Synthesis and application of polycaprolactone-glutamic acid block polymer

Abstract

The invention relates to synthesis and application of a polycaprolactone-glutamic acid block polymer, and provides polypeptide vesicles synthesized by initiating BLG polymerization by adopting PCL-NH2 through a self-designed catalytic system. Experiments prove that the constructed vesicles have dual functions, can significantly promote cell proliferation in the fermentation process of the cassava clear liquid, and can synchronously increase the sugar consumption rate at the same time. The system breaks through the traditional limitation in the field of biomedicine and biofuel application, the example of active regulation and control of microbial biosynthesis by polypeptide vesicles is shown for the first time, and a sustainable research platform is established for polypeptide nanoparticles and a biological refining technology.

Description

Technical Field

[0001] This invention relates to the field of yeast fermentation technology and its applications, and in particular to the synthesis and development of a polypeptide polymer that promotes the proliferation of yeast cells during fermentation. Background Technology

[0002] Saccharomyces cerevisiae, as one of the world's most critical industrial microorganisms, is not only the leading producer of ethanol in the alcoholic beverage and biofuel sectors, but also supports more than 80% of the world's renewable fuel supply. Its highly efficient fermentation capabilities form the foundation of the beer, wine, distilled spirits, and bioethanol industries, becoming an indispensable core element in the economic and energy strategies of many countries.

[0003] During fermentation, yeast cells typically require nitrogen and minerals such as magnesium / zinc as essential nutrients to maintain growth and metabolic activity. However, continuous fermentation often leads to nutrient depletion, triggering metabolic starvation, which in turn causes fermentation to stagnate or decrease in efficiency. Traditional optimization strategies usually rely on supplementing nitrogen sources (such as urea) or exogenous magnesium / zinc ions to enhance enzyme activity. However, while these methods improve fermentation performance, they have significant drawbacks. This is not only due to the increased risk of cytotoxicity from the use of additives with poor biocompatibility (especially in food-grade applications), but also because the need for continuous exogenous feeding significantly increases the process complexity and cost burden of industrial-scale production.

[0004] In contrast, introducing biocompatible additives at the initial stage of fermentation to activate cell viability by regulating organelle function is considered a more efficient optimization pathway, which can improve the overall fermentation efficiency. Existing research has found that certain mitochondrial-targeting peptides (MTPs) can enhance mitochondrial activity and thus regulate cellular life processes; however, these peptides require complex synthesis and purification steps due to their special structural motifs, which severely restricts their industrial application. Summary of the Invention

[0005] This invention relates to a polypeptide macromolecule that simultaneously promotes yeast cell proliferation and improves fermentation efficiency. The core of this invention lies in the convenient and efficient synthesis of polycaprolactone-glutamic acid block copolymers using a self-designed NCA polymerization catalyst. It was discovered that this polypeptide macromolecule, as a biocompatible additive, effectively promotes cell proliferation, thereby significantly improving yeast fermentation efficiency. The key to this invention is the use of a self-designed catalyst to initiate the NCA polymerization of polycaprolactone-NH2. This method overcomes the drawbacks of traditional polymerization methods, which require stringent polymerization conditions, enabling rapid polymerization in the presence of water and oxygen. The polymer formed, after deprotection at the ends, forms polypeptide vesicles. These vesicles exhibit excellent biocompatibility and can directly target mitochondrial organelles within the cell.

[0006] A method for synthesizing an NCA polymerization catalyst includes the following steps: Step 1: Synthesis of SiO2 nanospheres; Preparation of solution A: Mixing ammonia, ethanol and deionized water evenly; Preparation of solution B: Mixing triethoxysilane with ethanol; While vigorously stirring solution A, solution B is rapidly poured into solution A all at once. The mixture is stirred at room temperature, and the solid product is collected by centrifugation. After washing, it is centrifuged again, washed three times with ethanol, and finally dried in a vacuum oven to obtain SiO2 nanospheres. Step 2: Amination (SiO2-NH2) modification of SiO2 nanospheres; The obtained SiO2 nanospheres are dispersed in toluene solution, and then APTES is added. The mixture is heated to react. After the reaction, the solid product is collected by centrifugation, washed three times with ethanol, and finally dried in a vacuum oven. Step 3: Polymerization catalyst (SiO2@PBLG) initiated by SiO2-NH2; The synthesized SiO2-NH2 is dispersed in dichloromethane, and BLG monomer is added. SiO2-NH2 directly and rapidly initiates NCA polymerization to generate a silica polypeptide complex. This complex is dried under vacuum conditions. The dried solid particles are ground, and then acetic anhydride is added to remove excess surface amino groups. The resulting solid is washed with methanol and finally dried in a vacuum drying oven to obtain the silica polypeptide catalyst.

[0007] Optionally, the synthesized SiO2-NH2 is dispersed in dichloromethane and BLG monomer is added at a mass ratio of 1:1.

[0008] An NCA polymerization catalyst (SiO2@PBLG) synthesized using the aforementioned synthesis method.

[0009] A method for synthesizing a polycaprolactone-glutamic acid block polymer additive using the aforementioned NCA polymerization catalyst, characterized by comprising the following steps: Step 1: BLG-NCA was dissolved in dichloromethane. The SiO2@PBLG catalyst was pre-dispersed in dichloromethane by ultrasonic treatment and then added to the dichloromethane mixture and stirred. Then, the PCL-NH2 initiator was rapidly added to the reaction solution and stirred at room temperature. The conversion rate of NCA was monitored by Fourier transform infrared spectroscopy (FT-IR). After the reaction was completed, the reaction mixture was centrifuged, and the supernatant was added to methanol to precipitate the polypeptide product (PCL@PBLG). Step 2: The obtained PCL@PBLG product was added to an HBr-CH3COOH solution and stirred overnight at room temperature. Then, it was concentrated under vacuum. The crude product was then dried with an oil pump to remove excess acid. Finally, the residue was added to a DMF / H2O mixture at a ratio of 1:3 (volume ratio) and purified by dialysis in 30% NaHCO3 solution and pure water. The deprotected product was obtained by freeze-drying the dialysate.

[0010] A method for promoting vesicle proliferation using the aforementioned additive. Vesicle proliferation is promoted by intracellular targeting of mitochondrial organelles and reducing mitochondrial ROS levels.

[0011] Furthermore, this invention tested that these polypeptide vesicles can effectively promote cell proliferation, including yeast cells with fermentation and brewing functions.

[0012] This invention designs a readily synthesizable polypeptide vesicle that can promote yeast cell proliferation, thereby increasing the rate of raw material consumption during fermentation. This provides a new approach and solution for reducing industrial costs and addressing the bottleneck problems of low cell viability and low raw material consumption in yeast fermentation.

[0013] To address this challenge, this study innovatively employs a method developed by the research group. N The large-scale polymerization technology of carboxylic acid anhydrides (NCA) enables the rapid synthesis of polypeptide additives for fermentation. Specifically, this invention constructs an NCA polymerization catalytic system initiated by PCL-NH2, which can rapidly prepare polypeptide block compounds at room temperature. After further deprotection of the ends, polypeptide vesicles are formed in aqueous solution. These vesicles target mitochondria in cells, significantly promoting microbial proliferation and increasing ethanol yield in cassava broth fermentation. This strategy of integrating peptide nanomaterials with microbial systems successfully avoids the traditional contradiction between cell viability and metabolic output, and is the first example of polypeptide vesicles actively driving biosynthetic efficiency. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the overall process of the present invention; Figure 2 Schematic diagram of the design and synthesis of polymerization catalysts; Figure 3 Synthesis steps and characterization of polypeptide block polymers; Figure 4 Tests on the cell proliferation-promoting effects of polymer vesicles; Figure 5 Polymer vesicles target mitochondria and have the ability to clear ROS within mitochondria; Figure 6Tests on yeast cell concentration and sugar consumption rate during fermentation. Detailed Implementation

[0015] The technical solution of the present invention will be specifically described below through specific embodiments and in conjunction with the accompanying drawings. Unless otherwise specified, the components or devices in the following embodiments are all general standard parts or components known to those skilled in the art, and their structures and principles can be learned by those skilled in the art through technical manuals or conventional experimental methods.

[0016] Saccharomyces cerevisiae is one of the world's most important industrial microorganisms, supporting over 80% of renewable fuel production and serving as the core of the alcoholic beverage and bioethanol industries. However, continuous fermentation leads to nutrient depletion. While traditional methods of supplementing nitrogen or minerals can improve fermentation efficiency, they present challenges such as additive toxicity risks and complex processes. Research has found that activating organelle functions through biocompatible additives is more efficient, but existing mitochondrial-targeting peptide synthesis steps are complex and difficult to industrialize. Therefore, this study developed a polypeptide vesicle additive using rapid NCA polymerization technology. This material can target mitochondria, promoting yeast proliferation while simultaneously increasing ethanol production, successfully resolving the contradiction between cell viability and metabolic efficiency, and providing a new paradigm for biosynthetic optimization.

[0017] See Figure 1 The first objective of this invention is to synthesize a catalyst that can rapidly catalyze NCA polymerization in the presence of water and oxygen. This catalyst has a SiO2 sphere core on which a dense PBLG polypeptide is modified using NCA polymerization. The polypeptide α array anchored on the particle surface provides a self-accelerating environment for the polymerization reaction, thereby achieving the goal of catalyzing the NCA polymerization reaction.

[0018] The second objective of this invention is to synthesize a polycaprolactone-glutamic acid block polymer additive by using this catalytic polymerization method, with aminated polycaprolactone as an initiator, under the catalysis of an NCA polymerization catalyst.

[0019]

[0020] A third objective of this invention is to provide the aforementioned polycaprolactone-glutamic acid block polymer as a biocompatible additive in the fermentation process, promoting yeast cell proliferation and thereby improving fermentation efficiency.

[0021] The beneficial effects of this invention are as follows: The polypeptide molecule of this invention has a good effect on increasing ethanol yield and fermentation rate. Cell proliferation kinetics monitoring shows that this additive has a good cell proliferation-promoting effect; organelle co-localization experiments demonstrate the additive's ability to target mitochondria; monitoring of cell concentration and sugar consumption during yeast cell fermentation demonstrates that the additive can effectively promote the raw material consumption rate of yeast fermentation by promoting yeast cell proliferation. This polymer additive has advantages such as convenient synthesis, good biocompatibility, and ease of production, and has the potential to be used as a highly efficient and industrially applicable promoter of yeast fermentation.

[0022] Example 1: Synthesis of NCA polymerization catalyst The synthesis process of NCA polymerization catalyst is as follows: Figure 2 As shown. The specific synthesis steps are as follows: Step 1: Synthesis of SiO2 spheres Preparation of solution A: First, mix 9 mL of ammonia water, 16.25 mL of ethanol and 24.75 mL of deionized water evenly.

[0023] Preparation of solution B: Mix 4.5 mL of triethoxysilane with 45.5 mL of ethanol.

[0024] Reaction steps: Solution A was vigorously stirred at 1400 rpm while solution B was rapidly poured into solution A in one go. The mixture was then stirred at room temperature for 4 hours, yielding a milky white, turbid solution. The solid product was collected by centrifugation, centrifuged after each washing, washed three times with ethanol, and finally dried in a vacuum oven.

[0025] Step 2: Amination (SiO2-NH2) modification of SiO2 spheres The obtained SiO2 nanoparticles were dispersed in a toluene solution, and then APTES (5 eqv.) were added. The mixture was heated to 120 °C and reacted for 48 hours. After the reaction was completed, the solid product was collected by centrifugation, washed three times with ethanol (centrifuged after each wash), and finally dried in a vacuum oven.

[0026] Step 3: SiO2-NH2 initiates polymerization to generate a polymerization catalyst (SiO2@PBLG) The synthesized SiO2-NH2 was dispersed in dichloromethane, and BLG monomer (mass ratio 1:1) was added. SiO2-NH2 directly and rapidly initiated NCA polymerization to generate a silica polypeptide complex. This complex was dried under vacuum at 50°C. The dried solid particles were ground, and then acetic anhydride was added. Excess surface amino groups were reacted off at 80°C. The resulting solid was washed with methanol and finally dried in a vacuum drying oven to obtain the silica polypeptide catalyst.

[0027] Example 2: Synthesis of polycaprolactone-glutamic acid block polymer additive Synthesis process as follows Figure 3 As shown. The specific synthesis steps are as follows: The NCA polymerization reaction was carried out in a fume hood at room temperature. First, BLG-NCA (20 mg, 0.08 mmol) was dissolved in dichloromethane (2.0 mL). The previously mentioned SiO2@PBLG catalyst (20 mg) was pre-dispersed in dichloromethane (1.0 mL) for 20 min by sonication, then added to the dichloromethane mixture and stirred for 5 min. The PCL-NH2 initiator (M / I = 200:1) was rapidly added to the reaction solution and stirred at room temperature for 2 h. The conversion rate of NCA was monitored using Fourier transform infrared spectroscopy (FT-IR). After the reaction was complete, the reaction mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was added to methanol to precipitate the peptide product three times.

[0028] The obtained PCL@PBLG (13 mg) product was added to a 33% HBr-CH3COOH solution (3.0 mL), stirred overnight at room temperature, and then concentrated under vacuum. The crude product was then dried using an oil pump for 30 min to remove excess acid. Finally, the residue was added to a 1:3 (v / v) mixture of DMF / H2O and purified by dialysis in 30% NaHCO3 solution and pure water for 12 h. The deprotected product was obtained by freeze-drying the dialysate.

[0029] Example 3: Formation and characterization of polycaprolactone-glutamic acid polymer vesicles 10 mg of PCL-PGA was added to 3.0 mL of DMF, followed by 10 μL of CF3COOH to dissolve it. Then, 6.0 mL of H2O was added dropwise to the polymer solution over 10 min. After stirring for 2 h, the dialysate was treated with sodium bicarbonate aqueous solution for approximately 12 h, followed by treatment with deionized water for approximately 48 h. The dialysate was changed every 3 h. Finally, the dialysate was lyophilized to obtain polymer vesicles. The vesicles were characterized by GPC, DLS, and TEM. Figure 3 ).

[0030] Example 4: Effect of Polypeptide Additive on Promoting Cell Proliferation refer to Figure 4 Cells were placed in DMEM medium containing 512 μg / mL polypeptide vesicles (with 10% fetal bovine serum added) and cultured for 12 to 72 h. Cells without any added compounds served as a control group. After culture, the medium was removed, cells were washed once with PBS, and cell counts were determined manually under a microscope. Three replicates were set up for each data set. Notably, the vesicle-treated groups showed differences from the blank control group starting at 24 hours and exhibited statistically significant differences at 72 hours (p<0.01). Figure 4 These phenomena indicate that vesicles have cell proliferation-promoting properties, thus laying the foundation for subsequent applications.

[0031] Example 5: Investigation into the mechanism by which polypeptide vesicle additives promote cell proliferation—intracellular targeting of mitochondrial organelles and reduction of mitochondrial ROS levels refer to Figure 5 One × 10⁵ MCF-7 cells were placed in a confocal culture dish and cultured with F+D for 10–12 h. The culture medium was then replaced with 900 μL of DMEM and 100 μL of DMEM containing Cy3-peptide vesicles (0.2 mg / mL Cy3-peptide vesicles). Cells were cultured for another 3 h, followed by two washes with PBS. Then, 1 mL of DMEM solution containing 100 nmol Mito-Tracker (green) was added, and staining was performed for 40 minutes under cell culture conditions. The treated MCF-7 cells were washed twice with PBS and fixed with 4% formaldehyde solution. The cells were then imaged using a confocal microscope. Notably, the Cy3-labeled peptide vesicles showed significant co-localization with mitochondria. Figure 4(Figure a) This was confirmed by fluorescence microscopy. Mitochondria are double-membrane organelles in most eukaryotic cells and are the primary sites of aerobic respiration and energy production, often referred to as the cell's "energy factories." Based on these observations, this invention hypothesizes that the proliferative mechanism of these vesicles may be related to their involvement in mitochondrial energy metabolism or related bioenergetic processes. Inspired by this discovery, this invention further investigates whether these vesicles affect mitochondrial function. Based on reports that certain peptides targeting mitochondria can regulate mitochondrial activity by inhibiting reactive oxygen species (ROS) levels and mitigating oxidative damage, this invention tests the intracellular ROS levels after vesicle treatment. Specifically, this invention places 1×10^5 MCF-7 cells in a 24-well plate, co-cultures them with F+D for 10-12 h, then incubates them with the added polypeptide vesicles (0.5 mg / mL) for 3 h, washes them twice with PBS, and then incubates them with 10 μM DCFH-DA solution (1 mL) at 37°C for 20 min. Cells were collected after washing twice with PBS and resuspended in 0.5 mL PBS. Flow cytometry was used to detect the fluorescence intensity of intracellular DCF to quantify the amount of mitochondrial superoxide. The results showed that, compared with the control group, vesicle treatment significantly reduced intracellular reactive oxygen species (ROS). Figure 4 (Figure b in the figure) confirms the hypothesis of the present invention that vesicles may promote cell proliferation by protecting mitochondria.

[0032] Simultaneously, this invention systematically investigated the mechanism of vesicle internalization. This invention pretreated cells (1.5 × 10^5 MCF-7 cells cultured in 1 mL of medium) with different cell entry inhibitors for 30 minutes, followed by incubation with polypeptide vesicles for 3 hours, and then detected the cell entry effect. This invention found that vesicle uptake was significantly reduced only at 4°C (Figure 4c), a condition known to decrease membrane fluidity. Therefore, this invention hypothesizes that the cell entry mechanism is related to passive uptake due to membrane fluidity. Although the specific mechanistic details still need to be fully elucidated, these combined findings suggest that Cy3-polypeptide vesicles can bypass the lysosomal entry pathway and directly deliver bioactive macromolecules into the cytoplasm, a favorable characteristic for intracellular therapeutic applications.

[0033] Example 6: Polypeptide vesicle additive promotes yeast cell proliferation and fermentation refer to Figure 6Subsequently, this invention evaluated the effects of the polypeptide vesicle additive on cell growth and fermentation efficiency during the fermentation process. Polypeptide vesicle additive at a concentration of 500 mg / g yeast was added to fermentation broths with different initial sugar concentrations (50 g / L, 100 g / L, 220 g / L). Monitoring of cell concentration showed that, regardless of the initial sugar concentration, the polypeptide vesicle group exhibited a certain degree of proliferation-promoting effect. Comprehensive analysis of sugar consumption showed that the sugar consumption rate in all groups initially increased and then decreased during fermentation. The polypeptide-added groups consistently exhibited a higher sugar consumption rate than the control group, especially during the logarithmic growth phase (early stage of fermentation). The average sugar consumption rate in the early stage increased significantly, by 8.3%, 26.1%, and 16.7%, respectively, higher than the control group. This enhancement can be attributed to the higher cell concentration. Furthermore, during fermentation, the carbon source of the polypeptide group was essentially depleted within 50 hours. This rapid carbon source utilization shortens the fermentation cycle, which is beneficial for industrial production because it improves equipment utilization (the production cycle for industrial ethanol fermentation is approximately 70-80 hours) and ethanol yield. In summary, under the same sugar concentration, the average sugar consumption rate of the peptide group was higher than that of the control group, indicating that the peptides accelerate sugar consumption by promoting cell growth, especially in high-sugar fermentation broths, where this effect is more pronounced. These phenomena suggest that this peptide vesicle additive holds promise for application in industrial fermentation to improve substrate utilization, reduce costs, and increase efficiency.

[0034] Many specific details have been set forth in the foregoing description to provide a thorough understanding of the present invention. However, the above description is merely a preferred embodiment of the present invention, and the present invention can be implemented in many other ways different from those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed above. Furthermore, any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, using the methods and techniques disclosed above, without departing from the scope of the present invention. Any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the present invention, shall still fall within the protection scope of the present invention.

Claims

1. A method for synthesizing an NCA polymerization catalyst, characterized in that, Includes the following steps: Step 1: Synthesis of SiO2 nanospheres; Preparation of solution A: Mix ammonia, ethanol and deionized water thoroughly; Preparation of solution B: Triethoxysilane was mixed with ethanol; While vigorously stirring solution A, solution B was quickly poured into solution A all at once. The mixture was stirred at room temperature, and the solid product was collected by centrifugation. After washing, the solid product was centrifuged again, washed three times with ethanol, and finally dried in a vacuum oven to obtain SiO2 nanospheres. Step 2: Amination (SiO2-NH2) modification of SiO2 nanospheres; The obtained SiO2 nanospheres were dispersed in toluene solution, then APTES was added, and the mixture was heated to react. After the reaction was completed, the solid product was collected by centrifugation, washed three times with ethanol, and finally dried in a vacuum oven. Step 3: Generate a polymerization catalyst (SiO2@PBLG) by initiating polymerization with SiO2-NH2. The synthesized SiO2-NH2 was dispersed in dichloromethane, and BLG monomer was added. SiO2-NH2 directly and rapidly initiated the NCA polymerization reaction to generate a silica polypeptide complex. The complex was dried under vacuum conditions, and the dried solid particles were ground. Then, acetic anhydride was added to remove excess surface amino groups. The resulting solid was washed with methanol and finally dried in a vacuum drying oven to obtain a silica polypeptide catalyst.

2. The synthesis method according to claim 1, characterized in that, The synthesized SiO2-NH2 was dispersed in dichloromethane and BLG monomer was added at a mass ratio of 1:

1.

3. An NCA polymerization catalyst (SiO2@PBLG) synthesized using the synthesis method described in any one of claims 1-2.

4. A method for synthesizing a polycaprolactone-glutamic acid block polymer additive using the NCA polymerization catalyst described in claim 3, characterized in that, Includes the following steps: Step 1: BLG-NCA was dissolved in dichloromethane. The SiO2@PBLG catalyst was pre-dispersed in dichloromethane by ultrasonic treatment and then added to the dichloromethane mixture and stirred. Then, the PCL-NH2 initiator was rapidly added to the reaction solution and stirred at room temperature. The conversion rate of NCA was monitored by Fourier transform infrared spectroscopy (FT-IR). After the reaction was completed, the reaction mixture was centrifuged, and the supernatant was added to methanol to precipitate the polypeptide product (PCL@PBLG). Step 2: The obtained PCL@PBLG product was added to an HBr-CH3COOH solution and stirred overnight at room temperature. Then, it was concentrated under vacuum. The crude product was then dried with an oil pump to remove excess acid. Finally, the residue was added to a DMF / H2O mixture at a ratio of 1:3 (volume ratio) and purified by dialysis in 30% NaHCO3 solution and pure water. The deprotected product was obtained by freeze-drying the dialysate.

5. A method for promoting vesicle proliferation using the additive of claim 4.

6. The method according to claim 5, characterized in that, Promotes vesicle proliferation by targeting mitochondrial organelles intracellularly and reducing mitochondrial ROS levels.

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