阅读说明：本技术 一种静息细胞多轮发酵制备氢化可的松的方法 (Method for preparing hydrocortisone by multiple rounds of fermentation of resting cells ) 是由 徐明琴 付林 冯蕾 廖俊 田玉林 曾建华 于 2020-12-22 设计创作，主要内容包括：本发明公开了一种静息细胞多轮发酵制备氢化可的松的方法。本方法以蓝色犁头霉为生产菌种,经种子扩大培养后,在发酵液中投入底物RSA,转化生成氢化可的松；转化结束后离心收集菌丝体,再以水为发酵介质投料底物RSA进行发酵转化,以此类推进行多轮静息细胞转化。特别说明的是在静息细胞多轮转化工艺中,转化介质-水只需做简单的煮沸处理,不需要像培养基灭菌那样高温高压,也不需要做任何PH等方面的调整,既节省了菌种培养步骤,又节约了培养基的制备灭菌所需的物料和动力,并且提高了氢化可的松的转化率和比生产率。(The invention discloses a method for preparing hydrocortisone by multiple rounds of fermentation of resting cells. The method comprises the steps of taking Absidia coerulea as a production strain, after seed amplification culture, adding a substrate RSA into fermentation liquor, and converting to generate hydrocortisone; and after the conversion is finished, centrifugally collecting mycelia, feeding a substrate RSA (rivest-Adleman) by taking water as a fermentation medium for fermentation conversion, and performing multiple rounds of resting cell conversion by analogy. Particularly, in the multiple rounds of transformation process of resting cells, the transformation medium-water only needs to be subjected to simple boiling treatment, does not need high temperature and high pressure like culture medium sterilization, does not need any adjustment in aspects such as pH and the like, saves the strain culture step, saves materials and power required by preparation and sterilization of the culture medium, and improves the transformation rate and specific productivity of hydrocortisone.)

1. A method for preparing hydrocortisone by multiple rounds of fermentation of resting cells is characterized by comprising the following steps:

(1) adding RSA into the Absidia coerulea zymocyte liquid for fermentation and transformation;

(2) separating mycelium after the fermentation and the conversion are finished, adding the separated mycelium into water, and adding RSA again for resting cell conversion;

(3) and (3) repeating the step (2) to perform multiple rounds of conversion of the resting cells until the biological enzyme of the mycelium is exhausted, and ending the fermentation.

2. The method for preparing hydrocortisone through multiple rounds of fermentation of resting cells as claimed in claim 1, wherein the amount of RSA added in step (1) is 2.5-2.8 g/L.

3. The method for preparing hydrocortisone by multiple rounds of fermentation of resting cells according to claim 1 or 2, wherein the fermentation transformation in step (1) is performed by: the transformation was carried out at 25-29 ℃ and 160-200rpm for 30-33 h.

4. The method for preparing hydrocortisone through multiple rounds of fermentation of resting cells according to claim 1 or 2, wherein the volume ratio of the water used in step (2) to the Absidia coerulea fermented liquid in step (1) is 0.75: 1-1.1: 1.

5. the method for preparing hydrocortisone through multiple rounds of fermentation of resting cells as claimed in claim 1 or 2, wherein the amount of RSA added in step (2) is 1.5-2.5 g/L; the volume ratio of the water used in the step (2) to the Absidia coerulea zymocyte liquid in the step (1) is 1: 1.

6. the method for preparing hydrocortisone by multiple rounds of fermentation of resting cells according to claim 5, wherein the resting cells in step (2) are transformed by the following method: the transformation was carried out at 25-29 ℃ and 160-200rpm for 18-24 h.

7. The method for preparing hydrocortisone through multiple rounds of fermentation of resting cells according to claim 1, wherein the Absidia coerulea fermented liquid in step (1) is prepared by the following method: firstly, inoculating the Absidia coerulea to a slant for slant strain culture, then inoculating the cultured strain to a liquid culture medium for culture, and when the liquid culture reaches the pH value of 3.8-4.4, the culture reaches the end point to obtain the Absidia coerulea zymocyte liquid.

8. The method for preparing hydrocortisone by multiple rounds of fermentation of resting cells according to claim 7, wherein the slant culture is performed by: the culture medium is a PDA culture medium, the culture temperature is 25-29 ℃, the culture period is 7-9 days, and the spores are mature; the temperature of the culture in the liquid culture medium is 25-29 ℃, and the rotation speed is 160-200 rpm; the liquid culture medium comprises the following components: 1.0-1.2% of glucose, 1.0-1.5% of corn steep liquor, 0.3-0.5% of yeast extract, 0.4-0.6% of ammonium sulfate and the balance of water; pH 6.1-6.5.

9. The method for preparing hydrocortisone through multiple rounds of fermentation of resting cells according to claim 1, wherein the multiple rounds of transformation in step (3) are 2-10 rounds of transformation; the water of step (2) is sterilized before use.

10. The method for preparing hydrocortisone through multiple rounds of fermentation of resting cells according to claim 9, wherein the feeding manner of RSA in step (1) and step (2) is as follows: refluxing, dissolving and putting in cosolvent ethanol; the sterilization treatment is boiling sterilization at 100 ℃.

Technical Field

The invention belongs to the field of microbial steroid pharmacy and medical engineering, and particularly relates to a method for preparing hydrocortisone by multiple rounds of fermentation of resting cells.

Background

Steroids have important physiological activities and are second only to antibiotics. Hydrocortisone (HC) is also called as cortisol with a chemical name of 11 beta, 17 alpha, 21-trihydroxy pregn-4-ene-3, 20-dione, belongs to an adrenocortical hormone drug, is a variety with the largest yield in the current hormone drugs, has the effects of anti-inflammation, anti-allergy and the like, and is mainly clinically applied to the replacement treatment of adrenocortical insufficiency. Meanwhile, because it has certain influence on sugar metabolism, it is also used for treating diseases such as glucose and hyperglycemia, and can be used as a precursor of a plurality of steroid corticoid drugs in production. Since the microbial synthesis of glucocorticoids in the 60's and its commercialization, microbial transformation has become a key technology in the synthetic route for many steroidal drugs or drug intermediates, and the production of hydrocortisone utilizes 11 β -hydroxylation by microorganisms. For the synthesis of steroidal glucocorticoids, 11 β -OH is an essential group for anti-inflammatory drugs, and existing higher corticoid drugs all have C11 β -hydroxy group. The sterides C10 site and C13 site methyl easily causes the steric hindrance of C11 beta-hydroxyl to be larger than that of C11 alpha-hydroxyl, and causes the C11 beta-hydroxylation efficiency to be lower than that of C11 alpha-hydroxylation and more byproducts, thereby restricting the large-scale application of the steroid C10 site and C13 site. To increase the conversion of the product, many researchers have conducted extensive research including first stage mutagenesis or fermentation process improvement and second stage substrate conversion.

At present, 11 beta-hydroxylation strains for producing HC by domestic microbial transformation are mainly Absidia coerulea, and the strains take a compound RSA (chemical name is 17 alpha-hydroxy-pregn-4-ene-3, 20-diketone-21-acetic ester) as a substrate, generate RS (chemical name is 17 alpha, 21-dihydroxy-pregn-4-ene-3, 20-diketone) by deacetylation (hydrolysis), and generate HC by the catalysis of a C11 beta-hydroxylase system, and the reaction formula is as follows:

however, the specificity of the Absidia coerulea 11 beta-hydroxylase is poor, a plurality of byproducts are generated, the feeding concentration is low, and the HC yield is limited. Therefore, it is necessary to develop and optimize the fermentation process, improve the yield and quality of the product, reduce the use of manpower, material resources, power and raw materials, and achieve the purposes of reducing the cost and improving the yield, which is significant for the industrial production of hydrocortisone.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the invention provides a method for preparing hydrocortisone by multiple rounds of fermentation of resting cells. The invention separates and collects the mycelium after the fermentation and transformation of the prior art, then the mycelium is put into medium water, the water is taken as the transformation medium, the substrate RSA is added again for fermentation and transformation, and the rest cell transformation is carried out by analogy of the steps. The method is simple to operate, low in cost and capable of improving the yield.

Compared with the original process route, the mycelium multi-turn conversion process of the invention comprises the following steps:

the original process comprises the following steps: slant strain preparation, fermentation strain culture, substrate feeding conversion, fermentation liquor separation and extraction (mycelium waste)

The process of the invention comprises the following steps: slant strain preparation → fermentation strain culture → substrate feeding conversion → fermentation broth separation and extraction → mycelium and equal volume water (n times utilization) → substrate feeding conversion → fermentation broth separation and extraction → mycelium enzyme depletion, and fermentation is finished.

The purpose of the invention is realized by the following technical scheme:

a method for preparing hydrocortisone by multiple rounds of fermentation of resting cells comprises the following steps:

(1) adding RSA into the Absidia coerulea zymocyte liquid for fermentation and transformation;

(2) separating mycelium after the fermentation and the conversion are finished, adding the separated mycelium into water, and adding RSA again for resting cell conversion;

(3) and (3) repeating the step (2) to perform multiple rounds of conversion of the resting cells until the biological enzyme of the mycelium is exhausted, and ending the fermentation.

Preferably, the Absidia coerulea zymocyte liquid in the step (1) is prepared by the following method: firstly, inoculating the Absidia coerulea to a slant for slant strain culture, then inoculating the cultured strain to a liquid culture medium for culture, and when the liquid culture reaches the pH value of 3.8-4.4, the culture reaches the end point to obtain the Absidia coerulea zymocyte liquid.

Preferably, the slant culture method comprises: the culture medium is a PDA culture medium, the culture temperature is 25-29 ℃, the culture period is 7-9 days, and the spores are mature at the moment.

Preferably, the temperature for culturing in the liquid culture medium is 25-29 ℃, and the rotation speed is 160-200 rpm.

Preferably, the composition of the liquid medium is: 1.0-1.2% of glucose, 1.0-1.5% of corn steep liquor, 0.3-0.5% of yeast extract, 0.4-0.6% of ammonium sulfate and the balance of water; pH 6.1-6.5.

Preferably, the dosage of the RSA in the step (1) is 2.5-2.8 g/L.

Preferably, the fermentation conversion mode in the step (1) is as follows: the transformation was carried out at 25-29 ℃ and 160-200rpm for 30-33 h.

Preferably, the volume ratio of the water used in the step (2) to the Absidia coerulea fermented liquid used in the step (1) is 0.75: 1-1.1: 1, more preferably 1: 1.

preferably, the addition amount of the RSA in the step (2) is 1.5-2.5 g/L.

Preferably, the resting cells in step (2) are transformed by the following method: the transformation was carried out at 25-29 ℃ and 160-200rpm for 18-24 h.

Preferably, the conversion in the step (3) is carried out for 2-10 times.

Preferably, the RSA feeding mode in the step (1) and the step (2) is as follows: reflux dissolving with cosolvent alcohol.

Preferably, the water of step (2) is sterilized before use.

Preferably, the sterilization treatment of the water is boiling sterilization at 100 ℃.

The invention takes the Absidia coerulea as a production strain, and the Absidia coerulea is subjected to seed amplification culture and first round conversion of the original process, then waste mycelia in the original process are collected, and then water is used as a fermentation medium to perform multiple rounds of resting cell fermentation conversion. In particular, in the multiple rounds of transformation of resting cells, the transformation medium-water is simply boiled, high temperature and high pressure are not needed as in the case of medium sterilization, and any adjustment in pH and the like is not needed. Because water is used as a fermentation medium, the thallus cells are in a hungry state, and the phenomenon of contamination of the thallus cells can not occur. Therefore, the strain culture step is saved, the materials and power required by preparation and sterilization of the culture medium are saved, the conversion rate and specific productivity of hydrocortisone are improved, and the yield of products is improved. In the transformation of resting cells, no exogenous nutrient exists, so that the method is favorable for extracting and refining subsequent products. In multiple rounds of conversion of resting cells, the proportion of the main by-product epihydrocortisone is equal to the fermentation liquor conversion proportion in the original process, and the increase of the by-products caused by the reutilization of mycelium biological enzyme is avoided. The production method related by the invention is to repeatedly utilize hydrocortisone fermentation mycelium (namely biological enzyme) for many times, has simple operation, saves cost, shortens period and improves yield.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention provides a multi-round fermentation and transformation process of mycelium resting cells of hydrocortisone, which has the advantages that: in the resting cell multi-round transformation process, a plurality of links such as preparation of a culture medium, sterilization, pH adjustment, strain culture and the like are reduced, so that the process operation is greatly simplified, the material power is saved, and the production cost is reduced; the resting cell transformation process takes water as a transformation medium, so that the existence of nutrient substances is reduced, and the problem of bacterial contamination is not required to be considered; the resting cell transformation period is greatly shortened, and the production efficiency of the product is improved; the resting cells are transformed without exogenous nutrient substances, an emulsion layer is not easy to generate in the subsequent extraction process, and the extraction operation is more convenient. The production method related by the invention is to repeatedly utilize hydrocortisone fermentation mycelium (i.e. biological enzyme) for many times, and carry out multiple times of feeding and conversion, so that the bacterial biological enzyme can be fully utilized. The process has simple operation, low cost, short period and high yield.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the examples, the net conversion rate and the specific production rate of hydrocortisone were calculated by the following methods:

net conversion: the HC mass concentration sum/feeding concentration sum in n conversion rounds is%

Specific production rate: total HC concentration in n conversion rounds/total fermentation time (g/L/h)

Example 1

A method for preparing hydrocortisone by multiple rounds of fermentation of resting cells comprises the following steps:

preparing strains: inoculating the Absidia coerulea to a PDA solid slant culture medium under an aseptic condition, controlling the culture temperature to be 28 +/-0.5 ℃, and culturing for 7-9 days until gray spore silks and spores filled in a test tube grow out.

Fermentation culture: and (3) eluting the spores of the strains by using a certain amount of sterile water under the aseptic condition, inoculating the spores to a shake flask culture medium, culturing at the temperature of 28 +/-0.5 ℃ and the rotation speed of 200r/min until the pH value is 4.0, and culturing for 24h for a period to obtain the Absidia coerulea zymogen liquid.

The shake flask culture medium (by percentage) is: glucose 1.2%, corn steep liquor 1%, yeast extract 0.4%, ammonium sulfate 0.6%, and water in balance, wherein the pH is 6.3, and the mixture is sterilized at 121 ℃ for 30 minutes.

Substrate feeding conversion: when the liquid is cultured until the pH value is 4.0, adding the RSA which is dissolved in a backflow mode in advance, wherein the adding amount of the RSA is 2.8g/L, performing fermentation conversion, wherein the conversion period is 31h, and the fermentation liquid conversion is determined as the first conversion, namely the original fermentation process. The conversion rate of hydrocortisone is as follows: 1.35g/L/2.8 g/L48.2%; the specific productivity of hydrocortisone is 1.35g/L/(24+31) h which is 0.0245 g/L/h.

Resting cells were transformed in multiple rounds: and after the conversion is finished, centrifugally separating mycelium, adding the mycelium into a prepared shake flask, filling water with the same volume as the fermentation liquor into the shake flask, adding the RSA dissolved in a backflow mode, wherein the adding amount of the RSA is 1.5g/L, performing resting cell conversion, repeating the step, performing multiple rounds of conversion on resting cells, wherein the conversion period of each round is 20 hours, and performing 9 rounds of co-conversion. The net conversion rate of hydrocortisone is 9.007g/L/(2.8+1.5 x 9) g/L-55.2%; the specific productivity of hydrocortisone is 9.007g/L/(24+31+180) h-0.03833 g/L/h.

Example 2

A method for preparing hydrocortisone by multiple rounds of fermentation of resting cells comprises the following steps:

(1) preparing strains: inoculating the Absidia coerulea to a PDA solid slant culture medium under an aseptic condition, controlling the culture temperature to be 28 +/-0.5 ℃, and culturing for 7-9 days until gray spore silks and spores filled in a test tube grow out.

(2) Fermentation culture: then, the spores of the strains are eluted by a certain amount of sterile water under the aseptic condition, inoculated into a shake flask culture medium, cultured under the conditions that the temperature is 28 +/-0.5 ℃ and the rotating speed is 200r/min until the pH value is 4.0, and cultured for 24 hours to prepare the Absidia coerulea zymogen liquid.

The shake flask culture medium (by percentage) is: glucose 1.2%, corn steep liquor 1%, yeast extract 0.4%, ammonium sulfate 0.6%, and water in balance, wherein the pH value is 6.2, and the mixture is sterilized at 121 ℃ for 30 minutes.

(3) Substrate feeding conversion: when the liquid is cultured until the pH value is 4.0, adding the RSA which is dissolved in a backflow mode in advance, wherein the adding amount of the RSA is 2.5g/L, performing fermentation conversion, wherein the conversion period is 31h, and the fermentation liquid conversion is determined as the first conversion, namely the original fermentation process. The net conversion rate of hydrocortisone is 1.23g/L/2.5 g/L-49.2% and the specific productivity of hydrocortisone is 1.23g/L/(24+31) h-0.022364 g/L/h.

(4) Resting cells were transformed in multiple rounds: and after the conversion is finished, centrifugally separating mycelium, adding the mycelium into a prepared shake flask, filling water with the same volume as the fermentation liquor into the shake flask, adding the RSA dissolved in a backflow mode, wherein the adding amount of the RSA is 2.0g/L, performing resting cell conversion, repeating the step, performing multiple rounds of conversion on resting cells, wherein the conversion period of each round is 20 hours, and performing 9 rounds of co-conversion. The conversion rate of hydrocortisone is as follows: 10.961g/L/(2.5+2.0 × 9) g/L53.5%; the specific productivity of hydrocortisone is as follows: 10.961g/L/(24+31+20 × 9) h is 0.04664 g/L/h.

Example 3

A method for preparing hydrocortisone by multiple rounds of fermentation of resting cells comprises the following steps:

(1) preparing strains: inoculating the Absidia coerulea to a PDA solid slant culture medium under an aseptic condition, controlling the culture temperature to be 28 +/-0.5 ℃, and culturing for 7-9 days until gray spore silks and spores filled in a test tube grow out.

(2) Fermentation culture: then, the spores of the strains are eluted by a certain amount of sterile water under the aseptic condition, inoculated into a shake flask culture medium, cultured under the conditions that the temperature is 28 +/-0.5 ℃ and the rotating speed is 200r/min until the pH value is 4.0, and cultured for 24 hours to prepare the Absidia coerulea zymogen liquid.

The shake flask culture medium (by percentage) is: glucose 1.2%, corn steep liquor 1%, yeast extract 0.4%, ammonium sulfate 0.6%, and water in balance, wherein the pH is 6.4, and the mixture is sterilized at 121 ℃ for 30 minutes.

(3) Substrate feeding conversion: when the liquid is cultured until the pH value is 4.0, adding the RSA which is dissolved in a backflow mode in advance, wherein the adding amount of the RSA is 2.8g/L, performing fermentation conversion, wherein the conversion period is 31h, and the fermentation liquid conversion is determined as the first conversion, namely the original fermentation process. The conversion rate of hydrocortisone is as follows: the specific productivity of 47.9% hydrocortisone is 1.34g/L/2.8 g/L: 1.34g/L/(24+31) h is 0.02436 g/L/h.

(4) Resting cells were transformed in multiple rounds: and after the transformation is finished, centrifugally separating mycelium, adding the mycelium into a prepared shake flask, filling water with the same volume as the fermentation liquor into the shake flask, adding the RSA dissolved in a backflow mode, wherein the adding amount of the RSA is 2.0-2.5g/L, carrying out resting cell transformation, repeating the step, carrying out multiple rounds of transformation on the resting cells, wherein the transformation period is 20 hours per round, and carrying out 6 rounds of co-transformation. The conversion rate of hydrocortisone is as follows: 8.91g/L/(2.8+2.0+2.5 × 5) g/L51.5%, specific hydrocortisone production rate: 8.91g/L/(24+31+20 × 6) h 0.0509 g/L/h.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.