Method for producing gamma-polyglutamic acid embedded in extracellular polymeric substance of biological membrane through electric fermentation

Abstract

The invention provides a method for producing gamma-polyglutamic acid embedded with a biological membrane extracellular polymeric substance through electric fermentation, and relates to the technical field of fermentation engineering. The invention relates to a method for producing gamma-polyglutamic acid embedded into a biological membrane extracellular polymeric substance through electric fermentation. The method comprises the following steps: activating, culturing and producing a strain bacillus subtilis PB5760; an electric fermentation reaction is carried out, a biological membrane growing on the working electrode is triggered, and the activity of gamma-polyglutamic acid hydrolase is up-regulated; and separating the extracellular polymeric substance to obtain the gamma-polyglutamic acid. In the electric fermentation process, the gamma-polyglutamic acid hydrolase generated by triggering the expression of a biological membrane growing on a working electrode is increased, the gamma-polyglutamic acid hydrolase breaks amido bonds and shortens the chain length of the biopolymer, so that the increase of the gene expression quantity and activity of the gamma-polyglutamic acid hydrolase can lead to the reduction of the molecular weight of the gamma-polyglutamic acid; the purification process is simple, the production cost is low, and the molecular weight of the gamma-polyglutamic acid is accurately regulated and controlled.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, and in particular to a method for producing gamma-polyglutamic acid embedded in extracellular polymers of biological membranes by electric fermentation. Background Art

[0002] γ-polyglutamic acid (γ-PGA) is a water-soluble, anionic, biodegradable, non-immunogenic and environmentally friendly biopolymer. Unlike chemically synthesized α-PGA, γ-PGA is mainly produced by microbial fermentation, and its physicochemical properties depend on many factors, including the composition of the culture medium and the selection of microbial strains. The most important physicochemical properties of γ-PGA are molecular weight and stereochemical structure, because these two factors greatly affect its production cost, quality grade, biological function and application areas. Therefore, it is necessary to produce γ-PGA with a specific molecular weight, among which low molecular weight γ-PGA is used for drug delivery and other biomedical applications.

[0003] Conventional γ-PGA production methods involve the use of Cu 2+ or ethanol precipitation, but this will result in uneven product molecular weight. Culture condition optimization methods have also been used to control the molecular weight of γ-PGA, for example, by Fe 3+ concentration stress or the addition of Mn to the fermentation medium 2+ The concentration is controlled between 0 and 1.2 mM, and γ-PGA with a molecular weight of 400 to 2,000 kDa and a D-isomer content of 10% to 90% can be obtained. Another method of γ-PGA depolymerization that depends on culture conditions involves controlling the transfer rate of oxygen in the culture medium. These methods increase process costs and require the recovery or harmless treatment of toxic metals. More importantly, the molecular weight of γ-PGA cannot be precisely controlled.

[0004] In vitro enzymatic degradation by depolymerase can also be used for the production of different target molecular weights, however, the extraction, purification and subsequent hydrolysis of the depolymerase is very time-consuming and labor-intensive, and further increases the process cost. In addition, endogenous secreted γ-PGA hydrolase can also be used, which has mild reaction conditions, high product specificity, and is environmentally friendly, but the disadvantage of this method is that it has low hydrolysis activity, high purification cost, and is difficult to apply to large-scale production. Summary of the invention

[0005] The problem solved by the invention is how to solve the problem of high production cost of gamma-polyglutamic acid and inability to regulate the molecular weight of gamma-polyglutamic acid.

[0006] In order to solve the above problems, the present invention provides a method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation, comprising the following steps:

[0007] S1: Activated culture production strain Bacillus subtilis PB5760;

[0008] S2: Place the working electrode to conduct an electrofermentation reaction, trigger the biofilm growing on the working electrode, and upregulate the activity of γ-polyglutamate hydrolase;

[0009] S3: Put the working electrode into the extracellular polymer extract to separate the extracellular polymer and obtain γ-polyglutamic acid.

[0010] Optionally, the method further comprises step S4: purifying γ-polyglutamic acid by precipitation and dialysis at 4°C.

[0011] Optionally, the step of activating and culturing the production strain Bacillus subtilis PB5760 includes low-salt culture and growth culture, and the low-salt culture uses a low-salt medium to pre-culture the strain to adapt it to the growth medium.

[0012] Optionally, the growth culture is cultured in a growth medium at 25-35° C. for 48 hours to ensure sufficient biofilm and extracellular polymers are formed.

[0013] The growth medium includes glycerol, (NH4)2SO4, glutamic acid, MnSO4.H2O, FeCl3, CaSO4, MgSO4 and K2HPO4, and the pH value is adjusted to 7.5-8.5.

[0014] Optionally, during the electrofermentation reaction, the voltage is 0.2-0.4 V and the reaction is polarized for 48 hours.

[0015] Optionally, the molecular weight of γ-polyglutamic acid decreases from 13953±139 kDa (OCP) to 1227±315 kDa (the voltage during the electro-fermentation reaction is 0.2 V) or 2370±312 kDa (the voltage during the electro-fermentation reaction is 0.4 V).

[0016] Optionally, the relative expression level of γ-polyglutamate hydrolase in step S2 is 1.12±0.57 (open circuit potential), 2.74±0.55 (the voltage during the electric fermentation reaction is 0.2V) and 1.61±0.57 (the voltage during the electric fermentation reaction is 0.4V).

[0017] Optionally, the working electrode comprises a silver / silver chloride reference electrode, a platinum sheet electrode counter electrode and a stainless steel working electrode connected by a copper wire current collector.

[0018] Optionally, step S3 includes: washing the working electrode to remove unattached or loosely bound bacteria, and then placing the working electrode in an extracellular polymer extract, and completely separating the extracellular polymer attached to the working electrode by vortexing, ultrasonic oscillation and stirring.

[0019] Optionally, step S3 comprises: after separating the extracellular polymers, centrifuging the cells to precipitate the cells, and obtaining γ-polyglutamic acid in the supernatant.

[0020] The beneficial effects of the method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation of the present invention are as follows: activating and culturing the production strain Bacillus subtilis PB5760 to form sufficient biofilm and extracellular polymers, placing a working electrode, and performing an electrofermentation reaction. During the entire electrofermentation process, since the electrode (anode) acts as a stable and inexhaustible terminal electron acceptor, it can maintain a stable extracellular electron transfer rate, and under the influence of the applied electrochemical potential, the biofilm grown on the working electrode is triggered to express and produce γ-polyglutamate hydrolase, and the γ-polyglutamate hydrolase breaks the amide bond and shortens the chain length of the biopolymer. Therefore, the increase in the expression amount and activity of the γ-polyglutamate hydrolase gene can lead to a decrease in the molecular weight of γ-polyglutamate, thereby obtaining low-molecular-weight γ-polyglutamate. The degree of reduction in the molecular weight of γ-polyglutamate changes with the change in the applied potential. Therefore, the required molecular weight can also be obtained by applying different potentials within a specified time period. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a flow chart of a method for producing γ-polyglutamic acid embedded in extracellular polymers of biofilms by electrofermentation according to an embodiment of the present invention. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be interpreted as being limited to the embodiments described herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are only for exemplary purposes and are not intended to limit the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention;

[0024] The term "including" and its variations used in this article are open inclusions, that is, "including but not limited to"; the term "based on" is "based at least in part on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one other embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiments". The relevant definitions of other terms will be given in the following description. It should be noted that the concepts of "first" and "second" mentioned in the present invention are used to distinguish different objects, rather than to describe a specific order or a primary and secondary relationship. In addition, the terms "first" and "second" are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, the meaning of "multiple" is two or more.

[0025] In related technologies, researchers have used advanced genetic tools to genetically modify native or heterologous hosts to overexpress γ-PGA hydrolases, thereby improving the utilization of endogenous γ-PGA hydrolases. For example, by combining different promoters to produce γ-PGA hydrolases (poly-γ-glutamic (γ-PGA) hydrolases, PgdS) with different hydrolytic activities, γ-PGA with a wide range of molecular weights can be obtained; or by overexpressing homologous or heterologous PgdS hydrolases, the product molecular weight is reduced from 1000–1200 kDa to 600–800 kDa, or from 1,500 kDa to 20-30 kDa (Sha et al., 2019). The CRISPRi / CRISPR / Cas system can be used as an effective tool to regulate the expression of PgdS, and has shown good results for the expression of γ-PGA with customized molecular weights. Although some achievements have been made on a laboratory scale, the genetic instability of the engineered host and the loss of PgdS hydrolase activity remain bottlenecks in its application.

[0026] Although molecular biological techniques have been used to produce γ-PGA of a specific molecular weight, mainly by expressing γ-PGA synthases from different microbial sources in chassis strains, these methods do not provide a means for real-time detection of biologically active catalysts. The present invention indirectly reflects the respiration rate of the biocatalyst by measuring the current output, that is, taking the extracellular electron transfer rate as an indicator, thereby providing real-time information related to its metabolic state. In addition, currently applicable γ-PGA production methods also rely on soluble terminal electron acceptors such as oxygen and nitrates during the respiration process. In addition to greatly increasing production costs, these methods greatly reduce production efficiency due to the exhaustion of terminal electron acceptor concentrations. The present invention introduces a non-depleted terminal electron acceptor source into the production system, thereby effectively eliminating the tendency of the biocatalyst to be affected by fluctuations in the terminal electron acceptor concentration, especially in biofilm-based biological processes, because soluble electron acceptors such as oxygen and nitrates cannot reach the deep layers of the biofilm due to diffusion limitations and rapid reaction rates. The method proposed by the present invention can greatly improve production efficiency when the ventilation volume is as low as zero, especially enhance anode respiration.

[0027] In addition, since the biocatalytic process must regenerate cofactors (such as succinate, formate, acetate, lactate and gene) by synthesizing reduction products to stabilize the redox state, the energy generated in the fermentation process is limited. Although these cofactors are regarded as valuable raw materials and products for chemical industry production, the secretion of cofactors into the fermentation medium makes the downstream purification process more complicated. The method proposed in the present invention allows the regulation of redox electron flow by adjusting the potential of the system. More importantly, the separation method required for the production downstream of the present invention is more gentle and the purification step is simpler. In short, the above elements of the present invention are conducive to reducing the overall production cost.

[0028] Therefore, combined with genetic engineering modification technology, the electric fermentation method proposed in the present invention can provide higher stability for the engineered microbial chassis in the precise fermentation process, promote biocatalytic efficiency, and more importantly, achieve control and customization of the molecular weight of γ-PGA.

[0029] In view of the problems existing in the above-mentioned related technologies, this embodiment provides a method for producing γ-polyglutamic acid embedded in extracellular polymers of biofilm by electrofermentation.

[0030] like Figure 1 As shown, an embodiment of the present invention provides a method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers through electrofermentation, comprising the following steps:

[0031] S1: Activated culture production strain Bacillus subtilis PB5760;

[0032] S2: Place the working electrode to conduct an electrofermentation reaction, triggering the biofilm growing on the working electrode to express and produce γ-polyglutamate hydrolase;

[0033] S3: Put the working electrode into the extracellular polymer extract to separate the extracellular polymer and obtain γ-polyglutamic acid.

[0034] In this embodiment, the production strain Bacillus subtilis PB5760 is activated and cultured, and a working electrode is placed to perform an electrofermentation reaction. During the entire electrofermentation process, since the electrode (anode) acts as a stable and inexhaustible terminal electron acceptor, it can maintain a stable extracellular electron transfer rate. Under the influence of the applied electrochemical potential, the biofilm grown on the working electrode is triggered to express and produce γ-polyglutamate hydrolase. The γ-polyglutamate hydrolase breaks the amide bond and shortens the chain length of the biopolymer. Therefore, the increase in the expression amount and activity of the γ-polyglutamate hydrolase gene can lead to a decrease in the molecular weight of γ-polyglutamic acid, thereby obtaining low-molecular-weight γ-polyglutamic acid, with a simple purification process and low production cost. The degree of reduction in the molecular weight of γ-polyglutamic acid varies with the change in the applied potential. Therefore, the desired molecular weight can be obtained by applying different potentials within a specified time period, thereby achieving precise control of the molecular weight of γ-polyglutamic acid.

[0035] Specifically, in step S2, the laboratory-scale electrofermentation reaction of γ-PGA is carried out in a 100 mL bioelectrochemical reactor, which includes a silver / silver chloride reference electrode, a platinum electrode counter electrode, and a stainless steel working electrode connected to a copper wire current collector. The electrochemical cell is thoroughly purged with nitrogen for 30 minutes, and the corresponding electrode is connected to a multichannel constant potential instrument. In the electrochemical measurement, the working electrode is polarized at 0.2V or 0.4V (vs. silver / silver chloride) for 48 hours, and the current output is recorded.

[0036] Optionally, the method further comprises step S4: purifying γ-polyglutamic acid by precipitation and dialysis at 4°C.

[0037] Optionally, the step of activating and culturing the production strain Bacillus subtilis PB5760 includes low-salt culture and growth culture, and the low-salt culture uses a low-salt medium to pre-culture the strain to adapt it to the growth medium.

[0038] Specifically, low-salt culture: the γ-PGA production strain, Bacillus subtilis PB5760, was frozen in glycerol cryopreservative and stored at -80°C; after being taken out, it was subcultured in a nutrient broth medium supplemented with appropriate antibiotics and incubated overnight at 37°C with continuous shaking; subsequently, the culture was inoculated on a new nutrient agar plate and incubated at 37°C for 24 hours. Before activating the strain for production, a single colony of the strain was inoculated into a 50mL test tube and cultured with 5mL of basic low-salt culture medium at 37°C with shaking overnight. The culture was then centrifuged at 12,100rpm for 10 minutes, resuspended and washed twice with phosphate buffer (pH 7.2), and finally the optical density of the culture OD 600 Adjust to 1 to obtain a seed culture. The low-salt medium consists of a low-concentration fermentation medium, but does not contain isopropyl-β-D-thiogalactoside (IPTG). Therefore, the strain will not produce γ-polyglutamic acid during the pre-culture preparation stage with the low-salt medium. In addition, pre-culture in a low-salt medium allows the biocatalyst to first adapt to important components of the growth medium, such as carbon and nitrogen sources.

[0039] Growth culture: The seed liquid culture was inoculated into the growth medium at a ratio of 1% and cultured at 30° C. for 48 hours to ensure the formation of sufficient biofilm and extracellular polymeric substances (EPS) rich in γ-PGA.

[0040] Optionally, the growth culture is cultured in a growth medium at 25-35° C. for 48 hours to ensure sufficient biofilm and extracellular polymers are formed.

[0041] The growth medium includes glycerol, (NH4)2SO4, glutamic acid, MnSO4.H2O, FeCl3, CaSO4, MgSO4 and K2HPO4, and the pH value is adjusted to 7.5-8.5.

[0042] Specifically, the growth medium formula is: 2.5% glycerol, 3.2 g / L (NH4)2SO4, 25 g / L glutamic acid, 0.15 g / L MnSO4·H2O, 0.08 g / L FeCl3, 0.15 g / L CaSO4, 0.5 g / L MgSO4, 0.16 g / L K2HPO4, the pH value is adjusted to 8, and high-pressure steam sterilization is carried out at 121°C for 20 minutes.

[0043] Optionally, during the electric fermentation reaction, the voltage is 0.2-0.4 V and the reaction is polarized for more than 48 hours.

[0044] In this optional embodiment, the relative quantification of the mRNA transcript of γ-PGA hydrolase (pgdS) in reverse transcription quantitative PCR (RT-qPCR) represents the activity of γ-polyglutamate hydrolase, which increases more than two times at a voltage of 0.2 V (from 1.12±0.57 to 2.74±0.55) and increases slightly at a voltage of 0.4 V (from 1.12±0.57 to 1.61±0.57). It can be seen that the applied electrochemical potential triggers the increase in the expression of γ-polyglutamate hydrolase in the biofilm grown on the working electrode.

[0045] Specifically, the polydispersity index (Mz / Mn) of γ-polyglutamic acid produced by Bacillus subtilis PB5760 decreased from 2.310 to 1.957 after the reaction was polarized for 48 hours with an applied electrochemical potential of 0.4 V. The slight decrease in the polydispersity index can indicate that a more compact γ-polyglutamic acid is produced in the matrix during electrofermentation, i.e., a compact distribution of γ-polyglutamic acid.

[0046] Optionally, the molecular weight of γ-polyglutamic acid decreases from 13953±139 kDa (open circuit potential) to 1227±315 kDa (voltage during the electro-fermentation reaction is 0.2 V) or 2370±312 kDa (voltage during the electro-fermentation reaction is 0.4 V).

[0047] In this optional embodiment, low molecular weight γ-polyglutamic acid of a specific molecular weight is produced, and the molecular weight of γ-polyglutamic acid is precisely regulated. The low molecular weight γ-polyglutamic acid can be used for drug delivery and other biomedical applications.

[0048] Specifically, the molecular weight of γ-polyglutamic acid is 13953±139 kDa (open circuit potential), which means that the molecular weight of γ-polyglutamic acid is 13953±139 kDa when there is an open circuit potential. The open circuit potential (OCP) refers to the potential difference between the electrode and the electrolyte solution when the test electrode is not connected to any external circuit in an electrochemical test. It indicates the charge distribution state between the electrode and the electrolyte solution.

[0049] Optionally, the relative expression level of γ-polyglutamate hydrolase in step S2 is 1.12±0.57 (OCP), 2.74±0.55 (the voltage during the electric fermentation reaction is 0.2V) and 1.61±0.57 (the voltage during the electric fermentation reaction is 0.4V).

[0050] In this optional embodiment, γ-polyglutamate hydrolase breaks the amide bond and shortens the chain length of the biopolymer. Therefore, the increase in the expression level and activity of the γ-polyglutamate hydrolase gene can lead to a decrease in the molecular weight of γ-polyglutamate, thereby obtaining low molecular weight γ-polyglutamate with a molecular weight of 1227±315kDa (the voltage during the electric fermentation reaction is 0.2V) and 2370±312kDa (the voltage during the electric fermentation reaction is 0.4V).

[0051] Optionally, the working electrode comprises a silver / silver chloride reference electrode, a platinum sheet electrode counter electrode and a stainless steel working electrode connected to a copper wire by a copper wire current collector.

[0052] Optionally, step S3 includes: washing away unattached or loosely bound bacteria on the working electrode, and then placing the working electrode in an extracellular polymer extract, and completely separating the extracellular polymer attached to the working electrode by vortexing, ultrasonic oscillation and stirring.

[0053] Specifically, after the electrofermentation was completed, the working electrode was removed and sterile ultrapure water was used to wash away bacteria that were not attached or loosely bound to the electrode. After washing, the working electrode was placed in an EPS extract solution composed of 10 mM Tris, 10 mM EDTA, and 2.5% NaCl with a pH of 8, and after vigorous vortexing for 1 minute, ultrasonication was performed at 40 KHz for 20 minutes, and the leaching solution mixture was stirred at 250 rpm for 4 hours at 35 °C to completely separate EPS from the working electrode.

[0054] Optionally, step S3 comprises: after separating the extracellular polymers, centrifuging the cells to precipitate the extracellular polymers, removing the supernatant containing the extracellular polymers, and precipitating γ-polyglutamic acid from the supernatant.

[0055] Specifically, the cells were precipitated by centrifugation at 12,100 rpm for 10 minutes, and the supernatant containing EPS was sterilized by filtration to obtain natural γ-polyglutamic acid.

[0056] Example 1: A method for producing γ-polyglutamic acid embedded in extracellular polymers of biofilm by electrofermentation.

[0057] 1. Activation culture of inoculum: The γ-PGA production strain, Bacillus subtilis PB5760, was frozen in glycerol cryovial at -80°C, subcultured in nutrient broth medium supplemented with appropriate antibiotics, and incubated at 37°C overnight with continuous shaking. Subsequently, the culture was inoculated on a new nutrient agar plate and incubated at 37°C for 24 hours. Before activating the strain for production, a single colony of the strain was inoculated into a 50mL test tube and cultured with 5mL of basic low-salt medium at 37°C overnight with shaking. The culture was then centrifuged at 12,100 rpm for 10 minutes, resuspended and washed twice with phosphate buffer (pH 7.2), and finally the optical density of the culture OD600 Adjust to 1.

[0058] 2. Production medium formula: 2.5% glycerol, 3.2g / L (NH4)2SO4, 25g / L glutamic acid, 0.15g / L MnSO4.H2O, 0.08g / L FeCl3, 0.15g / L CaSO4, 0.5g / L MgSO4, 0.16g / L K2HPO4, pH adjusted to 8, high pressure steam sterilization at 121°C for 20 minutes. The seed culture prepared in step 1 was inoculated into the production medium at a ratio of 1%, and cultured at 30°C for 48 hours to ensure the formation of sufficient biofilm and EPS rich in γ-PGA.

[0059] 3. Electrofermentation reactor setup: Laboratory-scale γ-PGA was performed in a 100 mL bioelectrochemical reactor. The electrochemical reactor consisted of a silver / silver chloride reference electrode, a platinum electrode counter electrode, and an 11.2 cm copper wire current collector connected to the 2 The stainless steel working electrode was used and the applied electrochemical potential was 0.2-0.4 V. The electrochemical cell was thoroughly purged with nitrogen for 30 min and the counter electrode was connected to a multichannel potentiostat.

[0060] 4. Extraction of EPS: After the electrofermentation is completed, remove the working electrode and wash away the bacteria that are not attached or loosely bound to the electrode with sterile ultrapure water. After washing, place the working electrode in an EPS extract consisting of 10mM Tris, 10mM EDTA, 2.5% NaCl, and pH 8. After vigorous vortexing for 1 minute, ultrasonicate at 40KHz for 20 minutes. The leaching solution mixture is stirred at 250rpm for 4 hours at 35°C to completely separate the EPS, and then the solution is centrifuged at 12,100rpm for 10 minutes to precipitate the cells. After filtering and sterilizing the supernatant containing EPS, γ-PGA is purified by precipitation and dialysis at 4°C.

[0061] Example 2: Method for producing γ-polyglutamic acid embedded in extracellular polymers of biofilm by electrofermentation.

[0062] During the electrofermentation reaction, the electrochemical potential applied was 0.2-0.4 V, and other operations were the same as in Example 1.

[0063] Comparative Example, a method for producing gamma-polyglutamic acid embedded in biofilm extracellular polymers.

[0064] In the electrofermentation reaction, no electrochemical potential was applied, that is, the electrochemical potential was 0, and other operations were the same as in Example 1.

[0065] Effect Example

[0066] In Example 1, Example 2 and the comparative example, the molecular weight of γ-polyglutamate produced by Bacillus subtilis PB5760 and the relative expression level of pgdS were detected by applying different electrochemical potentials for reaction polarization for 48 hours. The results are shown in Table 1. The relative quantification of the mRNA transcript of pgdS in reverse transcription quantitative PCR (RT-qPCR) represents the activity of γ-polyglutamate hydrolase. It can be seen that the activity of γ-polyglutamate hydrolase increased by more than two times at 0.2V (from 1.12±0.57 to 2.74±0.55), and increased slightly at 0.4V (from 1.12±0.57 to 1.61±0.57). The results show that the activity of γ-polyglutamate hydrolase is affected by the applied electrochemical potential. After applying electrochemical potential, the molecular weight of γ-PGA in EPS decreased, more than threefold at 0.2 V (from 3953 ± 139 kDa to 1227 ± 315 kDa), and slightly at 0.4 V (from 3953 ± 139 kDa to 2370 ± 312 kDa). Furthermore, the amount of γ-PGA produced by the biofilm increased due to the decrease in molecular weight.

[0067] Table 1. Molecular weight of γ-PGA and relative expression of pgdS produced by applying different potentials

[0068] Fermentation potential γ-PGA molecular weight (kDa) Relative expression of pgdS 0 3953±139 1.12±0.57 0.2V 1227±315 2.74±0.55 0.4V 2370±312 1.61±0.57

[0069] The polydispersity index (Mz / Mn) of γ-polyglutamic acid produced by Bacillus subtilis PB5760 decreased from 2.310 to 1.957 when the electrochemical potential was applied at 0.4 V and the reaction was polarized for 48 hours. The change of polydispersity index was measured by size exclusion chromatography (SEC). The SEC-MALS system (1260 Infinity II HPLC (Agilent) combined with Dawn series multi-angle light scattering (Multi-angle Light Scattering, MALS) detector (Wyatt)) was equipped with a refractive index detector and an SB-806HQ column. The column temperature was maintained at 35 ° C, 0.9% NaCl was used as the mobile phase, and the injection volume was 100 μL. The slight decrease in polydispersity index can indicate that more dense γ-polyglutamic acid is produced in the matrix during electrofermentation, that is, the compact distribution of γ-polyglutamic acid.

[0070] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation, characterized in that: The following steps are involved: S1: Activated culture production strain Bacillus subtilis PB5760; S2: Place the working electrode to conduct an electrofermentation reaction, trigger the biofilm growing on the working electrode, and upregulate the activity of γ-polyglutamate hydrolase; S3: putting the working electrode into an extracellular polymer extract to separate the extracellular polymer to obtain γ-polyglutamic acid.

2. The method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation according to claim 1, characterized in that: The method further comprises step S4: purifying γ-polyglutamic acid by precipitation and dialysis at 4°C.

3. The method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation according to claim 1, characterized in that: The step of activating and culturing the production strain Bacillus subtilis PB5760 includes low-salt culture and growth culture. The low-salt culture uses a low-salt medium to pre-culture the strain so that it adapts to the growth medium.

4. The method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation according to claim 3, characterized in that: The growth culture is carried out using a growth medium and cultured at 25-35° C. for 48 hours to ensure the formation of sufficient biofilm and extracellular polymers. The growth medium includes glycerol, (NH4)2SO4, glutamic acid, MnSO4.H2O, FeCl3, CaSO4, MgSO4 and K2HPO4, and the pH value is adjusted to 7.5-8.

5.

5. The method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation according to claim 1, characterized in that: The voltage during the electric fermentation reaction is 0.2-0.4V and the reaction is polarized for 48 hours.

6. The method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation according to claim 5, characterized in that: The molecular weight of the γ-polyglutamic acid decreases from 13953±139 kDa (open circuit potential) to 1227±315 kDa (the voltage during the electrofermentation reaction is 0.2 V) or 2370±312 kDa (the voltage during the electrofermentation reaction is 0.4 V).

7. The method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation according to claim 5, characterized in that: The relative expression levels of the γ-polyglutamate hydrolase in step S2 are 1.12±0.57 (OCP), 2.74±0.55 (the voltage during the electric fermentation reaction is 0.2V) and 1.61±0.57 (the voltage during the electric fermentation reaction is 0.4V).

8. The method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation according to claim 1, characterized in that: The working electrode comprises a silver / silver chloride reference electrode, a platinum sheet electrode counter electrode and a stainless steel working electrode connected by a copper wire current collector.

9. The method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation according to claim 1, characterized in that: The step S3 comprises: washing the working electrode to remove unattached or loosely bound bacteria, and then placing the working electrode in an extracellular polymer extract, and completely separating the extracellular polymer attached to the working electrode by vortexing, ultrasonic oscillation and stirring.

10. The method for producing γ-polyglutamic acid embedded in biofilm extracellular polymers by electrofermentation according to claim 1, characterized in that: The step S3 comprises: after separating the extracellular polymer, centrifuging and precipitating the cells to obtain γ-polyglutamic acid in the supernatant.

Images