CN105628631A - Rapid detection method of biological catalytic conversion rate of hydrocortisone - Google Patents
Rapid detection method of biological catalytic conversion rate of hydrocortisone Download PDFInfo
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Abstract
The invention relates to a rapid detection method of the biological catalytic conversion rate of hydrocortisone. Due to the fact that when a principal product hydrocortisone and a byproduct epihydrocortisone obtained through fermentation and a substrate RSA react with concentrated sulfuric acid, different colors are displayed, according to the Lambert-Beer law, three-dimensional data modeling is combined, and the conversion rate of the hydrocortisone in fermentation liquor can be indirectly calculated through visible-light dual-wavelength detection. The rapid detection method is easy and rapid to operate, short in testing time, small in sample dosage, easy to achieve, capable of achieving parallel operation and high in accuracy. A repeatability error is within 10%, the conversion rate, capable of being detected, of the hydrocortisone is within 10%-95%, the detection method is easy and rapid to operate, the testing time of a single sample is within 20 min, but the time needed in a thin-layer chromatography method and a high-performance liquid chromatography method is longer.
Description
Technical field
The invention belongs to the check analysis field of bioconversion pharmacy Chinese traditional medicine output, it relates to the method for quick of a kind of hydrocortisone Biocatalytic Conversion rate, it is applicable to bio-conversion process process research and the detection of medicine.
Background technology
Hydrocortisone is a kind of adrenal cortex hormones drug, is widely used clinically. It still produces the basic material of the efficient corticosteroid drugs of other classes such as prednisone simultaneously.
The production of HC is that 11 ��-hydroxylations through microorganism are obtained by reacting by substrate of Compound RS (17-monohydric pregnant-4-alkene-3,20-diketone) or compound RSA (17-monohydric pregnant-4-alkene-3,20-diketone-21-acetic ester). Microbial strains is very big on the transformation efficiency of HC and the impact of receipts rate, and therefore the excellent species of breeding high-yield hydrocortisone is industrial goal in research always.
The excellent species of industrial breeding high-yield hydrocortisone is generally adopt selection by mutation mode. In selection by mutation process, the frequency producing superior strain through mutagenesis is very low, therefore all needs detection target bacterial classification from huge strain library. The detection of hydrocortisone transformation efficiency is generally first extracted by the fermented liquid composition of shake flask fermentation with organic solvent, then by thin layer chromatography, the output of HC is done qualitative detection. Eventually through high performance liquid chromatography, it is determined that bacterial classification is to the transformation efficiency of hydrocortisone.
Membrane chromatographic method and liquid phase chromatography are the methods that the detection of current domestic most factories is all using. , there is sampling amount big in membrane chromatographic method, extraction agent easily volatilizees, and operates loaded down with trivial details, quantitatively inaccurate, is subject to the problems such as ambient interference; Separately having liquid chromatography for measuring result accurate, but proving time is excessively long, program is complicated, and instrument price is expensive, is all unsuitable for the requirement of production scene quick test. Therefore, these points can not meet modern measure far away, and the requirement of high flux screening high conversion bacterial classification.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art part, it is provided that the method for quick of the hydrocortisone Biocatalytic Conversion rate that a kind of precision and sensitivity are all high.
The present invention realizes the technical scheme of object:
A method for quick for hydrocortisone Biocatalytic Conversion rate, step is as follows:
(1) utilize main product thing hydrocortisone, by product table hydrocortisone, substrate 17-monohydric pregnant-4-alkene-3,20-diketone-21-acetic ester meets the characteristic of the aobvious different colours of strong sulfuric acid response, ethyl acetate is used to be extracted by composition in fermented liquid, add the vitriol oil, itself and sulfuric acid are reacted, causes color generation respective change;
(2) first the HC/ vitriol oil, the EHC/ vitriol oil, the RSA/ vitriol oil are carried out full wavelength scanner, obtain the HC/ vitriol oil, the maximum absorption peak of the RSA/ vitriol oil be respectively 474nm, 535nm;
(3) combine, according to langbobier law, the calculation formula that three-dimensional data modeling second order correction draws hydrocortisone transformation efficiency;
(4) the composition passing through to measure in fermented liquid and the strong sulfuric acid response OD value under visible ray dual wavelength, can calculate the transformation efficiency of hydrocortisone in fermented liquid to be measured.
And, concrete steps are as follows:
(1) the fermented liquid that strain fermentation to be measured produces is down to after below 3.8, regulates pH to be 6.5, drops into the RSA of 0.25%, continues to be cultured to 72h;
(2) fermented liquid step (1) obtained adds the ethyl acetate of equivalent, fully gets 6 �� L upper organic phase after extraction and be placed in 96 hole quartz enzyme plates;
Until step (2) in 96 holes quartz enzyme plates in ethyl acetate volatilize after, add the 200 �� L vitriol oils, reaction 15min after use microplate reader to detect it in the OD value at 474nm place and 535nm place;
(4), after the standard substance of hydrocortisone, table hydrocortisone, RSA, RS being added anhydrous alcohol solution, it is configured to the stock solution that concentration is 0.5mg/mL respectively;
(5) HC, RSA, the EHC (4) step obtained, according to HC differentiated yields, maintenance total mass is 0.025mg, RSA, HC, EHC standard solution adding different volumes proportioning is in 96 hole quartz enzyme plates, after absolute ethanol volatilizes, adding the vitriol oil of 200 �� L, reaction 15min, measures absorbancy at 474nm, 535nm wavelength place respectively;
(6) the OD value that (5) step records corresponding 474nm and the 535nm place of different HC transformation efficiency arranges, according to (A474nm-A535nm)/A474nmCalculate the numerical value K as X=0.60; Fit to 3 D stereo orthographic plan by singmapolt software again, and thus draw transformation efficiency X and A474nm��A535nmBetween funtcional relationship;
By step (5) in sample to be tested at 474nm, 535nm place OD value substitution formula K=(A474nm-A535nm)/A474nm, very according to the K value that calculates, by transformation efficiency between 0-0.6 and separating between 0.6-1, then transformation efficiency X and the A that (6) step obtains474nm��A535nmBetween funtcional relationship, the transformation efficiency of HC in fermented liquid to be measured can be calculated, and then detect out the content of HC in fermented liquid fast.
And, described step (1) in throw material RSA help molten with the industrial spirit of 80%.
And, described step (2) in the ethyl acetate vitriol oil of fermented liquid and equivalent fully to be extracted, then left at room temperature for some time.
And, (3) get the bid time of product and strong sulfuric acid response of described step accurately controls at 15min.
Advantage and the positively effect of the present invention be:
1, the present invention is simple and quick, and the test duration is short, sample dosage is little, it is easy to realize parallel work-flow, accuracy height. Reproducibility error is within 10%; Can the transformation efficiency of detection hydrocortisone between 10%-95%, detection method is simple and quick, and the test duration of single sample is within 20min, and thin layer chromatography and high performance liquid chromatography required time are longer.
2, the method that the present invention proposes can detect the transformation efficiency of HC in fermented liquid fast, improves bacterial screening efficiency greatly, is the efficient bacterial strain of high flux screening, it is to increase the transformation efficiency of steroid drugs hydrocortisone provides reliable guarantee.
3, detection method test samples consumption provided by the invention is little, it is easy to realize parallel work-flow; The accuracy height of detection method, almost not by environmental influence, reproducibility error is within 5%. Precision and sensitivity are all higher than thin layer chromatography.
4, detection method provided by the invention is applicable to the high flux screening of the bacterial strain of high yield hydrocortisone, overcome that operational difficulty in tradition screening excellent species method, screening operation amount be big and the shortcoming of inefficiency, substantially reduce the screening cycle, it is to increase working efficiency.
Accompanying drawing explanation
Fig. 1 is the three-dimensional Dual Wavelength Absorbance data plot that hydrocortisone transformation efficiency is less than 60%;
Fig. 2 is the three-dimensional Dual Wavelength Absorbance data plot that hydrocortisone transformation efficiency is greater than 60%;
Fig. 3 is the HPLC detected result of No. 1 bacterial strain fermentation liquor composition;
Fig. 4 is the HPLC detected result of No. 2 bacterial strain fermentation liquor compositions;
Fig. 5 is the HPLC detected result of No. 3 bacterial strain fermentation liquor compositions.
Embodiment
The inventive method is described below by specific embodiment. Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art. In addition, embodiment is interpreted as illustrating property, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by claim book. To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change or the changes that carry out the material component in these embodiments and consumption also belong to protection scope of the present invention.
The present invention discloses the method for quick of a kind of hydrocortisone Biocatalytic Conversion rate, utilize main product thing hydrocortisone (HC), by product table hydrocortisone (EHC), substrate 17-monohydric pregnant-4-alkene-3,20-diketone-21-acetic ester (RSA) meets the characteristic of the aobvious different colours of strong sulfuric acid response, ethyl acetate is used to be extracted by composition in fermented liquid, add the vitriol oil in the proper ratio, itself and sulfuric acid are reacted, causes color generation respective change. First the HC/ vitriol oil, the EHC/ vitriol oil, the RSA/ vitriol oil are carried out full wavelength scanner, obtain the HC/ vitriol oil, the maximum absorption peak of the RSA/ vitriol oil be respectively 474nm, 535nm; The calculation formula that three-dimensional data modeling second order correction draws HC transformation efficiency is combined again according to langbobier law.
Experimental result shows: adopt visible ray double UV check method, three-dimensional data modeling is combined according to langbobier law, by OD value under visible ray dual wavelength of the composition that measures in fermented liquid and strong sulfuric acid response, the transformation efficiency going out roughly HC in fermented liquid to be measured can be calculated.
The method for quick of the transformation efficiency of concrete hydrocortisone, it is characterised in that: described method comprises the following steps:
(1) bacterial strain inclined-plane sterilized water makes spore suspension, gets 1mL and is inoculated in the 250mL triangular flask filling 30mL seed culture medium, 28 DEG C, 180r/min shaking table shaking culture 20h.
By step (1) in the seed culture fluid that obtains, be inoculated in the 500mL triangular flask filling 50mL fermention medium, shaking table shaking culture.
Detecting step (2) in fermented liquid be down to after below 3.8, regulate pH be 6.5, drop into 0.25% RSA, continue to be cultured to 72h.
(4) fermented liquid step (3) obtained adds the ethyl acetate of equivalent. Fully get 6 �� L upper organic phase after extraction and it is placed in 96 hole quartz enzyme plates.
Until step (4) in 96 holes quartz enzyme plates in ethyl acetate volatilize after, add the 200 �� L vitriol oils, reaction 15min after use microplate reader to detect it in the OD value at 474nm place and 535nm place.
(6), after the standard substance of hydrocortisone, table hydrocortisone, RSA, RS being added anhydrous alcohol solution, it is configured to the stock solution that concentration is 0.5mg/mL respectively.
(7) HC, RSA, the EHC (6) step obtained, according to HC differentiated yields, maintenance total mass is 0.025mg, RSA, HC, EHC standard solution adding different volumes proportioning is in 96 hole quartz enzyme plates, after absolute ethanol volatilizes, adding the vitriol oil of 200 �� L, reaction 15min, measures absorbancy at 474nm, 535nm wavelength place respectively.
(8) the OD value that (7) step records corresponding 474nm and the 535nm place of different HC transformation efficiency arranges, according to (A474nm-A535nm)/A474nmCalculate the numerical value K as X=0.60; Fit to 3 D stereo orthographic plan by singmapolt software again, and thus draw transformation efficiency X and A474nm��A535nmBetween funtcional relationship.
By step (5) in sample to be tested at 474nm, 535nm place OD value substitution formula K=(A474nm-A535nm)/A474nm, very according to the K value that calculates, by transformation efficiency between 0-0.6 and separating between 0.6-1, then transformation efficiency X and the A that (8) step obtains474nm��A535nmBetween funtcional relationship, the transformation efficiency of HC in fermented liquid to be measured can be calculated, and then detect out the content of HC in fermented liquid fast.
The concentration of described step (1) miospore suspension controls at n/mL-1=3 �� 106To 3 �� 107Between.
(2) middle inoculum size is between 5%-10% for described step, and shaking table culture condition is 28 DEG C, 180r/min; Incubation time is about 72h.
Described step (3) in throw material RSA help molten with the industrial spirit of 80%.
Described step (4) in the ethyl acetate vitriol oil of fermented liquid and equivalent fully to be extracted, then left at room temperature for some time.
(5) get the bid time of product and strong sulfuric acid response of described step accurately controls at 15min.
Described step (6) in the configuration of stock solution operate according to the following steps:
Accurately take hydrocortisone standard substance 5mg, surely it is dissolved in 10mL volumetric flask after adding anhydrous alcohol solution, be configured to the stock solution that concentration is 0.5mg/mL, stored refrigerated at 4 DEG C. The configuration of RSA, RS, table hydrocortisone standard solution is the same.
Described step (7) in RSA, HC, EHC standard solution of different volumes proportioning, can carry out according to following proportioning (transformation efficiency that wherein the content per-cent of HC is HC):
Described step (8) according to K=(A474nm-A535nm)/A474nm, calculate the numerical value K as X=0.60, and respectively to transformation efficiency between 0-60%, and the data of transformation efficiency between 60%-95% carry out matching, draw two transformation efficiency X and A474nm��A535nmBetween funtcional relationship formula.
Described step (9) in the OD value of sample to be tested at 474nm, 535nm place is substituted into formula: K=(A474nm-A535nm)/A474nm; As K < K0, transformation efficiency, between 0-0.6, is selected the formula one of transformation efficiency between 0-60%, is calculated the transformation efficiency of HC; Work as K > or=K0, transformation efficiency, between 0.6-1, is selected the formula two of transformation efficiency between 60%-95%, is calculated the transformation efficiency of HC; And then measure the transformation efficiency of HC in fermented liquid fast.
Embodiment 1
(1) the fresh inclined-plane sterilized water getting No. 1, No. 2, No. 3 bacterial strain with different HC transformation efficiency makes spore suspension, get 1mL and it is inoculated in the 250mL triangular flask filling 30mL seed culture medium, 28 DEG C, 180r/min shaking table shaking culture 20h.
(2) the seed culture fluid that will obtain, is inoculated in the 500mL triangular flask filling 50mL fermention medium, shaking table shaking culture.
(3) when the pH of fermented liquid is down to after below 3.8, regulate pH to be 6.5, drop into the RSA of 0.25%, continue to be cultured to 72h.
(4) fermented liquid step (3) obtained adds the ethyl acetate of equivalent. Fully get 6 �� L upper organic phase after extraction and it is placed in 96 hole quartz enzyme plates. Each part of corresponding three Duplicate Samples of sample to be tested.
(5) accurately take hydrocortisone standard substance 5mg, surely it is dissolved in 10mL volumetric flask after adding anhydrous alcohol solution, be configured to the stock solution that concentration is 0.5mg/mL, stored refrigerated at 4 DEG C. The configuration of RSA, RS, table hydrocortisone standard solution is the same.
(6) according to HC differentiated yields, maintenance total mass is 0.025mg, and RSA, HC, EHC standard solution adding different volumes proportioning is in 96 hole quartz enzyme plates. The volume proportion of RSA, HC, EHC standard solution is in table 1. For the sample parallel three parts of same HC transformation efficiency.
The content per-cent of table 1RSA, HC, EHC standard substance
Until step (4) in ethyl acetate, step (6) in dehydrated alcohol volatilize completely after, add the vitriol oil of 200 �� L simultaneously, reaction 15min, measures absorbancy at 474nm, 535nm wavelength place respectively, and result is such as table 2.
The mixing mark product absorbance measurement data of table 2 different content per-cent
(8), by the OD value arrangement at corresponding 474nm and the 535nm place of HC transformation efficiency different in table 2, fit to 3 D stereo orthographic plan by singmapolt software, and thus draw transformation efficiency X and A474nm��A535nmBetween funtcional relationship. The model of fit of HC transformation efficiency between 0-0.6 is shown in Fig. 1, and the formula one obtained is: X=4.4686+0.9341*A474nm-19.1657*A535nm-0.1161*(A474nm)2+19.5822*(A535nm)2. The model of fit of HC transformation efficiency between 0.6-1 is shown in Fig. 2, and the formula two obtained is: X=0.2275+8.5673*A474nm-16.6402*A535nm+3.8789*(A474nm)2+15.831*(A535nm)2��
(9) according to (A474nm-A535nm)/A474nmCalculate the numerical value K as X=0.60=0.53126. The OD value of sample to be tested in table 2 at 474nm, 535nm place is substituted into formula K=(A474nm-A535nm)/A474nm, according to the K value calculated and K0Relatively, by transformation efficiency between 0-0.6 and separating between 0.6-1, the transformation efficiency of HC in fermented liquid to be measured can be calculated by formula one, formula two, result is such as table 3. And then detect out the transformation efficiency of HC in fermented liquid fast.
The measurement result of hydrocortisone transformation efficiency in table 3 visible ray double UV check method fermented liquid
By step (4) in the fermented liquid that obtains carry out the transformation efficiency of high performance liquid chromatography detection HC, chromatographic condition: adopt C18 post (4.6 �� 250mm, 5 ��m), moving phase is V (methyl alcohol): V (water)=70:30, flow 0.5mL/min, sample size 20 �� L, post temperature 25 DEG C; UV242nm detects. Detected result is such as Fig. 3, Fig. 4, table 4.
The transformation efficiency measurement result of table 4 high performance liquid chromatography and visible ray double UV check hydrocortisone
| Bacterial strain | The transformation efficiency of visible ray double UV check HC | HPLC detects HC transformation efficiency | Error % |
| No. 1 | 69.80% | 71.90% | 2.9% |
| No. 2 | 54.40% | 52.81% | 2.9% |
| No. 3 | 32.70% | 30.94% | 5.7% |
Result shows, and the method visible ray double UV check method set up can measure roughly the transformation efficiency of HC in fermented liquid. Compared with official method high performance liquid chromatography, measurement result is basically identical. This method is easy and simple to handle, saves the time, efficiency height, the quick test being applicable in fermented liquid hydrocortisone transformation efficiency, and providing for the bacterial strain of high flux screening high yield hydrocortisone is one detection method fast and effectively.
Claims (5)
1. the method for quick of a hydrocortisone Biocatalytic Conversion rate, it is characterised in that: step is as follows:
(1) utilize main product thing hydrocortisone, by product table hydrocortisone, substrate 17-monohydric pregnant-4-alkene-3,20-diketone-21-acetic ester meets the characteristic of the aobvious different colours of strong sulfuric acid response, ethyl acetate is used to be extracted by composition in fermented liquid, add the vitriol oil, itself and sulfuric acid are reacted, causes color generation respective change;
(2) first the HC/ vitriol oil, the EHC/ vitriol oil, the RSA/ vitriol oil are carried out full wavelength scanner, obtain the HC/ vitriol oil, the maximum absorption peak of the RSA/ vitriol oil be respectively 474nm, 535nm;
(3) combine, according to langbobier law, the calculation formula that three-dimensional data modeling second order correction draws hydrocortisone transformation efficiency;
(4) the composition passing through to measure in fermented liquid and the strong sulfuric acid response OD value under visible ray dual wavelength, can calculate the transformation efficiency of hydrocortisone in fermented liquid to be measured.
2. the method for quick of hydrocortisone Biocatalytic Conversion rate according to claim 1, it is characterised in that: concrete steps are as follows:
(1) the fermented liquid that strain fermentation to be measured produces is down to after below 3.8, regulates pH to be 6.5, drops into the RSA of 0.25%, continues to be cultured to 72h;
(2) fermented liquid step (1) obtained adds the ethyl acetate of equivalent, fully gets 6 �� L upper organic phase after extraction and be placed in 96 hole quartz enzyme plates;
Until step (2) in 96 holes quartz enzyme plates in ethyl acetate volatilize after, add the 200 �� L vitriol oils, reaction 15min after use microplate reader to detect it in the OD value at 474nm place and 535nm place;
(4), after the standard substance of hydrocortisone, table hydrocortisone, RSA, RS being added anhydrous alcohol solution, it is configured to the stock solution that concentration is 0.5mg/mL respectively;
(5) HC, RSA, the EHC (4) step obtained, according to HC differentiated yields, maintenance total mass is 0.025mg, RSA, HC, EHC standard solution adding different volumes proportioning is in 96 hole quartz enzyme plates, after absolute ethanol volatilizes, adding the vitriol oil of 200 �� L, reaction 15min, measures absorbancy at 474nm, 535nm wavelength place respectively;
(6) the OD value that (5) step records corresponding 474nm and the 535nm place of different HC transformation efficiency arranges, according to (A474nm-A535nm)/A474nmCalculate the numerical value K as X=0.60; Fit to 3 D stereo orthographic plan by singmapolt software again, and thus draw transformation efficiency X and A474nm��A535nmBetween funtcional relationship;
By step (5) in sample to be tested at 474nm, 535nm place OD value substitution formula K=(A474nm-A535nm) /A474nm, very according to the K value that calculates, by transformation efficiency between 0-0.6 and separating between 0.6-1, then transformation efficiency X and the A that (6) step obtains474nm��A535nmBetween funtcional relationship, the transformation efficiency of HC in fermented liquid to be measured can be calculated, and then detect out the content of HC in fermented liquid fast.
3. the method for quick of hydrocortisone Biocatalytic Conversion rate according to claim 2, it is characterised in that: described step (1) in throw material RSA help molten with the industrial spirit of 80%.
4. the method for quick of hydrocortisone Biocatalytic Conversion rate according to claim 2, it is characterised in that: described step (2) in the ethyl acetate vitriol oil of fermented liquid and equivalent fully to be extracted, then left at room temperature for some time.
5. the method for quick of hydrocortisone Biocatalytic Conversion rate according to claim 2, it is characterised in that: (3) get the bid time of product and strong sulfuric acid response of described step accurately controls at 15min.
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| CN108504622A (en) * | 2017-02-28 | 2018-09-07 | 齐鲁制药有限公司 | A kind of high-flux cell suspension culture method |
| CN114136907A (en) * | 2021-11-26 | 2022-03-04 | 桂林医学院 | Method for determining total flavonoids of abelmoschus manihot |
| CN114813603A (en) * | 2022-03-09 | 2022-07-29 | 南京林业大学 | Method for rapidly representing halogenation conversion rate of phenolic hydroxyl compounds without standard substance |
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