Culture medium for producing penicillin G sodium degrading enzyme and culture method thereof

Abstract

The invention relates to a culture medium and a method for producing penicillin G sodium degrading enzyme. The culture medium is composed of the following components: 20 g / L of galactose, 8 g / L of yeast extract powder, 10 g / L of ammonium sulfate, 0.5 g / L of MgSO4. 7H2O, 0.5 g / L of NaCl, 0.5 g / L of KH2PO4, 1.5 g / L of K2HPO4, and 0.05% of Tween-80. According to the invention, 0.75 mmol / L of meropenem is creatively added to induce degrading enzyme when the strain is cultured for 9 hours. The method comprises the step of adjusting fermentation conditions such as seed culture time, pH value of a culture medium, inoculum size, culture temperature, rotating speed, liquid loading amount and the like so as to improve the yield of the penicillin G sodium degrading enzyme. The fermentation culture medium and the culture method provided by the invention have a proliferation promoting effect on the penicillin G sodium degrading enzyme, and have important significance on industrial production of the penicillin G sodium degrading enzyme by sphingobacterium sp. And harmless treatment of antibiotics in the environment.

Description

Technical Field

[0001] The present invention relates to the field of environmental microbial technology, and in particular to a culture medium and a culture method for increasing the yield of penicillin G sodium degrading enzyme. Background Art

[0002] Penicillin G sodium (PGNa) is a highly effective β-lactam antibiotic with broad-spectrum antimicrobial activity. Since its discovery in 1928, it has been widely used to treat bacterial infections in humans and animals. However, penicillin G sodium has low absorption and utilization in the body and rapid excretion, resulting in approximately 53–85% of penicillin G sodium being released into the environment through the parent compound or excreta after use. The overuse of antibiotics accelerates the accumulation of penicillin G sodium in the environment, inducing the emergence of numerous antibiotic-resistant bacteria (ARBs) and resistance genes (ARGs), leading to the serious global problem of antibiotic resistance. Pathogenic bacteria carrying ARGs can horizontally transfer ARGs between bacterial cells via mobile genetic elements such as plasmids, integrons, and transposons, posing a serious threat to public health and ecosystems. Effectively removing penicillin G sodium residues from the environment has become a critical environmental issue that requires urgent resolution.

[0003] Microbial degradation has become an effective method for addressing penicillin G sodium contamination due to its high efficiency, low cost, and simple operation. To date, only a few bacteria have been isolated capable of degrading penicillin G: Paracoccus KDSPL-02, Klebsiella pneumoniae Z-1, and Serratia marcescens R1. Current research on penicillin G sodium biodegradation remains limited to strain screening and cellular degradation, with limited studies at the enzymatic and molecular levels. Because enzymatic degradation does not induce resistant strains or transmit resistance genes, it represents a sustainable, environmentally friendly, and promising technology for the harmless treatment of antibiotic contamination. Given the challenges of penicillin G sodium's clinical prescription and overuse in livestock farming, as well as antibiotic residues in fermentation residues, the search for more efficient and environmentally friendly enzymes for the biodegradation of penicillin G sodium is of great significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a culture medium and a culture method for producing extracellular penicillin G sodium degrading enzyme. The culture medium and culture method of the present invention can significantly increase the yield of penicillin G sodium degrading enzyme produced by fermentation.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] In a first aspect, the present invention provides an optimized culture medium for producing sodium penicillin G degrading enzyme, comprising a carbon source, a nitrogen source, and metal ions. The main components and concentrations thereof are as follows: 5-30 g / L galactose, 1-10 g / L yeast extract, 1-10 g / L ammonium sulfate, 1-10 g / L MgSO4 . 7H2O 0.1-2.5g / L,NaCl 0.5-1.0g / L,KH2PO40.5-1.0g / L,K2HPO41.5-1.0g / L, Tween-80 0.00-0.25%.

[0007] Preferably, the optimized synthetic culture medium components are 20 g / L galactose, 8 g / L yeast extract, 10 g / L ammonium sulfate, 10 g / L MgSO 4. 7H2O 0.5g / L,NaCl 0.5g / L,KH2PO40.5 g / L,K2HPO41.5 g / L, Tween-80 0.05%.

[0008] In a second aspect, the present invention provides a method for inducing the secretion of penicillin G sodium degrading enzyme, the induction method comprising inoculating Sphingobacterium sphingosineum into the optimized synthetic culture medium as described in the first aspect and culturing the culture medium.

[0009] Furthermore, the inducer is meropenem, the addition time is 6-18 hours and the final concentration is 0-1 mmol / L.

[0010] The preferred inducer is meropenem, which is added for 9 hours and has a final concentration of 0.75 mmol / L.

[0011] In a third aspect, the present invention provides a method for culturing Sphingobacterium to produce penicillin G sodium degrading enzyme, the culturing method comprising inoculating Sphingobacterium into the culture medium and inducer as described in the first and second aspects for culturing.

[0012] Preferably, the initial pH value of the culture medium is 5-10, the seed age is 6-36h, the inoculation amount is 1-25% (v / v), the culture temperature is 15-40°C, the shaking speed is 140-240r / min, and the liquid volume is 10-110mL.

[0013] Preferably, the culture method is as follows: initial pH 7, inoculation amount 5%, culture temperature 30°C, rotation speed 200 r / min, inoculation age 24 hours and liquid volume 30 mL.

[0014] Preferably, the Sphingobacterium is isolated independently.

[0015] In summary, the present invention discloses the following technical effects:

[0016] When sphingobacterium is cultured using the novel culture medium of the present invention, the activity of penicillin G sodium degrading enzyme reaches a maximum of 682.82 U / mL in shake flask fermentation, which is increased by about 13 times.

[0017] This study selects the optimal carbon source, nitrogen source, and metal ion based on single-factor optimization screening, and optimizes the original initial culture medium composition using orthogonal experimental methods. Furthermore, the inductive agent meropenem is creatively added during the culture process, and a suitable culture method is proposed, which is of great significance for the industrial production of penicillin G sodium degrading enzyme by Sphingobacterium. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Effects of carbon source, nitrogen source and metal ion types on the production of penicillin G sodium degrading enzyme.

[0020] Reference numerals: glucose 1; galactose 2; fructose 3; xylose 4; arabinose 5; maltose 6; sucrose 7; lactose 8; soluble starch 9; peptone 10; yeast extract 11; beef extract 12; urea 13; soybean meal 14; ammonium sulfate 15; diammonium hydrogen citrate 16; ammonium chloride 17; corn steep liquor 18; MgSO4·7H2O 19; CoCl·6H2O 20; MnSO4·H2O 21; BaCl2 22; CaCl2 23; CuSO4 24; ZnSO4·H2O 25; FeSO4·7H2O 26; Al(NO3)3·9H2O 27;

[0021] Figure 2 Effects of carbon and nitrogen sources and metal ion concentrations and ratios on the production of penicillin G sodium degrading enzyme;

[0022] Figure 3 Effect of surfactant additive Tween-80 on the production of penicillin G sodium degrading enzyme;

[0023] Figure 4 Effects of inducer type, meropenem concentration, and addition time on the production of penicillin G sodium degrading enzyme;

[0024] Figure 5 Effects of culture methods on the production of penicillin G sodium degrading enzyme: pH, inoculum size, temperature, rotation speed, seed age and liquid volume; Figure 6Enzyme production diagrams of basal medium, optimized medium and after adding meropenem. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] The materials and methods used in the following examples generally include:

[0032] 1.1 Materials

[0033] 1.1.1 Starting strain

[0034] The experimental strain Sphingobacterium was isolated independently.

[0035] 1.1.2 Culture medium

[0036] Basic enzyme production medium: 0.5 g / L NaCl, 1.5 g / L KH2PO4, 0.5 g / L K2HPO4, 0.5 g / L MgSO 4. 7H2O, 10 g / L glucose, 5 g / L peptone.

[0037] LB medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl.

[0038] PBS substrate buffer solution: Penicillin G sodium was dissolved in 0.1 mol / L, pH 7 phosphate buffer solution prepared with NaH2PO 4. 4H2O and Na2HPO 4. 4H2O and Na2HPO

[0039] 1.2 Method

[0040] 1.2.1 Biomass OD 600

[0041] The fermentation broth stock solution was diluted to an appropriate multiple to determine the OD 600 on a spectrophotometer. OD is the measured absorbance value * dilution multiple.

[0042] 1.2.2 Penicillin G sodium degrading enzyme activity determination

[0043] 0.5 mL of 1200 mg / L penicillin G sodium substrate buffer solution was mixed with 0.4 mL of PBS buffer solution, incubated at 37°C for 5 min, 0.1 mL of enzyme solution filtered through a 0.22 μm filter was added, and shaken in a water bath at 37°C for 10 min, and finally 1 mL of acetonitrile was added to terminate the reaction. The residual amount of penicillin G sodium in the reaction system was detected by HPLC to calculate the activity of the penicillin G sodium degrading enzyme. In addition, 1 enzyme activity unit was defined as the amount of enzyme required to catalyze 1 μmol of penicillin G sodium per minute. The degradation enzyme activity calculation formula is as follows:

[0044]

[0045] Where C1 is the initial concentration of penicillin G sodium (mg / L), Ce is the residual concentration of penicillin G sodium in the solution (mg / L), Vt is the total volume of the reaction solution (mL), n is the dilution multiple of the enzyme solution, △t is the reaction time (min), and Ve is the volume of the enzyme solution (mL).

[0046] 1.2.3 Penicillin G sodium degrading enzyme production

[0047] Firstly, the strain S. phytometiculum was inoculated into 250 mL flask containing 50 mL LB medium, and incubated at 37 °C and 220 r / min for 24 h. The culture was centrifuged at 8000 r / min for 5 min, then washed with 0.9% sterile normal saline for 3 times, and added with normal saline again to prepare a bacterial suspension. Finally, 5.0 mL of the bacterial suspension was added into 250 mL flask containing 50 mL basal medium, and incubated at 37 °C in a shaking incubator at 220 r / min for 18 h.

[0048] Design of medium component optimization experiment for producing penicillin G sodium degrading enzyme by strain S. phytometiculum

[0049] On the basis of the original initial fermentation medium, the carbon source, nitrogen source and metal ions were changed, and nine different common carbon sources were selected: glucose, galactose, fructose, xylose, arabinose, maltose, sucrose, lactose and soluble starch to screen the best carbon source. Then the optimal carbon source was selected for concentration optimization, and the carbon source concentration was set to 5, 10, 15, 20, 25, 30 g / L; nine different common nitrogen sources were selected: peptone, yeast extract, beef extract, urea, soybean meal, ammonium sulfate, diammonium hydrogen citrate, ammonium chloride and corn steep liquor. The optimal nitrogen source was selected for concentration optimization, and the nitrogen source concentration was set to 1, 2, 4, 6, 8, 10 g / L. Since most microorganisms prefer to use complex nitrogen sources, the nitrogen sources that promote enzyme production were combined for use; nine different common metal ions were selected: MgSO4·7H2O, CoCl·6H2O, MnSO4·H2O, BaCl2, CaCl2, CuSO4, ZnSO4·H2O, FeSO4·7H2O and Al(NO3)3·9H2O. The optimal metal ion addition amount was optimized, and the addition amount gradient was set to: 0.1, 0.5, 1.0, 1.5, 2.0, 2.5 g / L

[0050] As shown in Figure 1 a, the best carbon source for S. phytometiculum to produce penicillin G sodium degrading enzyme was galactose. As shown in Figure 2 a-b, the enzyme activity measured when galactose was used as a single carbon source was higher than that when glucose was used as a single carbon source, and the combined carbon source of galactose and glucose. When the concentration of galactose was 15 g / L, the degrading enzyme activity reached a peak value of 193.25 U / mL. As shown in Figure 1 b, yeast extract, beef extract and ammonium sulfate could all promote the production of enzyme by strain SQW1. As shown in Figure 2 b, the combined nitrogen sources all promoted the production of enzyme and growth of the strain to different extents, among which the combination of yeast extract and ammonium sulfate more obviously promoted the secretion of degrading enzyme. As shown in Figure 1 c, the enzyme production was higher under the action of Mg 2+ . Figure 2 a shows that Mg 2+When the addition amount was 1.0 g / L, the enzyme production reached its peak.

[0051] Example 2 Orthogonal Experimental Design of Culture Medium Components

[0052] Based on the above experimental results, galactose was selected as the carbon source, yeast extract and ammonium sulfate as the nitrogen sources, and MgSO4·7H2O as the metal ion for orthogonal optimization of the carbon and nitrogen sources and metal ion addition content (Table 1).

[0053] Table 1 Orthogonal optimization of enzyme production medium components

[0054]

[0055] The results are shown in Table S1. A4C2B2D2 is the optimal level combination. The final optimized culture medium composition is 20g / L galactose, 8g / L yeast extract powder, 10g / L ammonium sulfate, 0.5g / LMgSO 4. 7H2O, 0.5g / LNaCl, 0.5g / L KH2PO4, 1.5g / LK2HPO4.

[0056]

[0057]

[0058] Example 3 Effects of surfactant additives and inducers on the secretion of degradative enzymes of bacterial strains

[0059] Based on the results of the culture medium optimization experiment, the effect of the surfactant Tween-80 on the secretion of degradative enzymes was investigated. Tween-80 was added at a concentration of 0-0.25%. The effects of 12 inducers (guaiacol, ferulic acid, penicillin G sodium, amoxicillin, ampicillin, cephalexin, ceftazidime, cephalothin, cefotaxime sodium, cefoxitin, aztreonam, and meropenem) at a final concentration of 0.5 mmol / L were also investigated. The effects of the time (6-18 hours) and concentration (0-1 mmol / L) of inducer addition on the secretion of degradative enzymes were also studied.

[0060] like Figure 3 As we know, 0.05% Tween-80 promoted the growth of Sphingobacterium and enhanced the activity of its degradation enzymes. Figure 4 As shown in a, compared with the blank treatment without the addition of inducer, meropenem can strongly induce the production of penicillin G sodium degrading enzyme, with the enzyme activity reaching 338.15U / mL, but it will inhibit the growth of bacteria to a certain extent. After 12 hours of incubation, the degrading enzyme activity showed a significant increase after adding 0.75mM meropenem for 6 hours, reaching 380.3U / mL ( Figure 4b). After 9 hours of incubation, the degradation enzyme activity increased significantly after adding meropenem for 6 hours, reaching 678.85 U / mL ( Figure 4 c).

[0061] Example 4: Effect of Culture Method Optimization on Degradation Enzyme Activity

[0062] Based on the above experimental results, the effects of culture conditions on the secretion of penicillin G sodium degrading enzymes were further investigated. The effects of different culture conditions, such as the initial pH of the culture medium (5-10), seed age (6-36 hours), inoculum size (1-25%, v / v), culture temperature (15-40°C), shaker speed (140-240 rpm), and liquid volume (10-110 mL), on the secretion of penicillin G sodium degrading enzymes were studied.

[0063] like Figure 5 The optimal culture method shown is: initial pH 7, inoculation size 5%, culture temperature 30°C, rotation speed 200 r / min, inoculation age 24 h and liquid volume 30 mL.

[0064] Determination of the curve of penicillin G sodium enzyme produced by Example 5

[0065] Based on the above experimental results, the seed liquid was inoculated into the culture medium and cultured for 24 hours. Samples were taken every 3 hours to measure the activity of the degradation enzyme, and enzyme production graphs of the basic culture medium, optimized culture medium and culture medium after adding meropenem were obtained.

[0066] like Figure 6 As shown, under optimized culture conditions, meropenem was added for induction after 9 hours of fermentation. After 3 hours, the degradation enzyme activity reached its peak, reaching 682.82 U / mL. Compared with basal medium and optimized medium, the induction-induced enzyme activity showed a significant increase, increasing the yield by approximately 13 times.

[0067] The applicant states that while the above-described embodiments illustrate a culture medium and method for producing penicillin G sodium-degrading enzyme, the present invention is not limited to these embodiments, nor does it necessarily rely on these embodiments for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for raw materials in the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0068] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A culture medium for producing penicillin G sodium degrading enzyme by fermentation, characterized in that: The culture medium is composed of 5-30 g / L galactose, 1-10 g / L yeast extract, 1-10 g / L ammonium sulfate, 1-10 g / L MgSO 4. 7H2O 0.1-2.5g / L,NaCl 0.5-1g / L,KH2PO40.5-1g / L,K2HPO41.5-1g / L, Tween-80 0.00-0.25%.

2. The method according to claim 1, wherein The culture medium used was composed of 20 g / L galactose, 8 g / L yeast extract, 10 g / L ammonium sulfate, 10 g / L MgSO 4. 7H2O 0.5g / L,NaCl 0.5g / L,KH2PO40.5 g / L,K2HPO41.5 g / L, Tween-80 0.05%.

3. A method for inducing the secretion of penicillin G sodium degrading enzyme, characterized in that: 0.75 mmol / L meropenem is added when the culture time of Sphingobacterium is 9 hours, and the strain seeds are inoculated into the culture medium of claim 2 for culture.

4. A method for culturing Sphingobacterium sphingomyelinum for producing penicillin G sodium degrading enzyme, characterized in that: The initial pH was 7, the inoculation size was 5%, the culture temperature was 30°C, the rotation speed was 200 r / min, the inoculation age was 24 h and the liquid volume was 30 mL.

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