CN1263153A - Method for preparing amino acylase by means of immobilization of Aspergillus oryzae and equipment for resolution and suspension of amino acid - Google Patents
Method for preparing amino acylase by means of immobilization of Aspergillus oryzae and equipment for resolution and suspension of amino acid Download PDFInfo
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- CN1263153A CN1263153A CN 99120925 CN99120925A CN1263153A CN 1263153 A CN1263153 A CN 1263153A CN 99120925 CN99120925 CN 99120925 CN 99120925 A CN99120925 A CN 99120925A CN 1263153 A CN1263153 A CN 1263153A
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Abstract
The method for preparing aminoacylation enzyme by immobilization of A spergillus oryzae and resolution equipment of amino acid belong to the field of optical active amino acid preparation technology by means of enzyme activity. The described immobilization process uses Aspergillus oryzae as enzyme source, utilizes the culture solution using soybean juice or potato juice as main component to culture and makes immobilization in gelatin-lthylenediamine-glutaraldehyde three-component system immobilizing fluid to prepare the invented product. Its enzyme activity retention rate is up to 50-80%. The described resolution equipment adopts enzyme reactor whose inlet and outlet are respectivly series-connected with ultrafilt ration membrane separation reactor, its resolution efficiency is high and operation is simple.
Description
The present invention relates to a kind of aspergillus oryzae immobilization and prepare the method for L-Aminoacylase and split the amino acid whose device of DL, it belongs to enzyme process and prepares photolytic activity amino acid technology.
Photolytic activity amino acid is the basal component that constitutes protein of life, is and the healthy closely-related basic substance of human life.Research and development preparation photolytic activity amino acid is significant.Produce the amino acid whose method of photolytic activity at present and have three kinds substantially: fermentation method, chemical synthesis and enzyme process.The reaction specificity is strong because enzyme process has, the product purity height of preparation, becomes present some the rare photolytic activity amino acid of production of generally acknowledging in the world and the best approach of alpha-non-natural amino acid.Adopt the amino acid whose reaction expression formula of L-Aminoacylase production L-as follows:
Adopt aspergillus oryzae to extract the existing many reports of technology of L-Aminoacylase, cultivate but majority is the employing solid phase, broken apart then extraction L-Aminoacylase, the refabrication carrier is fixed enzyme on it.The method of this immobilized enzyme, its weak point is: operational path is long, and the utilization ratio of enzyme is low.
In addition, at present being used for fixing enzyme split amino acid whose device most owing to enzyme contaminated or enzyme reason such as come off, cause the enzyme reactor cycle of operation short, operation operation complexity is unfavorable for the reduction of production cost.
Purpose of the present invention is to provide a kind of immobilization of aspergillus oryzae to prepare the method for L-Aminoacylase and split the amino acid whose device of DL.The method that immobilization proposed by the invention prepares L-Aminoacylase has that technology is simple, enzyme is lived the high characteristics of retention rate.The detachment device that is proposed has efficient height, simple to operate, the characteristics that the production cycle is long of splitting.
For achieving the above object, the present invention is realized by following technical proposals.Select for use aspergillus oryzae (as 3042 or No.9) as the enzyme source,, make concentration with sterilized water and be about 10 through after cultivating on the agar slant
8The bacteria suspension of spore/ml, then with this bacterial suspension inoculation in liquid phase medium, it is the aspergillus oryzae ball of 1-3 millimeter that the stirring fermentation culture becomes diameter, with the aspergillus oryzae ball with after nutrient solution separates, be placed on immediately and carry out crosslinked fixing in the liquid phase stationary liquid, can get the bacterial enzyme 200-1400U/g of being alive, the enzyme retention rate of living reaches the immobilized aminoacylase of 50-80%, and the composition weight volume ratio of nutrient solution is: soya bean juice 8-15%, glucose 1.5-4.5%, yeast extract paste 0.2-0.5%, KH2PO4 0.3-0.4%, MgSO4 0.05-0.2%, all the other are sterilized water; Operation control culture condition is: temperature 28-30 ℃, pH value 6-7.5, fermentation time 30-46 hour; The composition weight volume ratio of stationary liquid is: gelatin 0.8-1.2%, quadrol 1.8-3.0%, glutaraldehyde 0.8-1.5%, all the other are sterilized water; The control fixing condition is: temperature 20-60 ℃, reaction times 0.5-2 hour, pH value 4-7, thalline and stationary liquid volume ratio are 1: 10.
Above-mentioned nutrient solution or adopt following composition weight volume ratio to be: murphy juice 15-25%, yeast extract paste 0.15-0.5%, sucrose 1.5-2.5%, KH2PO4 0.2-0.4%, MgSO4 0.1-0.3%, all the other are sterilized water.
Adopt the immobilized aminoacylase catalyzer of preparation as stated above; be contained in the enzyme reactor; the device that resolution reaction adopted that carries out the amino acid raceme mainly comprises feed liquid handling machinery, feed liquid basin, enzyme reactor, product storage tank, is 40,000-70,000 ultra-filtration membrane separate reactor at the import and export series coupled molecular weight cut off of enzyme reactor.
The membrane module that above-mentioned membrane separation reactor adopts is a hollow-fibre membrane.
The membrane module that above-mentioned membrane separation reactor adopts is a flat sheet membrane.
Below in conjunction with drawings and Examples split process of the present invention is illustrated.
Fig. 1 splits the amino acid whose reaction process synoptic diagram of DL for the present invention adopts immobilized aminoacylase.
By shown in Figure 1; after the reaction substrate that desire is split is mixed with certain density solution; be placed in the feed liquid basin 2; by the nitrogen that discharges in the nitrogengas cylinder 1 with substrate solution force feed in the basin 2 to preposition membrane separation apparatus 3; solution after the separation feeds the enzyme reactor 4 that immobilized aminoacylase is housed from the bottom to top and carries out resolution reaction; the resolution reaction liquid that is overflowed by the top flows into rearmounted membrane separation reactor 5; proceed resolution reaction; reacted product flows in the product storage tank 6 behind membrane sepn, and enzyme is trapped within the membrane reactor.
Example 1: split N-acetyl-D, L-L-Ala
The 100-200 milliliter nutrient solution of packing in 1000 milliliters of triangular flasks, the component weightmeasurement ratio of this nutrient solution is: soya bean juice 10%, glucose 2.5%, yeast extract paste 0.3%, KH2PO40.3%, MgSO4 0.1%, all the other are sterilized water.After inserting a certain amount of bacteria suspension, in being with 100-200 rev/min speed oscillation on 1.0-3.0 centimetre the shaking table in rotation radius under 30 ℃, cultivated 30-45 hour, can obtain the 3.4g diameter is the aspergillus oryzae ball of 1-3 millimeter, via gelatin 0.8%, quadrol 1.8%, glutaraldehyde 0.85%, stationary liquid that all the other are formed for sterilized water crosslinked fixing after, the ratio enzyme of measuring immobilized aminoacylase is lived and is 420U/g; In packing this enzyme catalyst into the resolution process flow process shown in Figure 1, in diameter is 1.2 centimetres fixed bed enzyme reactor 4, adorn total enzyme and live and be the immobilized aminoacylase of 4600U.N-acetyl-D, the L L-Ala of 0.1M of packing in feed liquid basin 2 (contains CoCl25 * 10
-4M), PH7.0.Behind the pressure feeding feed liquid basin 2 of the nitrogen in the nitrogengas cylinder 1 with 0.1MPa; control different flow velocitys with N-acetyl-D, L-L-Ala solution force feed to preposition membrane separation apparatus 3; solution after the separation feeds the enzyme reactor 4 that immobilized aminoacylase is housed from the bottom to top and carries out resolution reaction; the resolution reaction liquid that is overflowed by the top flows into rearmounted membrane separation reactor 5, proceeds resolution reaction.Reacted product flows into behind membrane sepn in the product storage tank 6, and enzyme is trapped within the membrane reactor, and when flow velocity was lower than 10ml/h, its fractionation rate was 100%, and operate continuously is after 20 days, and the fractionation rate of enzyme reactor-film coupled reaction device is still more than 87%.
Example 2: split N-acetyl-D, L-phenylalanine
By the method for preparing immobilized aminoacylase described in the example 1, will making a certain amount of immobilized aminoacylase, to be placed on bed volume be 36.3cm
3Fixed bed enzyme reactor 4 in, in feed liquid basin 2, put into 0.1M N-acetyl-D, L-phenylalanine; Adjust PH=7 with phosphoric acid buffer, carry out resolution reaction by the described flow process of Fig. 1 at ambient temperature, when volumetric flow rate was 12ml/h, the fractionation rate was near 100%.Split 56 days continuously with this system, the enzyme of system retention rate alive still is 64.6%.To split liquid and transfer pH value to 5.0 with acetic acid, decompression inspissation is separated with ethanol, obtains white L-phenylalanine product, and resolution yield is 72.8%, product optical value [α]
D 20=-34.74 °, meet medical amino acid optically-active standard.
Example 3: split N-acetyl-D, L-methionine(Met)
The 3 liters of nutrient solutions of in 5 liters mechanical agitating fermentation tank, packing into, the composition weight volume ratio of this nutrient solution is: soya bean juice 12%, glucose 2.0%, yeast extract paste 0.4%, KH2PO4 0.3%, MgSO4 0.05%, and add NH3 water with stream and keep fermentating liquid PH value 6.5-7.0, insert behind a certain amount of bacteria suspension that the rotating speed with 100-200 rev/min stirs fermentation under 30 ℃, press 0.5-2 times of V simultaneously
8/ min gas speed feeds sterile air; can obtain the aspergillus oryzae spheroid that the 36g diameter is the 1-3 millimeter through fermentation in 30-45 hour; via gelatin 1.1%; quadrol 2.9%; the stationary liquid that glutaraldehyde 1.0% is formed crosslinked fixing after; measure its immobilized aminoacylase and split N-acetyl-D; the ratio enzyme of L-methionine(Met) is lived and is 450U/g; press the resolution process flow process of accompanying drawing; total enzyme of packing in fixed bed enzyme reactor 4 is lived and is the enzyme catalyst of 11400U; with 0.1M N-acetyl-D; the L-methionine(Met); solution PH 7.0; flow into fixed bed enzyme reactor 4-membrane separation reactor 5 with the pressure of 0.1MPa by basin 2 with nitrogen; when flow velocity is lower than 12 milliliters/hour; its fractionation rate is 100%; operate continuously 60 days, the enzyme of system are lived retention rate still greater than 60%.To split liquid and transfer to PH5.0 with acetic acid, decompression inspissation is separated with ethanol again, gets white L-methionine(Met), and resolution yield is 71%.Measuring the product optical value is [α]
D 20=+23.0 °, show to meet medical methionine(Met) optically-active standard.
Enforcement of the present invention; its gordian technique has 2 points: the one, and adopting more cheap soya bean juice or murphy juice is that main nutrient solution is cultivated balling-up with aspergillus oryzae; and carry out crosslinkedly as linking agent with gelatin-quadrol-glutaraldehyde ternary system, realize the immobilization of L-Aminoacylase.Adopting soya bean juice or murphy juice is that main nutrient solution is
An outstanding advantage of the present invention, it is that not only economy is cheap, the source is abundant for main nutrient solution than general employing extractum carnis, peptone, and institute's yield of enzyme height, thalline separate with nutrient solution easily, gelatin-quadrol-glutaraldehyde ternary system the stationary liquid that adopts, thalline after the immobilization its than enzyme work at 200-1400U/g, enzyme is lived retention rate up to 50-80%.The 2nd, adopt immobilized aminoacylase of the present invention carry out resolution reaction the enzyme reactor series coupled membrane separation reactor; preposition membrane separation apparatus has mainly played centrifugation; rearmounted membrane separation reactor not only plays centrifugation but also play further reactivity; so not only can effectively prolong the operational cycle of enzyme reactor; but and purified product, simplified the later separation operation.
Claims (5)
1, a kind of aspergillus oryzae immobilization prepares the method for L-Aminoacylase, and it is to select for use aspergillus oryzae as the enzyme source, through after cultivating on the agar slant, makes concentration with sterilized water and is about 10
8The bacteria suspension of spore/ml, then with this bacterial suspension inoculation in liquid phase medium, becoming diameter through the stirring fermentation culture is the aspergillus oryzae ball of 1-3 millimeter, with the aspergillus oryzae ball with after nutrient solution separates, be placed on immediately and carry out crosslinked fixing in the liquid phase stationary liquid, obtain the bacterial enzyme 200-1400U/g of being alive, enzyme retention rate alive reaches the immobilized aminoacylase of 50-80%, and it is characterized in that: the composition weight volume ratio of nutrient solution is: soya bean juice 8-15%, glucose 1.5-4.5%, yeast extract paste 0.2-0.5%, KH2PO40.3-0.4%, MgSO4 0.05-0.2%, all the other are sterilized water; Operation control culture condition is: temperature 28-30 ℃, pH value 6-7.5, fermentation time 30-46 hour; The composition weight volume ratio of stationary liquid is: gelatin 0.8-1.2%, quadrol 1.8-3.0%, glutaraldehyde 0.8-1.5%, all the other are sterilized water; The control fixing condition is: temperature 20-60 ℃, reaction times 0.5-2 hour, pH value 4-7, thalline and stationary liquid volume ratio are 1: 10.
2, said aspergillus oryzae immobilization prepares the method for L-Aminoacylase according to claim 1, and it is characterized in that: nutrient solution composition weight volume ratio is: murphy juice 15-25%, yeast extract paste 0.15-0.5%, sucrose 1.5-2.5%, KH2PO4 0.2-0.4%, MgSO40.1-0.3%, all the other are sterilized water.
3, a kind of with according to the prepared L-Aminoacylase of the said aspergillus oryzae process for fixation of claim 1; split the amino acid whose device of DL and mainly comprise feed liquid handling machinery, feed liquid basin, enzyme reactor, product storage tank, it is characterized in that: be 40,000-70,000 ultra-filtration membrane separate reactor at the import and export series coupled molecular weight cut off of enzyme reactor.
4, according to the amino acid whose device of the said fractionation DL of claim 3, it is characterized in that: the membrane module that membrane separation reactor adopts is a hollow-fibre membrane.
5, according to the amino acid whose device of the said fractionation DL of claim 3, it is characterized in that: the membrane module that membrane separation reactor adopts is a flat sheet membrane.
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| CN 99120925 CN1263153A (en) | 1999-09-24 | 1999-09-24 | Method for preparing amino acylase by means of immobilization of Aspergillus oryzae and equipment for resolution and suspension of amino acid |
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| CN 99120925 CN1263153A (en) | 1999-09-24 | 1999-09-24 | Method for preparing amino acylase by means of immobilization of Aspergillus oryzae and equipment for resolution and suspension of amino acid |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186944B (en) * | 2007-12-29 | 2011-09-28 | 河北科技大学 | Biological resolution method for amino acid |
| CN102409067A (en) * | 2011-12-10 | 2012-04-11 | 福州大学 | Culture medium for producing kojic acid by fermenting aspergillus oryzae, and fermentation method thereof |
| CN101671664B (en) * | 2009-03-20 | 2012-07-18 | 上海瀚鸿化工科技有限公司 | Preparation method of immobilized aminoacylase and product and application thereof |
| CN102925530A (en) * | 2012-11-04 | 2013-02-13 | 宁波市远发生物工程有限公司 | L-methionine preparation method |
| CN106244645A (en) * | 2016-08-31 | 2016-12-21 | 保龄宝生物股份有限公司 | A kind of method recycling aspergillus oryzae cell production oligofructose |
-
1999
- 1999-09-24 CN CN 99120925 patent/CN1263153A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186944B (en) * | 2007-12-29 | 2011-09-28 | 河北科技大学 | Biological resolution method for amino acid |
| CN101671664B (en) * | 2009-03-20 | 2012-07-18 | 上海瀚鸿化工科技有限公司 | Preparation method of immobilized aminoacylase and product and application thereof |
| CN102409067A (en) * | 2011-12-10 | 2012-04-11 | 福州大学 | Culture medium for producing kojic acid by fermenting aspergillus oryzae, and fermentation method thereof |
| CN102409067B (en) * | 2011-12-10 | 2013-12-04 | 福州大学 | Culture medium for producing kojic acid by fermenting aspergillus oryzae, and fermentation method thereof |
| CN102925530A (en) * | 2012-11-04 | 2013-02-13 | 宁波市远发生物工程有限公司 | L-methionine preparation method |
| CN106244645A (en) * | 2016-08-31 | 2016-12-21 | 保龄宝生物股份有限公司 | A kind of method recycling aspergillus oryzae cell production oligofructose |
| CN106244645B (en) * | 2016-08-31 | 2020-07-10 | 保龄宝生物股份有限公司 | Method for producing fructo-oligosaccharide by recycling aspergillus oryzae thallus |
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