Method for producing beta-glucuronidase from escherichia coli and application of beta-glucuronidase

Abstract

The invention provides a method for producing beta-glucuronidase from escherichia coli and application of the beta-glucuronidase, and relates to the technical field of microorganisms. The method provided by the invention comprises the following steps: inoculating Escherichia coli ATCC 25922 on a composite culture medium for pre-culture, and culturing in an electric field environment of 1-10N / C for 12-24h to obtain a mixture containing beta-glucuronidase; wherein the composite culture medium comprises 10-100 [mu] mol / L of ferric citrate, and the concentration of the ferric citrate is increased in stages in the pre-culture process. According to the method, the escherichia coli ATCC 25922 is cultured in the iron-containing culture medium and the low-intensity electric field environment, so that the synthesis of the beta-glucuronidase by the escherichia coli ATCC 25922 can be effectively improved, and the enzyme activity and the enzyme yield of the beta-glucuronidase are improved.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a method and application of producing beta-glucuronidase by Escherichia coli. Background Art

[0002] β-glucuronidase is a hydrolase that can specifically hydrolyze β-glucuronidyl bonds. In the production process of bezoar, bilirubin is converted into free bilirubin by hydrolyzing glucuronic acid, which is conducive to the free bilirubin combining with calcium ions under appropriate conditions to form bilirubin salt precipitation, which is also one of the traditional Chinese medicine ingredients of bezoar. Compared with the traditional method of extracting bezoar from bovine animals, the in vitro production of bezoar using β-glucuronidase has significant advantages. By precisely controlling the production conditions and the activity of β-glucuronidase, the production efficiency of bezoar can be greatly improved.

[0003] Escherichia coli ATCC 25922 can metabolize a large amount of β-glucuronidase in a fermentation environment, and a Chinese patent with publication number CN117562927A provides a preparation process for cultivating bezoar in vitro using β-glucuronidase, wherein β-glucuronidase is used to obtain free bilirubin and form bilirubin calcium precipitation with a calcium chloride solution, thereby simulating physiological activities similar to those in bovine animals in vitro, thereby improving the difference in composition between in vitro cultivated bezoar and natural bezoar. However, this method is highly dependent on the enzyme activity and enzyme yield of β-glucuronidase, so it is urgent to provide a solution to improve the enzyme activity and enzyme yield of β-glucuronidase produced by Escherichia coli ATCC 25922 in a fermentation environment. Summary of the invention

[0004] The object of the present invention is to provide a method and application of producing β-glucuronidase by Escherichia coli. By culturing Escherichia coli ATCC 25922 in a composite culture medium containing organic iron and a low-intensity electric field environment, the synthesis of β-glucuronidase by Escherichia coli ATCC 25922 can be effectively improved, thereby improving the enzyme activity and enzyme yield of β-glucuronidase.

[0005] In a first aspect, the present invention provides a method for producing β-glucuronidase by Escherichia coli, comprising: inoculating Escherichia coli ATCC 25922 on a composite culture medium for pre-culture, and then culturing for 12-24 hours in an electric field environment of 1-10N / C to obtain a mixture containing β-glucuronidase; wherein the composite culture medium comprises 10-100 μmol / L ferric citrate, and the concentration of the ferric citrate is increased in stages during the pre-culture process.

[0006] The method provided by the present invention can activate the metabolic pathway of Escherichia coli ATCC 25922 by pre-culturing Escherichia coli ATCC 25922 in a composite culture medium containing ferric citrate, and can pre-synthesize related proteins and protein factors. In addition, the concentration of ferric citrate is increased in stages during the pre-culturing process, which can avoid excessive stimulation of Escherichia coli ATCC 25922 by excessive ferric citrate concentration, and is beneficial to the absorption and utilization of ferric citrate by Escherichia coli ATCC 25922. At the same time, culturing under low-intensity electric field stimulation is beneficial to promoting the absorption of nutrients by Escherichia coli ATCC 25922, and can play a good synergistic role with the iron element absorbed in Escherichia coli ATCC 25922, regulate the gene expression of Escherichia coli ATCC25922 for synthesizing β-glucuronidase, and then improve the enzyme activity and enzyme yield of β-glucuronidase.

[0007] Optionally, when Escherichia coli ATCC 25922 is inoculated on a composite medium for pre-culture, an induction process, an absorption process and a consolidation process are included, and the concentration gradient of the ferric citrate during the induction process, the absorption process and the consolidation process is greater than or equal to 20 μmol / L.

[0008] Optionally, during the induction process, the initial concentration of the ferric citrate in the composite culture medium is 10-30 μmol / L; during the absorption process, the initial concentration of the ferric citrate in the composite culture medium is 30-80 μmol / L; during the consolidation process, the initial concentration of the ferric citrate in the composite culture medium is 80-100 μmol / L.

[0009] Alternatively, Escherichia coli ATCC 25922 was cultured at 10 4 -10 5 The density of cfu / mL was inoculated in a composite culture medium with a pH of 7.0-7.4, and pre-cultured for 45-50 hours in an environment of 35-40℃ and an oxygen content of 0.1-2%, including 10-12 hours in the induction process, 25-30 hours in the absorption process, and 8-10 hours in the consolidation process.

[0010] Optionally, the composite culture medium further comprises, by mass concentration, 8-12 g / L of tryptone, 3-6 g / L of yeast extract, 1-5 mg / L of vitamin B1, 0.1-0.5 mg / L of calcium salt and 8-12 g / L of sodium chloride.

[0011] Optionally, when cultured in an electric field environment of 1-10N / C for 12-24 hours, the electric field intensity of the electric field environment is increased in stages, including a stimulation stage and an enzyme production stage. During the stimulation stage, the electric field intensity is 1-5N / C, and during the enzyme production stage, the electric field intensity is 5-10N / C, and the electric field intensity gradient between the stimulation stage and the enzyme production stage is greater than or equal to 3N / C.

[0012] Optionally, when culturing for 12-24 hours under an electric field environment of 1-10N / C, the method includes: adjusting the initial concentration of ferric citrate to 80-100 μmol / L, culturing for a stimulation phase for 4-8 hours under an electric field strength of 1-5N / C, and then culturing for enzyme production for 8-16 hours under an electric field strength of 5-10N / C.

[0013] In a second aspect, the present invention also provides a use of β-glucuronidase produced by any of the above methods in cultivating bezoar. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should be the common meanings understood by people with general skills in the field to which the present invention belongs.

[0015] The present invention provides a method for producing β-glucuronidase by Escherichia coli, comprising: inoculating Escherichia coli ATCC25922 on a composite culture medium for pre-culture, and then culturing for 12-24 hours in an electric field environment of 1-10N / C to obtain a mixture containing β-glucuronidase. Specifically, Escherichia coli ATCC 25922 is purchased from Guangdong Huankai Biotechnology Co., Ltd. with a strain number of FSCC149002.

[0016] In fact, after the β-glucuronidase is produced, the mixture containing the β-glucuronidase can be directly mixed with bile, so that during the bezoar production process, the Escherichia coli ATCC 25922 remaining in the mixture can still produce a partial amount of β-glucuronidase. Similarly, after the β-glucuronidase is produced, the β-glucuronidase in the mixture can be separated and used by the commonly used purification and separation means in the art.

[0017] In fact, pre-culturing Escherichia coli ATCC 25922 is beneficial to increasing the number of Escherichia coli in ATCC 25922, and adjusting the elements of the composite culture medium can affect the gene expression of Escherichia coli in ATCC 25922. When culturing in an electric field environment, the electric field stimulation promotes the absorption of nutrients by Escherichia coli ATCC 25922, and regulates the gene expression of Escherichia coli ATCC 25922 for synthesizing β-glucuronidase, thereby increasing the enzyme activity and enzyme yield of β-glucuronidase.

[0018] Specifically, 10-100 μmol / L of ferric citrate is also included in the composite culture medium, and the concentration of ferric citrate is increased in stages during the pre-culture process. In fact, when Escherichia coli ATCC 25922 is pre-cultured, it can absorb the ferric citrate in the composite culture medium to promote its own metabolism, accelerate the growth rate so that Escherichia coli ATCC 25922 can reproduce faster, and after Escherichia coli ATCC 25922 absorbs ferric citrate, the iron element can participate in the composition of the antioxidant enzyme system, thereby alleviating oxidative stress. In addition, the concentration of ferric citrate is increased in stages, which can avoid excessive concentration causing growth stimulation to Escherichia coli ATCC25922.

[0019] In fact, when Escherichia coli ATCC 25922 is cultured in a composite culture medium and ingests ferric citrate, under low-intensity electric field stimulation, it can stimulate Escherichia coli ATCC 25922 to efficiently express the gene for producing β-glucuronidase, thereby effectively improving the enzyme activity and enzyme production of β-glucuronidase under electric field stimulation.

[0020] In some embodiments, when Escherichia coli ATCC 25922 is inoculated on a composite medium for pre-culture, the process includes an induction process, an absorption process, and a consolidation process, and the concentration gradient of ferric citrate during the induction process, the absorption process, and the consolidation process is greater than or equal to 20 μmol / L. In fact, by dividing the process during pre-culture, it is helpful to regulate the change of ferric citrate concentration during the pre-culture period, and avoid the inhibition of the growth of Escherichia coli ATCC 25922 by excessively high ferric citrate concentration during the initial culture process.

[0021] Specifically, the initial concentration of ferric citrate in the composite culture medium during the induction process is 10-30 μmol / L; the initial concentration of ferric citrate in the composite culture medium during the absorption process is 30-80 μmol / L; and the initial concentration of ferric citrate in the composite culture medium during the consolidation process is 80-100 μmol / L. In fact, by stimulating Escherichia coli ATCC 25922 with low concentration of ferric citrate in the initial stage, the iron transport-related genes activated by Escherichia coli ATCC 25922 can be promoted, while avoiding excessive stimulation of Escherichia coli ATCC 25922. In the absorption process, increasing the concentration of ferric citrate can promote the absorption of ferric citrate by Escherichia coli ATCC 25922, and absorption and consolidation can be performed during the consolidation process.

[0022] In some embodiments, Escherichia coli ATCC 25922 was cultured at 10 4 -10 5 cfu / mL was inoculated in a composite culture medium with a pH of 7.0-7.4, and pre-cultured for 45-50 hours in an environment of 35-40°C and an oxygen content of 0.1-2%. In fact, by adjusting the pH of the composite culture medium to 7.0-7.4, it is beneficial for the growth of Escherichia coli ATCC 25922. In addition, by regulating the inoculation amount of Escherichia coli ATCC 25922 in the composite culture medium, Escherichia coli ATCC 25922 can be promoted to grow rapidly during the pre-culture process, avoiding competition from too many Escherichia coli ATCC 25922, and can also promote Escherichia coli ATCC 25922 to take in more ferric citrate.

[0023] In fact, the induction process is cultured for 10-12 hours, the absorption process is cultured for 25-30 hours, and the consolidation process is cultured for 8-10 hours. By adjusting the culture time during the induction process, the absorption process, and the consolidation process, E. coli ATCC 25922 can be encouraged to fully absorb ferric citrate and reduce the growth stimulation of E. coli ATCC 25922.

[0024] In some embodiments, the composite culture medium includes 8-12 g / L of tryptone, 3-6 g / L of yeast extract, 1-5 mg / L of vitamin B1, 0.1-0.5 mg / L of calcium salt and 8-12 g / L of sodium chloride in terms of mass concentration. In fact, tryptone as a nitrogen source can provide Escherichia coli CICC 10421 with the required amino acids, short peptides and other nutrients, wherein the tryptone used can be obtained by digesting fresh beef and cattle bones with trypsin, and the main components of yeast extract are polypeptides, amino acids, nucleotides, B group microorganisms and other trace elements, which can provide rich nutrition for the growth of Escherichia coli ATCC 25922 and effectively promote the growth of Escherichia coli ATCC 25922, wherein the yeast extract uses protein-rich yeast as a raw material, and is refined through the steps of autolysis, enzymolysis, separation and concentration.

[0025] In fact, when Escherichia coli ATCC 25922 is inoculated on a composite culture medium for pre-culture, the composite culture medium is sterilized at high temperature and high pressure in advance, thereby avoiding the influence of foreign bacteria during the culture of Escherichia coli ATCC 25922, thereby improving the culture survival rate of Escherichia coli ATCC 25922.

[0026] In some embodiments, when cultured in an electric field environment of 1-10N / C for 12-24 hours, the electric field intensity of the electric field environment increases in stages, including a stimulation stage and an enzyme production stage. During the stimulation stage, the electric field intensity is 1-5N / C, and during the enzyme production stage, the electric field intensity is 5-10N / C, and the electric field intensity gradient between the stimulation stage and the enzyme production stage is greater than or equal to 3N / C.

[0027] In fact, by increasing the electric field intensity in the electric field environment in stages, it is beneficial to stimulate Escherichia coli ATCC 25922 at low intensity, thereby promoting the adaptation of Escherichia coli ATCC 25922 to the electric field environment. When electric field stimulation is carried out in the enzyme production stage, it is beneficial to activate the expression of the β-glucuronidase gene of Escherichia coli ATCC 25922, thereby increasing the enzyme activity and enzyme production of β-glucuronidase.

[0028] In some embodiments, when culturing for 12-24 hours under an electric field environment of 1-10N / C, the method includes: adjusting the initial concentration of ferric citrate to 80-100 μmol / L, culturing for a stimulation phase of 4-8 hours under an electric field strength of 1-5N / C, and then culturing for enzyme production for 8-16 hours under an electric field strength of 5-10N / C. This is conducive to maintaining the absorption and utilization of ferric citrate by Escherichia coli ATCC 25922 under the stimulation of the electric field, and then cooperating with the electric field to further improve the enzyme activity and enzyme yield of β-glucuronidase.

[0029] In fact, the present invention also provides an application of the purified β-glucuronidase produced in any of the above embodiments in the production of bezoar.

[0030] Example 1 This embodiment 1 provides a method for producing β-glucuronidase in Escherichia coli, comprising the following steps: S1. Escherichia coli ATCC 25922 was cultured at 5.5×10 4 cfu / mL is inoculated on a composite culture medium with a pH of 7.4, the initial concentration of ferric citrate in the composite culture medium is adjusted to 15 μmol / L, and then the culture is carried out for 10 hours of induction process in an incubator at 37°C and an oxygen content of 1%, the initial concentration of ferric citrate in the composite culture medium is adjusted to 50 μmol / L, and then the culture is carried out for 30 hours of absorption culture, and then the initial concentration of ferric citrate in the composite culture medium is adjusted to 90 μmol / L, and then the culture mixture is obtained after consolidation culture is carried out for 10 hours; wherein, the composite culture medium further includes 8 g / L of tryptone, 5 g / L of yeast extract, 3 mg / L of vitamin B1, 0.3 mg / L of calcium salt and 10 g / L of sodium chloride in terms of initial mass concentration; S2. After adjusting the initial concentration of ferric citrate in the culture mixture to 90 μmol / L, the mixture was stimulated and cultured for 5 hours in an incubator with an electric field strength of 3N / C and an oxygen concentration of 1% at 37°C. After that, the electric field strength was increased to 8N / C and the enzyme production culture was carried out for 12 hours to obtain a mixture containing β-glucuronidase.

[0031] Example 2 This embodiment 2 provides a method for producing β-glucuronidase in Escherichia coli, comprising the following steps: S1. Escherichia coli ATCC 25922 was cultured at 6.1×10 4 cfu / mL is inoculated on a composite culture medium with a pH of 7.4, and after adjusting the initial concentration of ferric citrate in the composite culture medium to 15 μmol / L, it is cultured in an incubator at 37°C and an oxygen content of 1% for 50 hours, and the concentration of ferric citrate in the composite culture medium is adjusted to 15 μmol / L every 10 hours during the culture process, and a culture mixture is obtained after the culture is completed; wherein the composite culture medium further includes 8 g / L of tryptone, 5 g / L of yeast extract, 3 mg / L of vitamin B1, 0.3 mg / L of calcium salt and 10 g / L of sodium chloride in terms of initial mass concentration; S2. After adjusting the initial concentration of ferric citrate in the culture mixture to 15 μmol / L, stimulate the mixture for 5 hours in an incubator with an electric field strength of 3N / C and an oxygen concentration of 1% at 37°C. After increasing the electric field strength to 8N / C and performing enzyme production culture for 12 hours, a mixture containing β-glucuronidase is obtained.

[0032] Example 3 This embodiment 3 provides a method for Escherichia coli to produce β-glucuronidase, comprising the following steps: S1. Escherichia coli ATCC 25922 was cultured at 4.2×10 4 cfu / mL is inoculated on a composite culture medium with a pH of 7.4, and after adjusting the initial concentration of ferric citrate in the composite culture medium to 90 μmol / L, the culture is carried out in an incubator at 37°C and an oxygen content of 1% for 50 hours, and the concentration of ferric citrate in the composite culture medium is adjusted to 90 μmol / L every 10 hours during the culture process, and a culture mixture is obtained after the culture is completed; wherein the composite culture medium further includes 8 g / L of tryptone, 5 g / L of yeast extract, 3 mg / L of vitamin B1, 0.3 mg / L of calcium salt and 10 g / L of sodium chloride in terms of initial mass concentration; S2. After adjusting the initial concentration of ferric citrate in the culture mixture to 90 μmol / L, the mixture was stimulated and cultured for 5 hours in an incubator with an electric field strength of 3N / C and an oxygen concentration of 1% at 37°C. After that, the electric field strength was increased to 8N / C and the enzyme production culture was carried out for 12 hours to obtain a mixture containing β-glucuronidase.

[0033] Example 4 This embodiment 4 provides a method for Escherichia coli to produce β-glucuronidase, comprising the following steps: S1. Escherichia coli ATCC 25922 was cultured at 5.3×10 4 cfu / mL is inoculated on a composite culture medium with a pH of 7.4, the initial concentration of ferric citrate in the composite culture medium is adjusted to 15 μmol / L, and then the culture is carried out for 10 hours of induction process in an incubator at 37°C and an oxygen content of 1%, the initial concentration of ferric citrate in the composite culture medium is adjusted to 50 μmol / L, and then the culture is carried out for 30 hours of absorption culture, and then the initial concentration of ferric citrate in the composite culture medium is adjusted to 90 μmol / L, and then the culture mixture is obtained after consolidation culture is carried out for 10 hours; wherein, the composite culture medium further includes 8 g / L of tryptone, 5 g / L of yeast extract, 3 mg / L of vitamin B1, 0.3 mg / L of calcium salt and 10 g / L of sodium chloride in terms of initial mass concentration; S2. After 5 hours of stimulation culture of the culture mixture in an incubator with an electric field strength of 3N / C and an oxygen concentration of 1% at 37°C, the electric field strength was increased to 8N / C and enzyme production culture was carried out for 12 hours to obtain a mixture containing β-glucuronidase.

[0034] Example 5 This embodiment 5 provides a method for Escherichia coli to produce β-glucuronidase, comprising the following steps: S1. Escherichia coli ATCC 25922 was cultured at 6.5×104 cfu / mL is inoculated on a composite culture medium with a pH of 7.4, the initial concentration of ferric citrate in the composite culture medium is adjusted to 15 μmol / L, and then the culture is carried out for 10 hours of induction process in an incubator at 37°C and an oxygen content of 1%, the initial concentration of ferric citrate in the composite culture medium is adjusted to 50 μmol / L, and then the culture is carried out for 30 hours of absorption culture, and then the initial concentration of ferric citrate in the composite culture medium is adjusted to 90 μmol / L, and then the culture mixture is obtained after consolidation culture is carried out for 10 hours; wherein, the composite culture medium further includes 8 g / L of tryptone, 5 g / L of yeast extract, 3 mg / L of vitamin B1, 0.3 mg / L of calcium salt and 10 g / L of sodium chloride in terms of initial mass concentration; S2. After adjusting the initial concentration of ferric citrate in the culture mixture to 90 μmol / L, the mixture was cultured in an incubator with an electric field strength of 3N / C and an oxygen concentration of 1% at 37°C for 17 hours to obtain a mixture containing β-glucuronidase.

[0035] Example 6 This Example 6 provides a method for Escherichia coli to produce β-glucuronidase, comprising the following steps: S1. Escherichia coli ATCC 25922 was cultured at 6.8×10 4 cfu / mL is inoculated on a composite culture medium with a pH of 7.4, the initial concentration of ferric citrate in the composite culture medium is adjusted to 15 μmol / L, and then the culture is carried out for 10 hours of induction process in an incubator at 37°C and an oxygen content of 1%, the initial concentration of ferric citrate in the composite culture medium is adjusted to 50 μmol / L, and then the culture is carried out for 30 hours of absorption culture, and then the initial concentration of ferric citrate in the composite culture medium is adjusted to 90 μmol / L, and then the culture mixture is obtained after consolidation culture is carried out for 10 hours; wherein, the composite culture medium further includes 8 g / L of tryptone, 5 g / L of yeast extract, 3 mg / L of vitamin B1, 0.3 mg / L of calcium salt and 10 g / L of sodium chloride in terms of initial mass concentration; S2. After adjusting the initial concentration of ferric citrate in the culture mixture to 90 μmol / L, the mixture was cultured in an incubator with an electric field strength of 8 N / C and an oxygen concentration of 1% at 37° C. for 17 hours to obtain a mixture containing β-glucuronidase.

[0036] Comparative Example 1 This comparative example 1 provides a method for producing β-glucuronidase in Escherichia coli, comprising the following steps: S1. Escherichia coli ATCC 25922 was cultured at 5.5×10 4cfu / mL was inoculated on a composite culture medium with a pH of 7.4, and cultured in an incubator at 37°C and an oxygen content of 1% for 50 hours to obtain a culture mixture; wherein the composite culture medium includes 8 g / L of tryptone, 5 g / L of yeast extract, 3 mg / L of vitamin B1, 0.3 mg / L of calcium salt, and 10 g / L of sodium chloride in terms of initial mass concentration; S2. After 5 hours of stimulation culture of the culture mixture in an incubator with an electric field strength of 3N / C and an oxygen concentration of 1% at 37°C, the electric field strength was increased to 8N / C and enzyme production culture was carried out for 12 hours to obtain a mixture containing β-glucuronidase.

[0037] Comparative Example 2 This comparative example 2 provides a method for producing β-glucuronidase in Escherichia coli, comprising the following steps: S1. Escherichia coli ATCC 25922 was cultured at 5.5×10 4 cfu / mL is inoculated on a composite culture medium with a pH of 7.4, the initial concentration of ferric citrate in the composite culture medium is adjusted to 15 μmol / L, and then the culture is carried out for 10 hours of induction process in an incubator at 37°C and an oxygen content of 1%, the initial concentration of ferric citrate in the composite culture medium is adjusted to 50 μmol / L, and then the culture is carried out for 30 hours of absorption culture, and then the initial concentration of ferric citrate in the composite culture medium is adjusted to 90 μmol / L, and then the culture mixture is obtained after consolidation culture is carried out for 10 hours; wherein, the composite culture medium further includes 8 g / L of tryptone, 5 g / L of yeast extract, 3 mg / L of vitamin B1, 0.3 mg / L of calcium salt and 10 g / L of sodium chloride in terms of initial mass concentration; S2. The culture mixture is cultured in an incubator at 37° C. and an oxygen concentration of 1% for 17 hours to obtain a mixture containing β-glucuronidase.

[0038] Comparative Example 3 This comparative example 3 provides a method for producing β-glucuronidase in Escherichia coli, comprising the following steps: S1. Escherichia coli ATCC 25922 was cultured at 5.5×10 4 cfu / mL was inoculated on a composite culture medium with a pH of 7.4, and cultured in an incubator at 37°C and an oxygen content of 1% for 50 hours to obtain a culture mixture; wherein the composite culture medium includes 8 g / L of tryptone, 5 g / L of yeast extract, 3 mg / L of vitamin B1, 0.3 mg / L of calcium salt, and 10 g / L of sodium chloride in terms of initial mass concentration; S2. The culture mixture is cultured in an incubator at 37° C. and an oxygen concentration of 1% for 17 hours to obtain a mixture containing β-glucuronidase.

[0039] Performance Testing The mixture containing β-glucuronidase prepared in Examples 1 to 6 and Comparative Examples 1 to 3 was tested for enzyme activity, as shown in Table 1 below; the purified β-glucuronidase prepared in Examples 1 to 6 and Comparative Examples 1 to 3 was used in bilirubin extraction, comprising the following steps: taking 1000 ml of fresh bile, adding a certain proportion of purified β-glucuronidase, fermenting at 37° C. for 48 h, taking the fermentation broth and detecting the bilirubin content of the fermentation broth according to the in vitro cultured bezoar semi-finished product part-bilirubin detection method in Part I of the Pharmacopoeia, and the results are shown in Table 1 below.

[0040] Table 1 Performance test of purified β-glucuronidase Enzyme activity (U / mL) Bilirubin content (%) Example 1 1298.83 0.013 Example 2 1095.37 0.012 Example 3 1196.45 0.012 Example 4 1187.62 0.012 Example 5 1193.28 0.011 Example 6 1182.67 0.010 Comparative Example 1 198.24 0.001 Comparative Example 2 283.46 0.002 Comparative Example 3 152.68 0.001 Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. A method for producing β-glucuronidase by Escherichia coli, characterized in that: include: After Escherichia coli ATCC25922 is inoculated on a composite culture medium for pre-culture, the mixture containing β-glucuronidase is prepared after culturing in an electric field environment of 1-10N / C for 12-24 hours; wherein the composite culture medium includes 10-100 μmol / L of ferric citrate, and the concentration of the ferric citrate is increased in stages during the pre-culture process.

2. The method according to claim 1, characterized in that When Escherichia coli ATCC 25922 is inoculated on a composite medium for pre-culture, the process includes an induction process, an absorption process and a consolidation process, and the concentration gradient of the ferric citrate during the induction process, the absorption process and the consolidation process is greater than or equal to 20 μmol / L.

3. The method according to claim 2, characterized in that During the induction process, the initial concentration of the ferric citrate in the composite culture medium is 10-30 μmol / L; during the absorption process, the initial concentration of the ferric citrate in the composite culture medium is 30-80 μmol / L; during the consolidation process, the initial concentration of the ferric citrate in the composite culture medium is 80-100 μmol / L.

4. The method according to any one of claims 2 to 3, characterized in that: Escherichia coli ATCC 25922 was cultured at 10 4 -10 5 The density of cfu / mL was inoculated in a composite culture medium with a pH of 7.0-7.4, and pre-cultured for 45-50 hours in an environment of 35-40℃ and an oxygen content of 0.1-2%, including 10-12 hours in the induction process, 25-30 hours in the absorption process, and 8-10 hours in the consolidation process.

5. The method according to claim 1, characterized in that The composite culture medium further comprises, by mass concentration, 8-12 g / L of tryptone, 3-6 g / L of yeast extract, 1-5 mg / L of vitamin B1, 0.1-0.5 mg / L of calcium salt and 8-12 g / L of sodium chloride.

6. The method according to claim 1, characterized in that When cultured in an electric field environment of 1-10N / C for 12-24 hours, the electric field intensity of the electric field environment is increased in stages, including a stimulation stage and an enzyme production stage. During the stimulation stage, the electric field intensity is 1-5N / C, and during the enzyme production stage, the electric field intensity is 5-10N / C, and the electric field intensity gradient between the stimulation stage and the enzyme production stage is greater than or equal to 3N / C.

7. The method according to claim 6, characterized in that When culturing for 12-24 hours under an electric field environment of 1-10N / C, it includes: adjusting the initial concentration of ferric citrate to 80-100 μmol / L, culturing for a stimulation phase for 4-8 hours under an electric field strength of 1-5N / C, and then culturing for enzyme production for 8-16 hours under an electric field strength of 5-10N / C.

8. Use of the β-glucuronidase produced by the method according to any one of claims 1 to 7 in cultivating bezoar.