A novel anti-diabetic factor and its use

Abstract

The application discloses a novel anti-diabetic factor and application thereof, and belongs to the technical field of biological medicine and microbial fermentation. The application first discovers a naturally existing glucose oxidase AnGOD-HHS from a specific and non-genetically modified Aspergillus niger ACCC 30177. The glucose oxidase has high specific activity, high thermal stability, and high activity under low oxygen conditions, effectively ensures the enzymolysis activity of the AnGOD-HHS under a low oxygen environment, reduces the risk of serious hypoglycemia caused by the sharp fluctuation of catalytic efficiency, and improves the safety and reliability of treatment.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and microbial fermentation technology, and more specifically to a novel antidiabetic factor and its application. Background Technology

[0002] Diabetes mellitus, a global chronic metabolic disease, is characterized by persistent hyperglycemia. Current treatment strategies primarily focus on promoting insulin secretion, improving insulin sensitivity, and exogenous insulin supplementation. However, these methods have many limitations, such as the risk of hypoglycemia, weight gain, gastrointestinal side effects, and the need for frequent injections. Therefore, developing novel antidiabetic drugs with novel mechanisms of action and high safety profiles has become a current research hotspot.

[0003] Glucose oxidase (GOD) is considered a promising new strategy for diabetes treatment because it directly catalyzes the conversion of glucose into gluconic acid in the body, thereby reducing blood sugar levels at the source. However, developing natural GOD into a therapeutic drug has long faced three major technological bottlenecks: First, its specific activity is generally low. The specific activity of existing commercially available GODs is usually below 10 U / mg, which means that to achieve an effective blood glucose-lowering concentration, a large amount of enzyme protein needs to be administered, increasing production costs and significantly raising the risk of immunogenicity caused by foreign proteins. Second, its thermal stability is poor. Natural GOD is sensitive to heat; for example, its enzyme activity is reduced by more than half after being treated at 70°C for several minutes. This poor stability severely restricts the drug's formulation process, long-term storage stability, and results in a short half-life in the body, making it impossible to maintain a lasting therapeutic effect. Third, and most critically, its catalytic activity is highly dependent on oxygen. GOD requires oxygen as a substrate, and its catalytic efficiency is strictly limited by the local oxygen partial pressure in the tissue. The oxygen concentration in the human body's internal environment (especially in diabetic lesion areas with microcirculatory disorders) is complex and may be low. This can cause drastic and unpredictable fluctuations in the catalytic rate of natural GOD, which can easily trigger fatal severe hypoglycemic events, posing the most significant safety hazard in its clinical translation.

[0004] To address these issues, current technologies often employ protein engineering techniques to modify the GOD gene in order to obtain mutants with improved performance. However, genetic engineering inevitably involves gene modification, which may introduce unpredictable safety issues and face complex biosafety regulations and ethical controversies. Its development path is lengthy, and its industrialization prospects are uncertain.

[0005] Therefore, how to screen wild-type GOD variants with inherently superior properties from natural microbial resources is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a novel anti-diabetic factor and its application. For the first time, a naturally occurring high-performance GOD variant (AnGOD-HHS) was discovered from a specific and ungenetically modified Aspergillus niger ACCC 30177, which has high specific activity, high thermal stability and high activity under low oxygen conditions.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] The primary objective of this application is to provide a novel antidiabetic factor, wherein the novel antidiabetic factor is glucose oxidase AnGOD-HHS, and the amino acid sequence of glucose oxidase AnGOD-HHS is shown in SEQ ID NO.1.

[0009] As a preferred technical solution, the glucose oxidase AnGOD-HHS is derived from Aspergillus niger ACCC 30177.

[0010] As a preferred technical solution, the glucose oxidase AnGOD-HHS is prepared by the following method:

[0011] (1) Activation culture of Aspergillus niger ACCC 30177;

[0012] (2) The activated Aspergillus niger ACCC 30177 was subjected to seed culture and fermentation culture respectively. After the culture was completed, glucose oxidase AnGOD-HHS was prepared by ultrafiltration concentration, precipitation and chromatography.

[0013] Another object of this application is to provide a pharmaceutical composition comprising the glucose oxidase AnGOD-HHS.

[0014] Another object of this application is to provide the use of the novel antidiabetic factor or the pharmaceutical composition thereof in the preparation of a drug for the prevention and / or treatment of diabetes.

[0015] Another object of this application is to provide: a DNA molecule that encodes the glucose oxidase AnGOD-HHS.

[0016] Another object of this application is to provide a recombinant expression vector comprising the aforementioned DNA molecule.

[0017] Another object of this application is to provide a host cell that contains the recombinant expression vector or has the DNA molecule integrated into its genome.

[0018] Another object of this application is to provide: the application of the said DNA molecule, the said recombinant expression vector, or the said host cell, wherein the application is in any of the following directions:

[0019] 1) Application in the artificial synthesis of glucose oxidase AnGOD-HHS;

[0020] 2) Use in the preparation of drugs for the prevention and / or treatment of diabetes.

[0021] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Original innovation: For the first time, a naturally occurring high-performance GOD variant (AnGOD-HHS) was discovered from a specific and unmodified Aspergillus niger ACCC 30177. Its superior characteristics are derived from the metabolic characteristics of the strain itself, avoiding the safety and ethical controversies brought about by gene modification.

[0023] (2) Superior performance: AnGOD-HHS simultaneously solves the three core defects currently faced by GOD as a diabetes treatment drug:

[0024] High specific activity means that less enzyme is needed to achieve the same blood sugar lowering effect, reducing treatment costs and potential immune burden.

[0025] High thermal stability: This greatly improves the storage stability and formulation feasibility of the drug, and is expected to extend its half-life in vivo.

[0026] Low oxygen and high activity: This characteristic is crucial, ensuring that AnGOD-HHS can maintain an effective and stable controllable glucose consumption rate in the complex oxygen environment of the human body (including the hypoxic environment of microcirculatory disorders and inflammatory sites), which greatly reduces the risk of severe hypoglycemia caused by drastic fluctuations in catalytic efficiency and improves the safety and reliability of treatment.

[0027] (3) Mature technology: The present invention adopts conventional microbial fermentation and protein purification technology, the process route is mature and easy to scale up to achieve industrial production. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] The Aspergillus niger ACCC 30177 used in the embodiments of this application was purchased from the China Agricultural Microbial Culture Collection Center.

[0030] The Sigma-G7141 glucose oxidase used in the embodiments of this application was purchased from Shanghai Beinuo Biotechnology Co., Ltd. (Sigma-G7141 glucose oxidase (Type XS, 100,000-250,000 units / G) 50KU original).

[0031] Example 1: Preparation, extraction and purification of AnGOD-HHS

[0032] (1) Activation of strain: Take Aspergillus niger ACCC 30177, inoculate it on PDA slant medium, and incubate at 28℃ for 5 days to activate it;

[0033] (2) Seed culture: The spores of Aspergillus niger ACCC30177 in PDA medium were washed off with sterile water and inoculated into seed culture medium. The culture was carried out at 30°C and 200 rpm for 24 h.

[0034] The components and composition of the seed culture medium are as follows:

[0035] 2% glucose;

[0036] 1% peptone;

[0037] Yeast extract 0.5%;

[0038] KH2PO4 0.1%

[0039] MgSO4·7H2O 0.05%

[0040] pH 6.0 (adjusted with 0.1 M HCl or NaOH).

[0041] (3) Fermentation culture: The seed liquid from step (2) was transferred into the fermentation medium at an inoculation rate of 8%, and fermented at 30°C, aeration rate of 1.0 vvm and stirring speed of 300 rpm until the mid-log growth stage. An inducing agent composition was added (to avoid inhibiting growth in the early stage) and cultured for 84 hours.

[0042] The components and composition of the fermentation medium are as follows:

[0043] 4% hydrolyzed sugar from wheat bran;

[0044] Soybean meal powder 2%;

[0045] (NH4)2SO4 0.5%;

[0046] KH2PO4 0.1%;

[0047] MgSO4·7H2O 0.05%;

[0048] CaCl2 0.01%;

[0049] Inducing composition: 1% lactose, 0.2% yeast extract, 0.005% MnSO4·H2O;

[0050] pH 6.0 (adjusted with 0.1 M HCl or NaOH).

[0051] (4) Purification of glucose oxidase AnGOD-HHS

[0052] 1) Preparation of crude enzyme solution: The culture medium after fermentation is centrifuged at 4℃ and 10000g for 30min using a high-speed refrigerated centrifuge, and the supernatant is collected as crude enzyme solution.

[0053] 2) Ultrafiltration concentration and buffer exchange: The crude enzyme solution obtained above is concentrated using an ultrafiltration membrane or ultrafiltration system with a molecular weight cutoff of 10 kDa at 4°C and 0.5-1.0 bar nitrogen pressure. During the concentration process, pre-cooled 20 mM sodium phosphate buffer (pH 6.5) is continuously added for buffer exchange (i.e., dialysis filtration to replace the buffer system). The solution is concentrated to 1 / 10-1 / 20 of its original volume and then set aside.

[0054] 3) Ammonium sulfate fractionation: Under continuous magnetic stirring and ice-water bath conditions, slowly and evenly add finely ground solid ammonium sulfate to the concentrated enzyme solution until it reaches 70% saturation; after the addition is complete, continue to stir gently at 4℃ for 6-8 hours or overnight to allow the protein to precipitate fully; centrifuge at 4℃ and 15000g for 40 minutes and collect the precipitate; redissolve the precipitate with the minimum volume of 20mM sodium phosphate buffer (pH 6.5) for later use;

[0055] 4) Ion exchange chromatography (DEAE-Sepharose)

[0056] Column equilibration: The redissolved protein sample was dialyzed thoroughly at 4°C with the same buffer used in the previous step to completely remove ammonium sulfate; then, the dialyzed sample was loaded onto a DEAE-Sepharose anion exchange column that had been fully equilibrated with the same buffer (20 mM sodium phosphate, pH 6.5).

[0057] Elution: After rinsing with equilibration buffer until the baseline stabilizes, elute with a continuously increasing linear gradient of NaCl (0→0.5M) for a total elution volume of approximately 5-10 column volumes (CV).

[0058] Collection and Detection: Collect the eluent tube by tube using an automated fraction collector. Take a small amount from each tube and analyze it using enzyme activity assays (e.g., o-anisidine method) and protein concentration assays (e.g., Bradford method) to plot the elution curve; combine the elution peaks with the highest enzyme activity for further purification.

[0059] 5) Gel filtration chromatography (Sephadex G-100)

[0060] Sample preparation: The active components from the ion exchange combined are concentrated to 1-5% of the column volume using ultrafiltration;

[0061] Sample loading and elution: Carefully and slowly load the concentrated sample onto a Sephadex G-100 gel column equilibrated with 20 mM sodium phosphate buffer (containing 0.15 M NaCl, pH 6.5); elute using constant pressure or constant flow with 20 mM sodium phosphate buffer (containing 0.15 M NaCl, pH 6.5);

[0062] Collection and detection: Collect the eluent in tubes and measure the enzyme activity and protein concentration in each tube. Combine the eluent fractions with enzyme activity at the main peak.

[0063] 6) Purity verification and preservation

[0064] Steps: Take the final merged purified sample and perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to verify the purity of the final product. After Coomassie brilliant blue staining, a single, clear band appears only at the position of molecular weight of about 73 kDa (monomer) (its apparent molecular weight is consistent with the theoretical molecular weight calculated based on the amino acid sequence), which proves that electrophoretically pure AnGOD-HHS has been obtained. Aliquot the purified enzyme solution, add 50% glycerol, and freeze at -20℃, or freeze-dry to make a dry powder for storage.

[0065] The amino acid sequence of glucose oxidase AnGOD-HHS was analyzed, and its amino acid sequence is as follows:

[0066] , SEQ ID NO.1.

[0067] Example 2, Determination of the enzymatic properties of AnGOD-HHS

[0068] Oxygen dependence determination

[0069] Enzyme activity was determined using the o-anisidine-coupled peroxidase method, with one enzyme activity unit (U) defined as the amount of enzyme required to catalyze the oxidation of 1 μmol of glucose per minute. The specific activity of purified AnGOD-HHS was determined to be 27.5 U / mg.

[0070] Thermal stability determination

[0071] AnGOD-HHS and commercially available GOD (sigma-G7141 glucose oxidase) were placed simultaneously in a 70℃ water bath, and residual enzyme activity was measured at different time points. The specific procedure is as follows:

[0072] (1) Dilute AnGOD-HHS and commercial GOD (sigma-G7141 glucose oxidase) with PBS (pH 7.4) to the same enzyme activity concentration of 1 U / mL;

[0073] (2) Heat treatment: Take an appropriate amount of diluted enzyme solution into a centrifuge tube and heat it in a 70°C constant temperature water bath;

[0074] (3) Sampling: Take a certain amount of sample at time points of 0, 5, 10, 15, 20, 25 and 30 minutes respectively, and immediately place it in an ice water bath to cool rapidly for 5 minutes to terminate thermal denaturation;

[0075] (4) Residual enzyme activity assay: The residual enzyme activity of the cooled samples at each time point was determined according to the above-mentioned "specific activity assay" method;

[0076] The enzyme activity of the unheated (0 min) sample is taken as 100%.

[0077] Residual enzyme activity (%) = (Enzyme activity of the heat-treated sample / Enzyme activity of the unheated sample) × 100%. Calculate the half-life (t½, the time required for enzyme activity to decrease to 50%).

[0078] Table 1 Thermal stability determination (heat treatment at 70℃)

[0079] Heat treatment time (minutes) AnGOD-HHS residual enzyme activity (%) Commercial GOD residual enzyme activity (%) 0 100.0 ± 2.5 100.0 ± 3.1 5 98.5 ± 2.1 65.4 ± 4.3 10 96.2 ± 1.8 45.2 ± 3.8 15 92.8 ± 2.0 32.1 ± 3.0 20 88.4 ± 1.7 24.6 ± 2.5 25 85.1 ± 1.9 20.8 ± 2.2 30 82.3 ± 1.5 18.5 ± 1.9 Half-life (t½) > 120 minutes ~8.5 minutes

[0080] Oxygen dependence determination

[0081] Results analysis: AnGOD-HHS has extremely excellent thermal stability, retaining 82.3% of its activity after treatment at 70℃ for 30 minutes, and its half-life is far more than 120 minutes; while commercial GOD is rapidly deactivated under the same conditions, with a half-life of only about 8.5 minutes.

[0082] The enzyme activity under different DO levels was measured by precisely controlling the dissolved oxygen (DO) concentration in the reaction system using an oxygen electrode, as detailed below:

[0083] (1) System calibration: The dissolved oxygen electrode (Clark type oxygen electrode) with a constant temperature device was calibrated at two points using oxygen-free water (with a small amount of sodium dithionite added) and air-saturated water (stirred vigorously to balance with air);

[0084] (2) Set initial DO: Add reaction mixture (without enzyme) to the reaction chamber, and precisely adjust and maintain the dissolved oxygen (DO) concentration at 8.0 mg / L by introducing a mixture of nitrogen (N2) and air (or oxygen O2) into the solution;

[0085] (3) Measurement of reaction rate: When the enzyme solution is added to start the reaction, the dissolved oxygen electrode will record the rate of decrease of the dissolved oxygen concentration in the solution in real time (-d[O2] / dt). This rate represents the enzyme reaction rate at this DO concentration.

[0086] (4) Change DO concentration: Repeat steps (2) and (3) to determine the reaction rate at different DO concentrations (6.0, 4.0, 2.0, 1.0 mg / L). Perform the same procedure on commercially available GOD (sigma-G7141 glucose oxidase).

[0087] The reaction rate was 100% (Vmax) at DO = 8.0 mg / L.

[0088] Relative enzyme activity (%) = (reaction rate at a certain DO concentration / reaction rate at DO = 8.0 mg / L) × 100%.

[0089] Table 2 Oxygen Dependence Determination

[0090] Dissolved oxygen (DO) concentration AnGOD-HHS relative enzyme activity (%) Commercial GOD relative enzyme activity (%) 8.0 100.0 ± 2.8 100.0 ± 3.3 6.0 95.2 ± 2.5 82.1 ± 3.0 4.0 88.6 ± 2.0 63.5 ± 2.8 2.0 72.1 ± 1.8 41.1 ± 2.5 1.0 50.3 ± 2.2 22.4 ± 1.9

[0091] Results Analysis: AnGOD-HHS exhibited significantly better adaptability to hypoxic environments than commercially available GOD. Under hypoxic conditions (DO = 2.0 mg / L), AnGOD-HHS maintained 72.1% of its relative enzyme activity, while commercially available GOD only retained 41.1%, indicating that AnGOD-HHS can function more stably and efficiently in the complex oxygen environment in vivo.

[0092] Example 3: Verification of the in vivo blood glucose lowering effect of AnGOD-HHS

[0093] (1) Construction of STZ-induced diabetic SD rat model

[0094] Healthy male Sprague-Dawley (SD) rats, weighing 180-220g, were selected and acclimatized for one week. Before modeling, rats were fasted for 12 hours and then intraperitoneally injected with freshly prepared streptozotocin (STZ) at a dose of 55-65 mg / kg (prepared with pre-cooled 0.1M citrate buffer, pH 4.5). 72 hours after STZ injection, fasting blood glucose (FBG) was measured via tail vein sampling. Rats with FBG ≥16.7 mmol / L were selected as successful diabetic models for subsequent experiments.

[0095] (2) Verification of hypoglycemic effect in vivo

[0096] First, the specific activity of the Sigma-G7141 glucose oxidase used was determined according to the method in Example 2, and its specific activity was found to be 15.2 U / mg. The specific activity of the AnGOD-HHS described in this invention is 27.5 U / mg.

[0097] Grouping: The successfully modeled diabetic rats were randomly divided into 4 groups (n=8-10), as follows:

[0098] Model control group: Intraperitoneal injection of an equal volume of physiological saline or enzyme buffer;

[0099] AnGOD-HHS low-dose group: intraperitoneal injection of 2.5 mg / kg body weight of AnGOD-HHS (prepared with normal saline or PBS, equivalent to 68.8 U / kg).

[0100] AnGOD-HHS high-dose group: intraperitoneal injection of 5.0 mg / kg body weight of AnGOD-HHS (prepared with normal saline or PBS, equivalent to 137.5 U / kg).

[0101] Commercial GOD control group: Intraperitoneal injection of 5.0 mg / kg body weight of commercial GOD (sigma-G7141 glucose oxidase) (prepared with physiological saline, equivalent to 76.0 U / kg).

[0102] All animals were fasted but allowed free access to water for 2 hours on the experimental day before administration of the drug, with a typical dosage of 5-10 mL / kg body weight. Blood was collected from the tail tip of rats before administration (0 h) and at 1, 2, 4, 6, and 8 hours after administration, and blood glucose levels were immediately measured using a portable glucometer. The blood glucose levels (mmol / L) of each group of rats at each time point were recorded, and the mean blood glucose level (Mean) and standard error (SEM) for each group at each time point were calculated. The area under the blood glucose curve (AUC) was also calculated. 0-8h The overall blood sugar lowering effect was comprehensively evaluated using an AUC of 100%. A smaller AUC indicates a better blood sugar lowering effect. Appropriate statistical methods (such as two-way repeated measures ANOVA) were used to compare differences between groups, with *p < 0.05 considered statistically significant.

[0103] Table 3. In vivo glucose-lowering effect (blood glucose concentration, mmol / L, Mean±SEM)

[0104] Time (hours) Model control group (saline) Commercially available GOD group (5 mg / kg, 76.0 U / kg) AnGOD-HHS low-dose group (2.5 mg / kg, 68.8 U / kg) AnGOD-HHS high-dose group (5 mg / kg, 137.5 U / kg) 0 21.5 ± 1.2 22.1 ± 1.0 21.8 ± 0.9 22.3 ± 1.1 1 22.0 ± 1.3 15.4 ± 1.1* 16.8 ± 0.8* 14.2 ± 0.7*# 2 22.8 ± 1.1 13.2 ± 0.9* 14.1 ± 0.7* 11.5 ± 0.6*# 4 23.1 ± 1.4 18.9 ± 1.2* 15.3± 0.8*# 12.0 ± 0.5*# 6 22.5 ± 1.0 20.8 ± 1.3 16.9± 0.9*# 13.8 ± 0.7*# 8 22.0 ± 1.2 21.5 ± 1.1 18.5± 1.0*# 15.9 ± 0.8*# <![CDATA[AUC 0-8 ]]> 175.6 146.2* 138.5*# 115.8*#

[0105] Note: * indicates p < 0.05 compared to the model control group; # indicates p < 0.05 compared to the commercial GOD group; AUC (area under the curve) is calculated in mmol·h / L.

[0106] Results analysis:

[0107] Overall efficacy advantages: Under the premise of the same amount of protein administered, the hypoglycemic effect of both the high- and low-dose AnGOD-HHS groups was significantly better than that of the commercial GOD control group. It is worth noting that the enzyme activity of the low-dose AnGOD-HHS group (2.5 mg / kg, 68.8 U / kg) was even slightly lower than that of the commercial GOD group (5.0 mg / kg, 76.0 U / kg), but its hypoglycemic effect (AUC value) was significantly better (P<0.05). This strongly demonstrates the excellent in vivo stability and catalytic efficiency of AnGOD-HHS, rather than simply relying on a higher enzyme dosage.

[0108] Exceptional durability: The commercially available GOD group showed a sharp decline in glucose-lowering effect 4 hours after administration, with blood glucose levels rising back to near the model group level. In contrast, the AnGOD-HHS group maintained a stable and significant glucose-lowering effect throughout the entire 8-hour experimental period, further demonstrating its excellent in vivo stability, which is corroborated by the results of in vitro thermal stability experiments.

[0109] Clear dose-response: The high-dose group of AnGOD-HHS (5 mg / kg) showed better hypoglycemic effect than its low-dose group (2.5 mg / kg), exhibiting good dose dependence.

[0110] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0111] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A novel anti-diabetic factor, characterized in that, The novel antidiabetic factor is glucose oxidase AnGOD-HHS, and the amino acid sequence of glucose oxidase AnGOD-HHS is shown in SEQ ID NO.

1.

2. The novel anti-diabetic factor according to claim 1, characterized in that, The glucose oxidase AnGOD-HHS was derived from Aspergillus niger ACCC 30177.

3. A pharmaceutical composition, characterized in that, Includes the glucose oxidase AnGOD-HHS as described in any one of claims 1-2.

4. The use of the novel antidiabetic factor according to any one of claims 1-2 or the pharmaceutical composition according to claim 3 in the preparation of a drug for treating diabetes.

5. A DNA molecule, characterized in that, The DNA molecule encodes the glucose oxidase AnGOD-HHS as described in any one of claims 1-2.

6. A recombinant expression vector, characterized in that, Includes the DNA molecule as described in claim 5.

7. A host cell, characterized in that, The host cell contains the recombinant expression vector of claim 6, or its genome is integrated with the DNA molecule of claim 5.

8. The application of the DNA molecule of claim 5, the recombinant expression vector of claim 6, or the host cell of claim 7, characterized in that, The application is in any of the following directions: 1) Application in the artificial synthesis of glucose oxidase AnGOD-HHS; 2) Application in the preparation of drugs for treating diabetes.