo area was large, and the soil was hard.

[0175] Example 8: Chelating performance test of polyglutamic acid rhamnosyl ester and polyglutamic acid fucoidan ester for calcium.

[0176] Materials: 7% calcium chloride solution, 7% ammonium sulfate solution, 5% polyglutamic acid rhamnosyl ester aqueous solution, 5% polyglutamic acid fucoidan aqueous solution.

[0177] Methods: 1) Add 50 ml of calcium chloride solution to 3 beakers, and add 0.5 ml of polyglutamic acid rhamnosyl ester aqueous solution and 0.5 ml of polyglutamic acid fucoidan aqueous solution to 2 beakers respectively, and stir well; 2) Add 50 ml of ammonium sulfate solution and let stand for observation; 3) Use calcium solution with added water as a control and observe in the same way.

[0178] like Figure 14 As shown, after 1 minute, a large amount of calcium sulfate precipitate was produced in the water (without added polyglutamic acid rhamnosyl ester and polyglutamic acid fucoidan ester), while the two polyglutamic acid ester groups were clear and stable.

[0179] Example 9: pH buffering effect test of polyglutamic acid rhamnosyl ester

[0180] Materials: 5 g / L potassium sulfate solution (pH 3.6), 60 g / L urea solution (pH 9.2), 5% polyglutamic acid rhamnosyl ester aqueous solution, and water.

[0181] Methods and Results:

[0182] Adjusting the acidic solution: 1) Measure the initial pH value of potassium sulfate solution as 3.6; 2) Add 0.5 ml of polyglutamic acid rhamnosyl ester aqueous solution, stir well, and measure the pH value as 4.61.

[0183] Adjusting the alkaline solution: 1) The initial pH value of the urea solution is 9.2; 2) Add 0.5 ml of polyglutamic acid rhamnosyl ester aqueous solution, stir well, and measure the pH value to be 7.11.

[0184] The buffering effect is shown in Table 6.

[0185] Table 6 Buffering effect test results

[0186]

[0187] As shown in Table 6, the polyglutamic acid rhamnosyl ester provided in this application has a pH buffering effect.

[0188] This application proposes an iterative approach to the traditional chemical catalytic method for preparing polyglutamic acid rhamnosyl esters. The resulting polyglutamic acid ester products exhibit a narrower molecular weight distribution and higher uniformity, achieving a higher quality product update and iteration. This is of great significance for improving the market competitiveness of polyglutamic acid ester products and has important industrial application value.

[0189] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method for preparing polyglutamic acid sugar esters by bio-fermentation, characterized in that, The preparation method includes a step of bio-fermentation using engineered bacteria with glutamic acid or its salt and rhamnosaccharide as substrates. The engineered bacteria are controlled to first express pgsB, pgsC and pgsA to synthesize polyglutamic acid or its salt from glutamic acid or its salt; After controlling the expression of the engineered bacteria, Pseudomonas aeruginosa Acyltransferase RhlA and glycosyltransferase RhlB are used to synthesize the polyglutamic acid glycoester with polyglutamic acid or its salt and rhamnooligosaccharide.

2. The method for preparing polyglutamic acid sugar ester by bio-fermentation according to claim 1, characterized in that, The acyltransferase and glycosyltransferase share a common promoter, and a linker is present between them.

3. A method for preparing polyglutamic acid sugar esters by bio-fermentation, characterized in that, The preparation method includes a step of bio-fermentation using engineered bacteria with polyglutamic acid or its salt and rhamnosaccharide as substrates. The engineered bacteria expressed from Pseudomonas aeruginosa Acyltransferase RhlA and glycosyltransferase RhlB.

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