CN103805577A - Method for efficiently producing hydroxysteroid dehydrogenase with testosterone comamonas - Google Patents
Method for efficiently producing hydroxysteroid dehydrogenase with testosterone comamonas Download PDFInfo
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Abstract
The invention relates to a method for efficiently producing hydroxysteroid dehydrogenase with testosterone comamonas. The method comprises steps of bacteria culture, seeding tank culture, fermentation tank culture and the like, and can produce hydroxysteroid dehydrogenase efficiently. The hydroxysteroid dehydrogenase has remarkable effects on crops in preventing disease, promoting growth and increasing yield. Furthermore, if being used as a novel fodder enzyme preparation in cultivation in stock farming, the hydroxysteroid dehydrogenase can effectively promote pigs and broiler chickens to grow, improves the laying rate of laying hens, reduces disease of beasts and birds, and has obvious effect in promoting growth. According to the method, viable bacteria in per milliliter of fermentation liquor is improved to be 30 billion from 0.1 billion, the enzyme activity in per gram of dry powder is improved to be 300 thousand from 1 thousand, so that the product cost is greatly lowered, and the method can be used for industrial production.
Description
Technical field
The present invention relates to a kind of method that adopts Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase.
Background technology
Any plant individual, all histocyte and its inside and outside enzyme by plant self, the molectron of microorganism, that is to say that enzyme and microorganism itself are exactly the part in plant, wherein enzyme plays very important effect in vital movement, the various biochemical reactions of enzyme catalysis at normal temperatures and pressures, produce Multiple Classes of Antibiotics, somatropin, activating soil discharges a large amount of trace elements, can increase backbone root and the capillary radical amount of plant, widen the transport pipe of nutrient, allow the reasonable Balance Absorption nutriment of crop, promote plant growth, precocious, increase income, strengthen resistance, disease resistance, fighting drought etc.If there is no these enzymes, crop just can not grow.
China is a large agricultural country, the annual financial loss being caused by diseases and pests of agronomic crop is huge, farm crop continuous cropping disease is also one of disease the most serious in agriculture production, for prevention and elimination of disease and pests causes the abuse of chemical pesticide, the sick worm of result is more and more stronger to the resistance of chemical pesticide, the amount of application of agricultural chemicals is increasing, concentration is more and more higher, disease is refractory more and more, add organic fertilizer and use minimizing year by year, chemical fertilizer excessively uses, cause soil compaction, chemicals, hormone, the objectionable impurities severe overweights such as nitrite, ecotope has also suffered very big destruction, environmental pollution is also more and more serious, not only person poultry poisoning's event occurs again and again, and cause the resistance against diseases of farm crop greatly to decline, cause crop failure, the quality of agricultural-food is subject to very big impact, seriously undermine the competitive power of agricultural products in China in world market, along with agricultural products in China export volume increases day by day, improving constantly of living standards of the people, no matter be domestic or world market, agricultural-food are produced to matter output to be required more and more stricter, green agriculture production is had higher requirement.
Soil is made up of mineral substance, organic matter and microorganism three parts, it is the basis of crop growthing development, enzyme in soil and microorganism are participated in matter and energy in soil directly and transform, adopt artificial mode to increasing useful enzyme and microorganism, quantity in soil, just can strengthen the enzymic activity in soil, thereby increase soil fertility.
Soil is more serious from malicious phenomenon at present, just refer at the residual body of preceding crop and rotting can produce some toxin in decomposition course from malicious phenomenon, as: aldehyde, alcohol, hydro carbons, also have drug residue and the steroid hormone etc. of accumulation, second stubble crop is had to significant restraining effect, affect self growing of crop, add the amount reproduction of pathogenic soil microorganism, quantity rises year by year, a little less than seedling resistivity, be easy to catch an illness, nutritive element wane or unbalance, soil nutrient elements presents physiological imbalance, directly cause Crop nutrient balance imbalance, crop root rots, root system is undeveloped, affect the normal physiological metabolism of crop, causing crop to drop in production over a large area even has no harvest.
According to above existing technical problem, the present invention, through research for many years, successfully develops a kind of novel agricultural zymin, hydroxysteroid dehydrogenase, and according to the results show, this enzyme has significant diseases prevention, growth-promoting, volume increase function to crop.In addition, enzyme is a kind of biological catalyst, in nature, all life phenomenon all has relation with the activity of enzyme, and the application of enzyme product in aquaculture entering Rapid development stage, becomes gradually the main direction of livestock breeding industry new technology revolution and the key that benefit promotes.Along with the sustained and rapid development of China's economic, the continuous expansion of Derived from Agricultural Modernization, will certainly increase the market requirement of enzyme product greatly.The present invention catches this opportunity, bring into play the research advantage of self, be applied to the cultivation aspect of livestock industry using hydroxysteroid dehydrogenase as a kind of novel fodder enzyme preparation, through multiple batches of evidence, this enzyme can effectively promote the growth of pig, broiler chicken, improve laying rate of laying hen, reduce livestock and poultry, growth-promoting effect is remarkable.
Summary of the invention
For overcoming above-mentioned technical problem, the invention provides a kind of method that adopts Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase, it comprises following steps:
Preparation LB substratum: by 10 grams of peptones, 5 grams of yeast soak powder and 5 grams of sodium-chlor are put in 1000 ml waters regardless of front and back order, pH value nature, after stirring and dissolving, be sub-packed in multiple diameter 25mm, in the test tube of length 200mm, in each test tube, fill 5 milliliters, described multiple test tubes are put in medical disinfecting, adopt 0.12-0.14 MPa sterilizing 20 minutes;
Inoculation: open freeze-drying pipe Comamonas testosteroni kind under aseptic condition, then add 1 milliliter of the LB substratum that above sterilizing is good to described freeze-drying pipe Comamonas testosteroni kind the inside, it is for subsequent use that shake makes freeze-drying pipe bacteria suspension after dissolving, then get 5 milliliters of the substratum of the bacterium of having gone out above, under aseptic condition, open test tube, and add 0.3 milliliter of described freeze-drying pipe bacteria suspension in test tube, again by test tube good seal, put shake in bottle swingging machine and make expansion strain liquid;
First order seed is cultivated:
In first order seed culture medium prescription, the weight percent of each component is:
Yeast soaks powder 0.15%-0.25% peptone 0.25%-0.45% sodium-chlor 0.1%-0.3%
Plant sterol 0.01%-0.15% Vitamin D3 500,000 I.U/GM 0.01%-0.15% ammonium sulfate 0.25%-0.5%
Surplus is water ph value nature
In water by the each raw material in described first order seed culture medium prescription regardless of adding of front and back order of described first order seed culture medium prescription, start stirrer stirring and make first order seed nutrient solution; Described first order seed nutrient solution is measured to institute's consumption to be joined in triangular flask, sterilizing after sealing, put in super quiet worktable and lower the temperature being equipped with through the triangular flask of the nutrient solution of sterilizing, in the time that temperature drops to 27-31 ℃, in described cultured expansion strain liquid being received to triangular flask under aseptic condition, go, the expansion strain liquid of 100 milliliters of first order seed nutrient solutions access is 2 milliliters, after put in bottle swingging machine, make first class inoculum liquid;
The pipeline of fermentation equipment, sterile air filtering system are carried out to sterilising treatment, and seeding tank and fermentor tank are carried out to slack tank sterilising treatment;
Seed tank culture:
In seed tank culture based formulas, the weight percent of each component is:
Yeast soaks powder 0.1%-0.4% peptone 0.2%-0.5% sodium-chlor 0.15%-0.3%
Plant sterol 0.1%-0.3% Vitamin D3 500,000 I.U/GM 0.02%-0.2% ammonium sulfate 0.2%-0.6%
Surplus is water ph value nature
In water by the each raw material in described seed tank culture based formulas regardless of adding of front and back order of described seed tank culture based formulas, after high-temperature sterilization and cooling processing, described cultured first class inoculum liquid is inoculated in the seeding tank of sterilising treatment under aseptic condition, the amount that 100 kilograms of seed tank culture bases access described first class inoculum liquid is 1 kilogram, then build the inoculation cap on tank, make seeding tank strain liquid by aseptic aerlbic culture;
Fermentor cultivation:
In fermentor cultivation based formulas, the weight percent of each component is:
Yeast soaks powder 0.1%-0.3% peptone 0.15%-1.5% sodium-chlor 0.1%-0.5%
Plant sterol 0.1%-0.4% Vitamin D3 500,000 I.U/GM 0.01%-0.1% ammonium sulfate 0.2%-0.7%
Vitamin-E 0.01%-0.15% surplus is water ph value nature
First the water in described fermentor cultivation based formulas is put in the fermentor tank of sterilising treatment, then start stirrer, to in fermentor tank regardless of the each raw material in adding of front and back order described fermentor cultivation based formulas, after high-temperature sterilization and cooling processing, access bacterial classification, starts fermentation culture;
The extraction of hydroxysteroid dehydrogenase:
First adopt tubular-bowl centrifuge, turn/per minute of 8000-16000, controls 1000 kilograms per hour of flow, abandons supernatant, collects wet thallus; Then by the wet thallus of collection by 1 kilogram of wet thallus 3-10 kilogram that adds water, stir and make bacteria suspension, adding gross weight is the N,O-Diacetylmuramidase of 0.01-0.1% and the helicase of 0.02-0.2% again, under the condition of 4 ℃-35 ℃, stir 1 hour, then put into-40 ℃ freezing, freeze in fact until freeze, after taking-up, dissolve ice, and then put into-40 ℃ and freeze, so repeatedly can reach the best effect of hydroxysteroid dehydrogenase described in extraction for 3-6 time.
the function that this enzyme possesses and characteristic
1, this enzyme is the composition of an enzyme system, organic composition in soil can be transformed and resolves into organic matter, decompose nitrogen compound, the materials such as the protein in organic matter, fat, nucleic acid can be decomposed into again to amino acid that plant very easily absorbs, lipid acid, polypeptide etc., improve the ability of Crop nutrient, Promoting plant growth.
2, promote to do the synthetic of object endoenzyme, manufacture nutrient by the growth needs of crop, effectively suppress pathogenic bacteria, rhizospheric microflora of soil is restored balance, thereby reach inhibition harmful bacteria, diseases prevention, healthy tree, the effect of volume increase.
3, improve the activity of making plurality of enzymes in object, make crop metabolism vigorous, sugar, dry-matter accumulation increases, and strengthens crop disease-resistant ability, significantly improves crop yield and quality.
4, impel beneficial microorganism to breed at crop root, thereby activating soil, make Soil Nutrient Transformation, discharge the effective constituent that increases soil, improve utilization rate of fertilizer and soil fertility, keep soil from packing together, improve rhizospheric environment, decomposing pesticide is residual, and phenol, hormone, hydro carbons suppress the generation of the diseases such as pythium spp, sickle-like bacteria, nematode.
5, can promote fast crop rhizosphere cell fission, Sheng Xingen, increases root amount, improves improving activity of root system, forms strong root system, and has significantly and take root, and supports root, protects root effect, effectively solves rotten, takes root slowly, makes plant growth stalwartness.
6, hydroxysteroid dehydrogenase system can promote crop propagation catalase and chitinase; catalase is a kind of protective enzyme; plant disease-resistant is play a part positive; can make the susceptible rear entirety of crop obtain disease resistance; chitinase can decompose the chitin in fungi, bacteria cell wall, fungi, bacteria cell wall is divided and dead, and chitin also can activate crop cell; start crop immunity system, strengthen greatly the crop defensive ability/resistance ability of poor environment to external world.
7, stimulate organic releasing nutrients, abundant organic matter is by after enzyme activity, can constantly discharge the needed nutritive element of plant, reaches the object that fertilizer efficiency is lasting, and the fertilizer conservation of loosening the soil, improves environment, reaches fertility and waits lastingly unusual effect.
technical advance:
One, Comamonas testosteroni is human body, animal body, the bacterium existing in soil, (genus intracellular enzyme) hydroxysteroid dehydrogenase, there is the ability of the many hard degradation compounds of degraded, we study discovery for many years, the enzyme system that this bacterium produces, can powerfully decompose moving, plant protein, steroid hormone, hydro carbons, phenol, the objectionable impuritiess such as nonyl phenol, we find that in experiment hydroxysteroid dehydrogenase can significantly improve soil and microorganism structure, promote crop growth, it is a kind of desirable resistance successive crop, anti-soil compaction, soil aeration is good, the novel enzyme preparation of volume increase.
Two, hydroxysteroid dehydrogenase has very high using value in agricultural, but the digestion degraded of the enzyme in this bacterium to pollutent and corresponding substrate, transform, speed is very slow, limit this bacterium (enzyme) in soil biological restoration, if it is very limited to depend merely on this bacterium breeding amount of occurring in nature, therefore produced enzyme rareness, be difficult to play a role, therefore develop hydroxysteroid dehydrogenase and there is long-range economic benefit and social benefit.
Three, to really make hydroxysteroid dehydrogenase performance unusual effect, first will be to the substratum of fermented bacterium, culture condition, fermention medium, a series of technology such as the extraction of enzyme are furtherd investigate, otherwise can not tell on, because this bacterium is wild type strain, the enzyme producing belongs to cold-adapted enzyme system in born of the same parents, expect effectively, valuable, enzyme cheaply, first to see the thalline of living in fermented liquid is how many, utilize after former wild strain fermentation, in every milliliter of fermentation, only have 100,000,000 viable bacterias, the present invention is through continual effort for many years, thousand culture experiment, viable bacteria body in fermented liquid is brought up to 30,000,000,000 by original 100,000,000 every milliliter, 300 times are improved, we have selected nearly 100 kinds of these enzyme liberating substrates, last repeated screening goes out three kinds of substrates, and to induce this enzyme be that enzyme significantly improves comprehensively, rising to 300,000 enzymes of every gram of present dry powder by 1000 enzyme work in every gram of original dry powder lives, greatly reduce production cost, can enter suitability for industrialized production, this is our research from various substratum, fermentation pH value, sterilization time, raw material ratio, inoculum size, fermentation time, fermentor tank rotating speed, ventilation (because being aerobic bacteria) etc., carry out all-round exploration, the achievement of research.
Four, produce commercially available bacterium number: the ATCC11996 that obtains of bacterial classification that this enzyme uses
Five, produce the equipment using: fermentor tank, tubular-bowl centrifuge, pulverizer, premixer, vacuum drying oven, vacuum freeze drier, biochemical cultivation case, shaking table, Bechtop, air dry oven, mediclave
accompanying drawing explanation
Nothing.
Embodiment
A kind of method that adopts Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase of the present invention, it comprises following steps:
spawn culture:
Buy the enlarged culturing of bacterial classification, first configure 10 grams of LB substratum, peptones, yeast soaks 5 grams, powder, 5 grams, sodium-chlor, 1000 milliliters, water, pH value nature
Operation: the raw materials such as above peptone are put into and are sub-packed in multiple diameter 25mm in the water in formula after stirring and dissolving regardless of front and back order, in the test tube of length 200mm, every pipe fills 5 milliliters, puts in medical disinfecting 0.12-0.14 MPa sterilizing 20 minutes, and medium sterilization is complete
Inoculation: open the freeze-drying pipe bacterial classification of buying under aseptic condition, then add 1 milliliter of the substratum that above sterilizing is good to the inside, shake is dissolved rear for subsequent use, this is called freeze-drying pipe bacteria suspension, then get 5 milliliters of the substratum of the bacterium of having gone out above, under aseptic condition, open test tube, then to adding 0.3 milliliter of the freeze-drying pipe bacteria suspension that dissolved in test tube, again by test tube good seal, put in bottle swingging machine 27-31 ℃, 150-210 rev/min, 8-40 hour, this claims to expand strain liquid
one, first order seed is cultivated:
Formula: the weight percent of the each component of substratum
Yeast soaks powder 0.15%-0.25% peptone 0.25%-0.45% sodium-chlor 0.1%-0.3%
Plant sterol 0.01%-0.15% Vitamin D3 500,000 I.U/GM 0.01%-0.15% ammonium sulfate 0.25%-0.5%
Surplus is water ph value nature
Operation:
After above-mentioned raw materials is weighed up, regardless of the pouring in the water in formula of front and back, start stirrer and stir 20 minutes, this is called first order seed nutrient solution
The first order seed nutrient solution being stirred is measured to institute's consumption to be joined in triangular flask, then the 8 layers of additional one deck kraft paper of gauze tying, put into medical pressure kettle sterilizing, before sterilizing, first open the vent valve on sterilizer, close when having a small amount of steam to discharge, in the time that sterilizer pressure reaches 0.05 MPa, open drain tap exhaust 6-8 minute, then close drain tap, continue to heat, in the time that reaching 0.12-0.14 MPa, vapor pressure in sterilizer starts timing, sterilization time 20 minutes, after allow it naturally lower the temperature, in the time that the tensimeter on sterilizer makes zero, open sterilizer, take out the substratum of sterilizing, the triangular flask that substratum is housed is put in super quiet worktable and lowered the temperature, in the time that temperature drops to 27-31 ℃, in above-mentioned cultured expansion strain liquid being received to triangular flask under aseptic condition, go, inoculum size is 2% to be advisable, be 100 milliliters of first order seed nutrient solutions, expanding strain liquid is 2 milliliters, after put temperature 27-31 ℃ in bottle swingging machine, turn/per minute of 150-210, 6-20 hour, this is called first class inoculum liquid.
the sterilizing of the pipeline of fermentation equipment and sterile air filtering system
Operation: open valve and the sterile air pipeline valve of each inlet and outlet piping, pass into the steam of 0.12-0.14 MPa, allow steam and pipeline valve UNICOM have steam discharge, ventilate 10 minutes, then close each drain tap, allow the interior vapor pressure of pipeline keep 0.12-0.14 MPa, keep 40 minutes.
the slack tank sterilizing of seeding tank and fermentor tank
Close each valve, open the sewage draining valve of tank interlayer, open open steam valve to the steam that passes into 0.12-0.14 MPa in tank, in the time reaching 121 ℃ ± 1 ℃ in tank, start timing, sterilization time is 40 minutes, then close pot bottom wash water valve, in tank, temperature drops between 25 ℃-35 ℃ for subsequent use.
seed tank culture:
Formula: the weight percent of the each component of substratum
Yeast soaks powder 0.1%-0.4% peptone 0.2%-0.5% sodium-chlor 0.15%-0.3%
Plant sterol 0.1%-0.3% Vitamin D3 500,000 I.U/GM 0.02%-0.2% ammonium sulfate 0.2%-0.6%
Surplus is water ph value nature
Concrete operations:
First the water using in formula is put in sterilized seeding tank, then start stirrer, add respectively raw material required in formula (addition sequence is regardless of front and back), pass into steam heating, while utilizing jacket steam to be heated to 95 ℃, use again open steam heating instead, in the time that vapor pressure in tank reaches 0.12-0.14 MPa, be incubated and within 35 minutes, reach sterilising effect, then steam off, start cooling, for subsequent use in the time that temperature in tank drops to 27-31 ℃, this is called seed tank culture.Above cultured first class inoculum liquid is inoculated in seeding tank under aseptic condition, inoculum size is 1% of the interior substratum of tank, 100 kilograms of seed tank culture bases access 1 kilogram of first class inoculum liquid, then build the inoculation cap on tank, start aseptic aerlbic culture, sterile air consumption is 0.05 cube of every 100 kilograms of seed culture medium per minutes sterile air, pH value nature, turn/per minute of stirring velocity 140, tank pressure 0.05 MPa, 30 ℃ of temperature, incubation time 10 hours, this is called seeding tank strain liquid
fermentor cultivation
Formula: the weight percent of the each component of substratum
Yeast soaks powder 0.1%-0.3% peptone 0.15%-1.5% sodium-chlor 0.1%-0.5%
Plant sterol 0.1%-0.4% Vitamin D3 500,000 I.U/GM 0.01%-0.1% ammonium sulfate 0.2%-0.7%
Vitamin-E 0.01%-0.15% surplus is water ph value nature
Concrete operations:
First the water of required use in formula is put in sterilized fermentor tank, then start stirrer, in fermentor tank, drop into the various materials (addition sequence is regardless of front and back) in formula, then pass into steam heating, after utilizing jacket steam to be heated to 95 ℃, use again open steam heating instead, in the time that vapor pressure in tank reaches 0.12-0.14 MPa, be incubated and can reach sterilising effect in 25 minutes, then start cooling, in the time that temperature in tank is down to 30 ℃, start to access bacterial classification, when inoculation, first improve the pressure tank of seeding tank, reach 0.1 MPa, fermentor tank pressure maintains 0.05 MPa, then open the valve of inoculation pipeline, bacterium liquid good seed tank culture is transported in fermentor tank and is gone, then close inoculation pipeline valve and start fermentation culture, culture condition: temperature 27-31 ℃, turn/per minute of 80-140, the every 100 kilograms of nutrient solution per minutes of ventilation pass into sterile air, front 5 hours 0.07 cubic metre, 5 hours is 0.1 cubic metre to fermenting complete, pH value nature, fermentation time is to finish for 16-24 hour.
Centrifugal collection thalline:
Adopt tubular-bowl centrifuge, turn/per minute of 8000-16000, controls 1000 kilograms per hour of flow, abandons supernatant, collects wet thallus (mud)
The extraction of hydroxysteroid dehydrogenase:
The wet thallus (mud) of collecting is added water to 5 kilograms by 1 kilogram of wet thallus, stir and make bacteria suspension, then add the helicase of gross weight 0.01% N,O-Diacetylmuramidase and 0.02%, under the condition of 4 ℃-35 ℃, stir 1 hour, then put into-40 ℃ freezing, freeze in fact until freeze, after taking-up, dissolve ice, and then put into-40 ℃ freeze, so repeatedly can reach the best effect of extraction 3-6 time.
The lyophilize of hydroxysteroid dehydrogenase
To the trehalose that adds 3-10% in the enzyme liquid of broken wall, 8-20% skim-milk, the glycerol of 3-6%, the vitamins C of 1.5-5%, the maltodextrin (addition sequence is regardless of front and back) of 3-10%, under the condition of turn at 80-160/per minute, stir 1 hour, then divide and install to pre-freeze in lyophilize dish, pre-freeze temperature-40 ℃, 5 hours time, till freeze over freezes in fact, then vacuumize dry, below vacuum tightness 15 handkerchiefs, temperature-65 ℃, freeze-drying time generally 40-50 time between, moisture content 3%-5%, then pulverize with Lowtemperaturepulverizer, fineness 60-80 order, then lyophozyme powder plastic bag sealing collection being crushed is good, putting into 4 ℃ of refrigerators preserves, matching while using.
Embodiment recited above is described the preferred embodiment of the present invention; not the spirit and scope of the present invention are limited; do not departing under the prerequisite of design concept of the present invention; various modification and improvement that in this area, common engineering technical personnel make technical scheme of the present invention; all should fall into protection scope of the present invention; the technology contents of request protection of the present invention, has all been documented in claims.
Claims (12)
1. adopt a method for Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase, it is characterized in that comprising following steps:
Preparation LB substratum: by 10 grams of peptones, 5 grams of yeast soak powder and 5 grams of sodium-chlor are put in 1000 ml waters regardless of front and back order, pH value nature, after stirring and dissolving, be sub-packed in multiple diameter 25mm, in the test tube of length 200mm, in each test tube, fill 5 milliliters, described multiple test tubes are put in medical disinfecting, adopt 0.12-0.14 MPa sterilizing 20 minutes;
Inoculation: open freeze-drying pipe Comamonas testosteroni kind under aseptic condition, then add 1 milliliter of the LB substratum of the above bacterium of having gone out to described freeze-drying pipe Comamonas testosteroni kind the inside, it is for subsequent use that shake makes freeze-drying pipe bacteria suspension after dissolving, then get 5 milliliters of the substratum of the bacterium of having gone out above, under aseptic condition, open test tube, and add 0.3 milliliter of described freeze-drying pipe bacteria suspension in test tube, again by test tube good seal, put shake in bottle swingging machine and make expansion strain liquid;
First order seed is cultivated:
In first order seed culture medium prescription, the weight percent of each component is:
Yeast soaks powder 0.15%-0.25% peptone 0.25%-0.45% sodium-chlor 0.1%-0.3%
Plant sterol 0.01%-0.15% Vitamin D3 500,000 I.U/GM 0.01%-0.15% ammonium sulfate 0.25%-0.5%
Surplus is water ph value nature
In water by the each raw material in described first order seed culture medium prescription regardless of adding of front and back order of described first order seed culture medium prescription, start stirrer stirring and make first order seed nutrient solution; Described first order seed nutrient solution is measured to institute's consumption to be joined in triangular flask, sterilizing after sealing, put in super quiet worktable and lower the temperature being equipped with through the triangular flask of the nutrient solution of sterilizing, in the time that temperature drops to 27-31 ℃, in described cultured expansion strain liquid being received to triangular flask under aseptic condition, go, the expansion strain liquid of 100 milliliters of first order seed nutrient solutions access is 2 milliliters, after put in bottle swingging machine, make first class inoculum liquid;
The pipeline of fermentation equipment, sterile air filtering system are carried out to sterilising treatment, and seeding tank and fermentor tank are carried out to slack tank sterilising treatment;
Seed tank culture:
In seed tank culture based formulas, the weight percent of each component is:
Yeast soaks powder 0.1%-0.4% peptone 0.2%-0.5% sodium-chlor 0.15%-0.3%
Plant sterol 0.1%-0.3% Vitamin D3 500,000 I.U/GM 0.02%-0.2% ammonium sulfate 0.2%-0.6%
Surplus is water ph value nature
In water by the each raw material in described seed tank culture based formulas regardless of adding of front and back order of described seed tank culture based formulas, after high-temperature sterilization and cooling processing, described cultured first class inoculum liquid is inoculated in the seeding tank of sterilising treatment under aseptic condition, the amount that 100 kilograms of seed tank culture bases access described first class inoculum liquid is 1 kilogram, then build the inoculation cap on tank, make seeding tank strain liquid by aseptic aerlbic culture;
Fermentor cultivation:
In fermentor cultivation based formulas, the weight percent of each component is:
Yeast soaks powder 0.1%-0.3% peptone 0.15%-1.5% sodium-chlor 0.1%-0.5%
Plant sterol 0.1%-0.4% Vitamin D3 500,000 I.U/GM 0.01%-0.1% ammonium sulfate 0.2%-0.7%
Vitamin-E 0.01%-0.15% surplus is water ph value nature
First the water in described fermentor cultivation based formulas is put in the fermentor tank of sterilising treatment, then start stirrer, to in fermentor tank regardless of the each raw material in adding of front and back order described fermentor cultivation based formulas, after high-temperature sterilization and cooling processing, access bacterial classification, starts fermentation culture;
The extraction of hydroxysteroid dehydrogenase:
First adopt tubular-bowl centrifuge, turn/per minute of 8000-16000, controls 1000 kilograms per hour of flow, abandons supernatant, collects wet thallus; Then by the wet thallus of collection by 1 kilogram of wet thallus 3-10 kilogram that adds water, stir and make bacteria suspension, adding gross weight is the N,O-Diacetylmuramidase of 0.01-0.1% and the helicase of 0.02-0.2% again, under the condition of 4 ℃-35 ℃, stir 1 hour, then put into-40 ℃ freezing, freeze in fact until freeze, after taking-up, dissolve ice, and then put into-40 ℃ and freeze, so repeatedly can reach the best effect of hydroxysteroid dehydrogenase described in extraction for 3-6 time.
2. the method for employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as claimed in claim 1, wherein said expansion strain liquid is at the temperature of 27-31 ℃, adopt the rotating speed of 150-210 rev/min, in bottle swingging machine, shake and make for 8-40 hour.
3. the method for employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as claimed in claim 1, the sterilizing of wherein said first order seed nutrient solution, that described first order seed nutrient solution is put into medical pressure kettle sterilizing, before sterilizing, first open the vent valve on sterilizer, close when having a small amount of steam to discharge, in the time that sterilizer pressure reaches 0.05 MPa, open drain tap exhaust 6-8 minute, then close drain tap, continue to heat, in the time that reaching 0.12-0.14 MPa, vapor pressure in sterilizer starts timing, sterilization time 20 minutes, after allow it naturally lower the temperature, in the time that the tensimeter on sterilizer makes zero, open sterilizer, take out the nutrient solution after sterilizing.
4. the method for employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as claimed in claim 1, wherein said first class inoculum liquid is at the temperature of 27-31 ℃, adopt under the rotating speed of turn at 150-210/per minute of bottle swingging machine, shake and make for 6-20 hour.
5. the method for employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as claimed in claim 1, the sterilizing of the pipeline of wherein said fermentation equipment and sterile air filtering system is to pass through: valve and the sterile air pipeline valve of opening each inlet and outlet piping, pass into the steam of 0.12-0.14 MPa, allow steam and pipeline valve UNICOM and have steam discharge, ventilate 10 minutes, then close each drain tap, allow the interior vapor pressure of pipeline keep 0.12-0.14 MPa, keep 40 minutes.
6. the method for employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as claimed in claim 1, the slack tank sterilizing of wherein said seeding tank and fermentor tank is to pass through: close each valve, open the sewage draining valve of tank interlayer, open open steam valve to the steam that passes into 0.12-0.14 MPa in tank, in the time reaching 121 ℃ ± 1 ℃ in tank, start timing, sterilization time is 40 minutes, then closes pot bottom wash water valve, and in tank, temperature drops between 25 ℃-35 ℃ for subsequent use.
7. the method for employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as claimed in claim 1, high-temperature sterilization in wherein said seed tank culture program and cooling are processed, be: pass into steam heating, while utilizing jacket steam to be heated to 95 ℃, then use open steam heating instead, in the time that vapor pressure in tank reaches 0.12-0.14 MPa, be incubated and within 35 minutes, reach sterilising effect, then steam off, starts cooling, for subsequent use in the time that temperature in tank drops to 30 ℃.
8. the method for employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as claimed in claim 1, in the wherein said process that makes seeding tank strain liquid by aseptic aerlbic culture, sterile air consumption is 0.05 cube of every 100 kilograms of seed culture medium per minutes sterile air, pH value nature, turn/per minute of stirring velocity 80-180, tank pressure 0.05 MPa, temperature 27-31 ℃, incubation time 6-20 hour.
9. the method for employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as claimed in claim 1, high-temperature sterilization in wherein said fermentor cultivation step and cooling are processed and are: pass into steam heating, after utilizing jacket steam to be heated to 95 ℃, use again open steam heating instead, in the time that vapor pressure in tank reaches 0.12-0.14 MPa, be incubated and can reach sterilising effect in 25 minutes, then start cooling, in the time that temperature in tank is down to 27-31 ℃.
10. the method for employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as claimed in claim 1, in wherein said fermentor cultivation step, the process of described access bacterial classification is: the pressure tank that first improves seeding tank when inoculation, reach 0.1 MPa, fermentor tank pressure maintains 0.05 MPa, then the valve of opening inoculation pipeline, is transported to bacterium liquid good seed tank culture in fermentor tank and goes, and then closes inoculation pipeline valve.
The method of 11. employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenases as claimed in claim 1, the culture condition of wherein said fermentation culture is: temperature 27-31 ℃, turn/per minute of 80-140, the every 100 kilograms of nutrient solution per minutes of ventilation pass into sterile air, front 5 hours 0.07 cubic metre, 5 hours is 0.1 cubic metre to fermenting complete, pH value nature, fermentation time is to finish for 16-24 hour.
The method of the employing Comamonas testosteroni High-efficient Production hydroxysteroid dehydrogenase as described in 12. claims as arbitrary in claim 1-11, wherein the freeze-drying method of hydroxysteroid dehydrogenase is, in the hydroxy steroid desaminase liquid of broken wall, be the trehalose of 3-10% regardless of the weight ratio that adds of front and back order, the skim-milk of 8-20%, the glycerol of 3-6%, the vitamins C of 1.5-5% and the maltodextrin of 3-10%, under the condition of turn at 80-160/per minute, stir 1 hour, then divide and install to pre-freeze in lyophilize dish, pre-freeze temperature-40 ℃, 5 hours time, till freeze over freezes in fact, then vacuumize dry, below vacuum tightness 15 handkerchiefs, temperature-65 ℃, freeze-drying time is generally between 40-50 hours, moisture content 3%-5%, then pulverize with Lowtemperaturepulverizer, fineness 60-80 order, then lyophozyme powder plastic bag sealing collection being crushed is good, putting into 4 ℃ of refrigerators preserves, matching while using.
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Application publication date: 20140521 |