CN1353193A - Enzyme method for preparing dextral glycoanhydride - Google Patents
Enzyme method for preparing dextral glycoanhydride Download PDFInfo
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- CN1353193A CN1353193A CN 00130268 CN00130268A CN1353193A CN 1353193 A CN1353193 A CN 1353193A CN 00130268 CN00130268 CN 00130268 CN 00130268 A CN00130268 A CN 00130268A CN 1353193 A CN1353193 A CN 1353193A
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Abstract
An enzyme method for preparing dextran includes such steps as culturing enzyme-generating bacteria, growing and reproducing the said bacteria, and producing dextran sucrase; culturing high-yield and high-quality bacteria by the gene mutation techinque, preparing special culture medium to increase the yield of dextran sucrase by one time and over, after centrifugal processing for removing bacteria, adding enzyme liquid to cane sugar buffering liquid at 5.2-5.3 of pH value, intermittently stirring at 230-26 deg.C for 18-24 hr, and then deposition in alcohol. Its advantages include controllable fermentation procedure, low cost and output increased by over 20%.
Description
The present invention relates to other polyoses and the production method of a kind of polyose and derivative thereof, relate in particular to a kind of dextran and derivative thereof and production method.
Dextran (claiming dextran again) is a kind of nontoxic biopolymer material.Because it has special physiological function and chemically reactive, therefore, can derive a series of important novel products again.Just because of this, it has been widely used in every field such as industry, agricultural, medicine and scientific research.Become one of biotechnology class important in the mankind nowadays life.
No matter the production of dextran in decades domestic, external, is still followed traditional fermentation using bacteria process basically.This process is uncontrollable in itself in fact.Because in this course, from bacterial growth, arrive and produce enzyme (dextransucrase), and all same bio-reactor, carry out, and each required optimum condition of step is far from it from the enzymic synthesis dextran.The output that causes therefrom and the unstable of quality can't overcome.
Just because of this, scientists is devoted to the research of the synthetic dextran of enzyme process always.But this is an engineering that wastes time and energy, and it is easier said than done especially more to be applied to suitability for industrialized production.
The objective of the invention is to: develop a kind of advanced method of easy, quick, High-efficient Production dextransucrase, and set up the industrial process of the synthetic dextran of enzyme process, to replace traditional uncontrollable fermentation using bacteria process.
The present invention is achieved in that
1, the new production method of dextransucrase
The seed selection of A, product enzyme bacterium
The bacterium (the bright string of goldbeater's skin shape coccus) that will be in the growth animated period is centrifugal, after the precipitation, washing, with Guanidinium nitrate solution (concentration is 100 mcg/ml) processing 90 minutes, supernatant liquor was removed in centrifugation then, washed three times with physiological saline again.With these microbionations of handling on slant medium.Through repeated screening.Therefrom choose the high yield and high quality bacterial strain.
B, bacterial growth and breeding
With the high-quality microbionation on the above-mentioned slant medium in 500 milliliters of liquid substratum.This substratum composed as follows:
Sucrose 2.0% yeast extract paste 0.5% dipotassium hydrogen phosphate 0.5% soya bean extract 0.3%
Inorganic salt liquid 0.5% adding distil water is to 100%PH7.3-7.6
Described inorganic salt liquid composition is sulfur acid magnesium 4 grams in per 100 milliliters, each 0.2 gram of ferrous sulfate, manganous sulfate and sodium-chlor.Liquid nutrient medium is necessary autoclave sterilization before inoculation, and inoculation is placed on 25 ℃ of water-soluble middle cultivations 8 to 12 hours of constant temperature, promptly can 10 multiple proportions examples continue to be inoculated in the new substratum, and amplifies step by step.
The production of C, dextran sucrase
At first be a kind of special culture medium that is suitable for the Production by Bacteria enzyme of preparation, it is composed as follows:
Sucrose 3.0% yeast extract paste 1.0% soya bean extract 0.3% dipotassium hydrogen phosphate 1.5%
Inorganic salt liquid 1.0% adding distil water is to 100%PH7.3-7.8
Described inorganic salt liquid consists of: in per 100 milliliters, and sal epsom 2 grams, calcium chloride 0.5 gram, each 0.1 gram of other ferrous sulfate, manganous sulfate and sodium-chlor.
Autoclave sterilization behind the above-mentioned substratum mixing, be cooled to 23-25 ℃ standby.
During production, under aseptic condition, in culture volume 10% ratio, the bacterium liquid of above-mentioned growth animated period is inoculated wherein, holding temperature is about 23-25 ℃, and the gap stirring, about 24 hours, measures PH and enzymic activity.When PH drops to the 4.5-5.0 enzymic activity and reaches the peak, get final product stopped reaction, blowing is centrifugal.Centrifugal speed is generally 3000-5000 rev/min, about 15 minutes of time.After centrifugal precipitation is discarded, supernatant liquor is collected in the container of cleaning sterile, and after adding a cover, it is standby to deposit in 4 ℃ of cold houses, and measures enzymic activity.In 4 ℃ of environment, enzymic activity generally can be stablized for 3 months.
2, the enzyme process route of synthesis of dextran
Get the enzyme liquid that aforesaid method is produced, calculate, be added in the sucrose damping fluid by every milliliter 10 unit.For example sucrose damping fluid volume is 1000 milliliters, then need add 10 * 1000=10, the enzyme liquid of 000 unit.Suppose that again enzymic activity is 100 units per ml, then in fact should add 100 milliliters of enzyme liquid.
Being prepared as follows of sucrose damping fluid:
Sucrose 15%
Calcium ions acetate solution 10%
Adding distil water to 100%
And regulate PH to 5.2-5.3 with 30% acetic acid.
Consisting of of calcium ions acetate buffer contains sodium-acetate 6.8 grams, calcium chloride 0.5 gram in per 1000 milliliters.
After enzyme liquid is added on the sucrose damping fluid, stir immediately, and regulate PH between 5.2-5.3, holding temperature is at 24-26 ℃ and be interrupted stirring.When treating that viscosity reaches peak value (generally at 18-24 hour), promptly available equal-volume 95% alcohol precipitation.Throw out is dextran (claiming thick acid anhydride again).
The present invention is compared with the prior art the advantage that has:
(1) advanced technology
Enzyme process is synthetic, dextran production is jumped by traditional noncontrol system one and becomes brand-new may command process.Changed backward state in decades, the forward position in the field, the world of marching toward.
(2) increase output
Facts have proved, this technology, enzyme-added by sugar, fixed supply, and also in last bio-reactor, composition is simple, does not disturb, thereby guarantees high yield, stable yields.Increase by 20% approximately with the traditional zymotic mean yield of comparing.
(3) production cost reduces
Because it is above that this technology can make the output of dextransucrase double, add in the sucrose damping fluid of producing dextran to add other nutritive substances that this just reduces production costs widely.
(4) improve the quality of products
Because this new technology bacterium is separated, so the quality of product is than the outstanding improvement of traditional zymotic Faxian.
Realize the best form of invention
Embodiment 1
Laboratory scale production
In the sterilising liq substratum with slant medium microbionation to 500 milliliter.It consists of sucrose 10 grams, yeast extract paste 2.5 grams, dipotassium hydrogen phosphate 2.5 grams, soya bean extract 1.5 grams, 2.5 milliliters of inorganic salt liquids, adding distil water make molten, volume is added to 500 milliliters again, PH7.2-7.7 (inorganic salt consist of in 100 milliliters each 0.2 gram of sulfur acid magnesium 4 grams, ferrous sulfate, manganous sulfate and sodium-chlor).
Postvaccinal liquid nutrient medium is placed 23-25 ℃ the water-soluble cultivation of constant temperature about 8-10 hour, when treating bacterial growth vigorous (PH is 4.8 to 5.2), promptly can be transferred in the nutrition base that produces enzyme.Its process is as follows:
Prepare nutritional medium earlier, method is to take by weighing sucrose 15 grams, yeast extract paste 5 grams, and soya bean extract 1.5 grams, dipotassium hydrogen phosphate 7.5 grams, 5 milliliters of inorganic salt liquids, adding distil water makes molten, adds to 500 ml volumes again, and autoclave sterilization, is chilled to 23-25 ℃ then.(inorganic salt consist of sulfur acid magnesium 2 grams in per 100 milliliters, calcium chloride 0.5 gram, each 0.1 gram of other ferrous sulfate, manganous sulfate and sodium-chlor).
Secondly 50 milliliters of aforesaid liquid substratum bacterium liquid are shifted in the product enzyme nutritional medium that is inoculated into 500 milliliters, be positioned over behind the mixing 23-25 ℃ of constant temperature water-soluble in, about 24 hours, when treating that PH reduces to 4.5-5.0, can take out, centrifugal (3000-5000 rev/min) 15 minutes carefully draws supernatant liquor, measure enzymic activity, it is standby to put 4 ℃ of refrigerators.
Above-mentioned what make is dextransucrase (thick enzyme), introduces how to utilize this kind of enzyme below, synthetic dextran in the laboratory.At first prepare the sucrose damping fluid.Method is to take by weighing sucrose 150 gram, adds 800 ml distilled waters and makes moltenly, adds sodium-acetate 0.68 gram then, and calcium chloride 0.05 gram is with 30% acetic acid adjusting PH to 5.2-5.3.Add 10,000 unit dextransucrase liquid then, and adding distil water to cubic capacity is 1000 milliliters, re-adjustment PH to 5.2-5.3.Keep solution temperature at 24-26 ℃, and be interrupted and stir, after 24 hours, add 1000 milliliter of 95% concentration alcohol precipitation, can obtain dextran.
Embodiment 2
Small Scale Industry is produced
Method is identical with embodiment 1, and just bacterial growth breeding will carrying out secondary amplifies, and every grade all with 10 times of increases, promptly expands 10 liters to so that 100 liters by 500 milliliters * 2.But the composition of substratum and embodiment 1 are just the same.Produce the enzyme nutritional medium and be extended to 1000 liters.Enlarge though produce the dextran volume by enzyme, the composition of sucrose damping fluid and synthetic condition are all same with embodiment 1.Now that its Production Flow Chart diagram is as follows:
Slant medium → 500 milliliters of liquid substratum → 10 liter seeding tanks → 100 liters of seeding tank → 1000 liter product enzyme jar → tubular type continuous centrifuge → 20,000 liter dextrans synthesize.
Claims (2)
1, a kind of enzyme method for preparing dextral glycoanhydride is characterized in that: may further comprise the steps:
1) production method of dextransucrase:
The seed selection of A, product enzyme bacterium:
To be in the growth animated period the centrifugation of the bright string of bacterium goldbeater's skin shape coccus bacterium, wash after, with concentration is the Guanidinium nitrate solution-treated 90 minutes of 100 mcg/ml, centrifugation then, remove supernatant liquor, again with physiological saline washing three times, these microbionations of handling on slant medium, through repeated screening, are therefrom chosen the high yield and high quality bacterial strain;
B, bacterial growth and breeding:
With the high-quality microbionation on the above-mentioned slant medium in 500 milliliters of liquid substratum, the consisting of of this substratum:
Sucrose 2.0% yeast extract paste 0.5% dipotassium hydrogen phosphate 0.5%
Soya bean extract 0.3% inorganic salt liquid 0.5%
Adding distil water is to 100%PH7.3-7.6
Described inorganic salt liquid composition is sulfur acid magnesium 4 grams in 100 milliliters, each 0.2 gram of ferrous sulfate, manganous sulfate and sodium-chlor, liquid nutrient medium is essential autoclave sterilization before inoculation, inoculation is placed in 25 ℃ of water-baths of constant temperature and cultivated 8 to 12 hours, promptly can 10 multiple proportions examples continue to be seeded in the new substratum, and amplify step by step;
The production of C, dextransucrase:
At first be the special culture medium of a kind of suitable Production by Bacteria enzyme of preparation, it is composed as follows:
Sucrose 3.0% yeast extract paste 1.0% soya bean extract 0.3% dipotassium hydrogen phosphate 1.5%
Inorganic salt liquid 1.0%, adding distil water is to 100%PH7.3-7.8
Described inorganic salt liquid consists of: sal epsom 2 grams in per 100 milliliters, and calcium chloride 0.5 gram, each 0.1 gram of other ferrous sulfate, manganous sulfate and sodium-chlor,
High pressure detoxicating behind the above-mentioned substratum mixing, be cooled to 23-25 ℃ standby;
During production, under aseptic condition,, the bacterium liquid of above-mentioned growth animated period is inoculated wherein in culture volume 10% ratio, holding temperature is about 23-25 ℃, and the gap stirring, about 24 hours, measure PH and enzymic activity, when PH drops to the 4.5-5.0 enzymic activity and reaches the peak, get final product stopped reaction, blowing is centrifugal, and centrifugal speed is generally 3000-5000 rev/min, about 15 minutes of time, after centrifugal precipitation is discarded, supernatant liquor is collected in the container of cleaning sterile, after adding a cover, it is standby to leave 4 ℃ of cold houses in, and the mensuration enzymic activity, in 4 ℃ of environment, enzymic activity generally can be stablized for 3 months.
2) the enzyme process route of synthesis of dextran:
Get the enzyme liquid that aforesaid method is produced, calculate, be added in the sucrose damping fluid by every milliliter of 10 unit.
2, enzyme method for preparing dextral glycoanhydride according to claim 1 is characterized in that: described sugarcane
Being prepared as follows of sugar damping fluid:
Sucrose 15% calcium ions acetate solution 10% adding distil water to 100%, and regulate PH5.2-5.3 with 30% acetic acid, consisting of of described calcium ions acetate buffer contains sodium-acetate 6.8 grams in per 1000 milliliters, calcium chloride 0.5 gram after enzyme liquid is added in the sucrose damping fluid, stirs immediately, and regulate PH between 5.2-5.3, holding temperature is at 24-26 ℃ and be interrupted to stir, available equal-volume 95% alcohol precipitation when treating that viscosity reaches peak value, and throw out is dextran.
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| CN 00130268 CN1353193A (en) | 2000-11-02 | 2000-11-02 | Enzyme method for preparing dextral glycoanhydride |
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| CN 00130268 CN1353193A (en) | 2000-11-02 | 2000-11-02 | Enzyme method for preparing dextral glycoanhydride |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100363373C (en) * | 2005-01-14 | 2008-01-23 | 庄茅 | Preparation method of isomalto loigosaccharide sulphate (IMOS) |
| CN101363009B (en) * | 2007-10-17 | 2011-04-06 | 合肥工业大学 | Expression of dextran sucrase genetic engineering bacteria, construction method and use thereof |
| CN102392063A (en) * | 2011-10-24 | 2012-03-28 | 邹平旭阳棉制品有限公司 | Production process of biological sewage treating agent dextran 1000 |
| CN102796783A (en) * | 2012-08-23 | 2012-11-28 | 江南大学 | Bioprocessing method of functional glucose having polymerization degree of 3-8 |
| CN103243052A (en) * | 2013-05-22 | 2013-08-14 | 广西大学 | Breeding method for excellent strain capable of producing dextranum sucrase |
| CN107074985A (en) * | 2014-11-05 | 2017-08-18 | 纳幕尔杜邦公司 | The gelation dextran of enzymatic polymerization |
-
2000
- 2000-11-02 CN CN 00130268 patent/CN1353193A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100363373C (en) * | 2005-01-14 | 2008-01-23 | 庄茅 | Preparation method of isomalto loigosaccharide sulphate (IMOS) |
| CN101363009B (en) * | 2007-10-17 | 2011-04-06 | 合肥工业大学 | Expression of dextran sucrase genetic engineering bacteria, construction method and use thereof |
| CN102392063A (en) * | 2011-10-24 | 2012-03-28 | 邹平旭阳棉制品有限公司 | Production process of biological sewage treating agent dextran 1000 |
| CN102392063B (en) * | 2011-10-24 | 2013-07-31 | 山东旭阳生物科技有限公司 | Production process of biological sewage treating agent dextran 1000 |
| CN102796783A (en) * | 2012-08-23 | 2012-11-28 | 江南大学 | Bioprocessing method of functional glucose having polymerization degree of 3-8 |
| CN102796783B (en) * | 2012-08-23 | 2015-02-11 | 江南大学 | Bioprocessing method of functional glucose having polymerization degree of 3-8 |
| CN103243052A (en) * | 2013-05-22 | 2013-08-14 | 广西大学 | Breeding method for excellent strain capable of producing dextranum sucrase |
| CN107074985A (en) * | 2014-11-05 | 2017-08-18 | 纳幕尔杜邦公司 | The gelation dextran of enzymatic polymerization |
| CN107074985B (en) * | 2014-11-05 | 2020-07-24 | 纳幕尔杜邦公司 | Enzymatically polymerized gelled dextran |
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