CN1072957A - A kind of fixed yeast cell and the application on adenosine triphosphate is produced thereof - Google Patents
A kind of fixed yeast cell and the application on adenosine triphosphate is produced thereof Download PDFInfo
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- CN1072957A CN1072957A CN 92113813 CN92113813A CN1072957A CN 1072957 A CN1072957 A CN 1072957A CN 92113813 CN92113813 CN 92113813 CN 92113813 A CN92113813 A CN 92113813A CN 1072957 A CN1072957 A CN 1072957A
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Abstract
The invention discloses a kind of preparation method and the application in adenosine triphosphate (ATP) production thereof of fixed yeast cell.Adopt fixed yeast cell of the present invention adenosine or adenylic acid (AMP) can be changed into adenosine triphosphate quantitatively, transformation efficiency can reach more than 99%.
Description
The present invention relates to a kind of preparation method of fixed yeast cell, and with the production technique of fixed yeast cell biosynthesizing adenosine triphosphate.
As everyone knows, adenosine triphosphate (being called for short ATP) has been widely used as the medicine in amyotrophy, myocardial infarction, hepatitis and the various first aid illness.The end of the sixties, people utilized the free yeast cell to produce adenosine triphosphate by adenosine (being called for short A) or adenylic acid (AMP) (being called for short AMP).But since this method not only yeast cell utilize low (only use once), and, brought the difficulty of separating, so the actual recovery of ATP is not high because the free yeast cell mixes in product.For this reason, since the seventies, many biological workers are devoted to develop the research that fixed yeast cell is produced ATP, for example: Ado, people such as Y disclose on " J.Solid-phase Biochem, 1979,4; 43 " magazine and a kind of the quick-frozen yeast cell have been fixed on method in the ethyl cellulose microcapsule, continuous production ATP in column type reactor, the transformation efficiency of the ATP during its initial reaction can reach 70%, drops to later on about 50%; People such as Zhang Yuanliang disclose the research of a kind of immobilized saccharomyces cerevisiae cell biosynthesizing ATP in " He'nan University's journal is on the second phase in 1985 ", and its initial conversion can reach 80%, but the immobilized saccharomyces cerevisiae cell access times are few.In a word, up to the present, with fixed yeast cell biosynthesizing ATP, because its transformation efficiency is low far beyond free yeast cell method, it is shorter work-ing life to add fixed yeast, so fail to realize industrialization so far.
But the objective of the invention is to prepare a kind of fixed yeast cell of life-time service, the transformation efficiency of its biosynthesizing ATP can reach more than 99%.
Design of the present invention is such:
The contriver finds that there is the following defective in prior art in " fixed yeast cell and the biosynthesizing ATP thereof " process that studies for a long period of time:
When (1) preparing fixed yeast cell, temperature is too high, causes the activity of yeast cell to reduce;
When (2) using glutaraldehyde solution to use fixed yeast cell to strengthen, temperature is too high, causes the activity of fixed yeast cell to reduce;
(3) be adsorbed on the glutaraldehyde aldehyde radical that is not closed on the fixed yeast cell and exist, influence the activity of fixed yeast cell;
(4) fixed yeast cell causes sulfydryl enzyme deactivation in the part glycolytic ferment system in strengthening process, influences the overall activity of fixed yeast cell;
(5) in the process of using fixed yeast cell biosynthesizing ATP, wherein contained nadide constantly runs off, and causes the activity of immobilized cell to reduce, and shows the rapid decline of the transformation efficiency of ATP.
In a word, the factor of above-mentioned aspects causes not only that the fixed yeast cell initial activity is lower (to be compared with the free yeast cell, its initial activity only is 80%), and, influenced industrialized prospect because the loss of nadide in biosynthetic process has caused the rapid decline of ATP transformation efficiency.
The present invention is directed to the above-mentioned defective of prior art, proposed to overcome the technical measures of these defectives:
Temperature when (1) reducing the preparation fixed yeast cell is selected to be suitable for the microenvironment that yeast cell is survived, and yeast cell is in the most suitable activation environment is immobilized;
(2) reduce temperature when fixed yeast cell is crosslinked to be strengthened, reduce the linking agent glutaraldehyde the active disadvantageous effect of fixed yeast cell;
(3), make the free aldehyde sealing that is adsorbed on the glutaraldehyde on the fixed yeast cell, to eliminate of the detrimentally affect of free glutaraldehyde aldehyde radical to fixed yeast cell with the solution washing fixed yeast cell that contains Methionin;
(4), make the sulfydryl reduction activation of the sulfydryl enzyme in the glycolytic ferment system of inactivation in immobilization process with containing the solution washing fixed yeast cell of sodium bisulfite;
(5) employing increases the permeability of fixed yeast cell to the method for fixed yeast cell low-temperature quick-freezing, reduces time of enzymatic reacting;
(6) in reaction solution (substrate), add micro-nadide (VITAMIN B4 niacinamide dinucleotides can be called for short NAD), to replenish the loss of fixed yeast cell nadide in the enzymatic reaction process.
The present invention also is achieved in that
One. the preparation of high activity immobilization yeast cell:
(1) be 0.1mol/L at first with concentration, PH be 5.8 phosphoric acid buffer to be mixed with concentration be 12～18%(Wt) gelatin solution, be fixation support solution;
(2) above-mentioned solution container will be housed then and place 33～37 ℃ of water-bath constant temperature, add the yeast of constant temperature in 15～37 ℃ of water-baths, mix, pour into and be paved into the thick film of 2～4mm in the dish, place refrigerator (4 ℃) to solidify 1～3 hour, take out the back by screen cloth be cut into 10 order sizes particle (10 sieve apertures/centimetre
2);
(3) again the particle of above-mentioned 10 order sizes is placed 0～10 ℃, concentration is 1.5%(Wt) crosslinked reinforcement 20～25 minutes in the glutaraldehyde water solution, take out the back and clean with deionized water, obtain rough fixed yeast cell;
(4),, make the glutaraldehyde aldehyde radical sealing of crosslinked traces of unreacted on fixed yeast cell earlier with the solution washing that contains 0.1～1%(Wt) Methionin with rough fixed yeast cell; Again with containing 0.2～2%(Wt) aqueous solution of sodium bisulfite washing, make the sulfydryl reduction activation of the sulfydryl enzyme in the glycolytic ferment system of inactivation in the cross-linking process; After cleaning with deionized water at last, place quick-frozen under-25～-40 ℃ of low temperature, obtain the said fixed yeast cell of the present invention.Preserve standby at low temperatures.
By the fixed yeast cell that aforesaid method makes, can keep whole vigor of 20 plurality of enzymes in the yeast cell, and the gained fixed yeast cell has excellent mechanical intensity.
Wherein: carrier soln and zymic ratio of mixture (Wt) during the preparation fixed yeast cell are 1: 1～2, and the most suitable ratio of mixture is 1: 1.0～1.5.
The most suitable temperature of fixation support solution is 34～36 ℃; The most suitable temperature of zymic is 18～25 ℃.
The most suitable concentration of carrier is 15%(Wt).The most suitable temperature was 4 ℃ when fixed yeast cell was strengthened with glutaraldehyde cross-linking, and crosslinking time is 22 minutes.
Two. the technology with the present invention's said fixed yeast cell biosynthesizing adenosine triphosphate (ATP) mainly comprises following content:
(1) preferred reaction liquid (or claiming substrate aqueous solution), its mole ratio of components is:
A adenosine or adenylic acid (AMP): 30～70 mMol/L,
B glucose: 100～300 mMol/L,
C sal epsom: 4～20 mMol/L,
The d nadide: 0～1 mMol/L,
The e phosphoric acid buffer: 150～350 mMol/L,
PH=6.5～7.5
F water: all the other.
(2) fixed yeast cell (Wt) is 1: 1～2 with the ratio of reaction solution (V).
(3) preferred enzymatic range of reaction temperature is: 26～38 ℃.
(4) preferred time of enzymatic reacting is: 1.5～3 hours.
Wherein: the optimum molar ratio of components of reaction solution is:
A adenosine or adenylic acid (AMP): 35～45 mMol/L,
B glucose: 150～200 mMol/L,
C sal epsom: 6～10 mMol/L,
The d nadide: 0.2～0.4 mMol/L,
The e phosphoric acid buffer: 200～300 mMol/L,
PH=7
F water all the other
Said nadide is the abbreviation of VITAMIN B4 niacinamide dinucleotides.
Reaction solution with fixed yeast cell of the present invention and above-mentioned composition, ratio in solid-to-liquid ratio 1: 1～2 adds in the reactor, feed 30～38 ℃ thermostat(t)ed water in the chuck of reactor, enzymatic reaction is maintained in this temperature range to be carried out, feed the nitrogen of compression at reactor bottom, make fixed yeast cell be fluidization, reaction was carried out 1.5～3.0 hours, and the sampling in 0.5 hour of every interval is once, with the transformation efficiency of conventional gelose electrophoresis technique determining ATP, and sampling between 1.5～2.5 hours, the transformation efficiency of ATP can reach 90～99%, 3 hours sampling has detected the existence less than adenosine or adenylic acid (AMP), and as seen all adenosines or adenylic acid (AMP) have all changed into ATP, and reaction finishes, open baiting valve, bleed off the feed liquid in the reactor, add fresh reaction solution again, repeat aforesaid operations.But fixed yeast cell then life-time service goes down.The feed liquid of emitting from reactor can be passed through a D296 resin anion(R.A) exchange column, and the ATP in the feed liquid is that D296 anionite-exchange resin is adsorbed, behind the water wash-out impurity, again with the aqueous solution wash-out that contains Nacl, collects the level separatory of ATP, adds alcohol.Make ATP precipitation, after filtration, vacuum-drying, can get ATPNa
22H
2The O product.
Wherein: the enzymatic reaction temperature is good during with 34～36 ℃.The ratio of fixed yeast cell (Wt) and reaction solution (V) is preferably 1: 1～and 1.5.
Further illustrate content of the present invention below in conjunction with embodiment:
Embodiment 1
Take by weighing the 3g gelatin, add one and fill 17ml, PH is 5.8, concentration is in the Erlenmeyer flask of 0.1Mol/L phosphoric acid buffer, place 35 ℃ of waters bath with thermostatic control, until dissolving fully, mix with the 30g yeast that is incubated in 18 ℃ of water-baths then, stir evenly, pour into and be paved into the thick film in the 2mm left and right sides in the porcelain dish, place refrigerator (4 ℃) to solidify again 2 hours, take out the back cuts into 10 order sizes by a stainless steel mesh particle, under 4 ℃, deposit in 1.5% glutaraldehyde solution crosslinked 22 minutes, take out the back and use earlier deionized water wash,, continue with the solution washing that contains sodium bisulfite again with the solution washing that contains Methionin, clean with deionized water more at last, place-40 ℃ cryogenic refrigerator quick-frozen then, make the said fixed yeast cell of the present invention, and under this low temperature, preserve standby.
In the 250ml Erlenmeyer flask, according to fixed yeast cell (Wt): reaction solution (V)=ratio of 1: 1 adds the reaction solution of 50 gram fixed yeast cells and 50ml, the ratio of components of reaction solution is: adenylic acid (AMP) 40mMol/L, glucose 150mMol/L, sal epsom 8mMol/L, nadide 0.2mMol/L, phosphoric acid buffer (PH=7.0) 250mMol/L, all the other are water., take a sample after 2 hours 35 ℃ of following oscillatory reactions.Use the gelose cataphoretic determination, the transformation efficiency of ATP reaches 96%, inclines then to reacted feed liquid, adds new reaction solution 50ml again, continues to repeat the aforesaid operations step 35 ℃ of following oscillatory reactions.The transformation efficiency of ATP can remain at 96%.But fixed yeast cell life-time service.
By D296 resin anion(R.A) exchange column (volume of wet resin is 40ml), ATP is stayed in the post by resin is adsorbed in the feed liquid with reacted feed liquid 200ml.Wash with water assorted after, the aqueous solution wash-out of forming with 0.7mol/L Nacl-0.01mol/L Hcl is again collected the fraction 60ml of ATP, uses alcohol precipitation then.Filtration, vacuum-drying can get 3.2 gram ATP Na
22H
2The O product.
Embodiment 2
Take by weighing the 0.5kg gelatin, add one and fill 2.83l, PH is 5.8, and concentration is in the container of 0.1Mol/L phosphoric acid buffer, places 40 ℃ of waters bath with thermostatic control until dissolving fully.Be chilled to 37 ℃ of following constant temperature one hour then, add the 4.5kg yeast that in 25 ℃ of water-baths, has been incubated again, after mixing, pour in the enamel tray, be paved into the thick film of 2～4mm, place 4 ℃ of refrigerators to solidify 2 hours, cut into the particle of 10 order sizes after the taking-up, pouring temperature again into is 4 ℃, concentration is 1.5%(Wt), volume is in the glutaraldehyde solution of 10l, and placed in 4 ℃ the refrigerator crosslinked 22 minutes, spend ion-cleaning after the taking-up earlier,, continue with the solution washing that contains sodium bisulfite again with the solution washing that contains Methionin, clean with deionized water more at last, place the cryogenic refrigerator quick-frozen below-25 ℃ then, make fixed yeast cell of the present invention, and under this low temperature, preserve standby.
At a volume is in the fluidized-bed reactor of 10l, adds above-mentioned fixed yeast cell 4kg and reaction solution 6l, and the ratio of components of reaction solution is with embodiment 1.In the chuck of reactor, feed 36 ℃ of thermostat(t)ed waters, make system under temperature constant state, carry out enzymatic reaction, feed the nitrogen (nitrogen is by the air filter degerming) of compression in the bottom of reactor, make that fixed yeast cell is fluidization in the reactor, react after 3 hours, sampling, use the gelose electrophoresis technique determining, nearly all adenosine (or adenylic acid (AMP)) all changes into ATP, and promptly transformation efficiency is more than 99%, so reaction 3 hours suited as the termination time, open baiting valve, bleed off the feed liquid that reaction finishes, add the fresh reaction solution of 6l again, continue to repeat above-mentioned enzymatic reaction.But fixed yeast cell life-time service of the present invention, the transformation efficiency of its ATP can remain on about 99%.Extract, separate the method for ATP with embodiment 1.
Therefore: be used for biosynthesis adenosine triphosphate (ATP) according to the prepared fixed yeast cell of method of the present invention and have following advantage:
1. the activity of fixed yeast cell of the present invention and free yeast cells is quite active, and when being used for biosynthesis adenosine triphosphate (ATP), its conversion ratio can reach more than 99%, is much higher than 80% of prior art.
2. suitable condition of cure makes the said fixed yeast cell of the present invention have enough intensity, can satisfy the long-term use in the industrial production.
3. because the present invention has added micro-DPN in reaction system, run off thereby suppressed fixed yeast cell DPN in the enzymatic reaction process, make 20 plurality of enzymes in the fixed yeast cell when participating in enzymatic reaction, all keep good overall activity.
4. fixed yeast cell of the present invention is applicable to that reactant liquor contains the condition of adenosine or the adenylate of higher concentration.
5. when adopting fixed yeast cell biosynthesis adenosine triphosphate of the present invention (ATP), its time of enzymatic reacting can foreshorten to 1.5～3 hours, reduces half than prior art at least.
In a word, because fixed yeast cell of the present invention has above-mentioned remarkable advantage, thereby has widely prospects for commercial application.
Obviously, for any technical staff who is engaged in adenosine triphosphate production, adopt this Invent said fixed yeast cell, can also (be 10～20mMol/L) to change into ATP such as adenosine or determination of adenosine phosphates in mouse, this reaction time can be less than 1.5 hours with containing the lower reactant liquor of adenosine or adenylate concentration.
In addition, the said yeast of the present invention can be fresh yeast, can also be the freeze-drying yeast, and after they were immobilized, under condition of the present invention, the conversion ratio of ATP can reach 96～99%.
Claims (9)
1, a kind of is the preparation method of the fixed yeast cell of carrier with the gelatin, it is characterized in that:
(1) said carrier system to adopt concentration be 0.1Mol/L, PH is that 5.8 phosphoric acid buffer is mixed with the gelatin solution of 12～18% (wt);
(2) place 33～37 ℃ water bath with thermostatic control to be incubated (1) described gelatin solution, then in the ratio of carrier and yeast 1:1～2 will be in 15～37 ℃ of water-baths the homothermic yeast join and go in the carrier, pour into behind the stirring and evenly mixing and be paved into the thick film of 2～4mm in the dish, place 4 ℃ refrigerator to solidify again 1～3 hour, be cut into the particle of 10 order sizes after the taking-up;
(3) particle of (2) described 10 order sizes being placed 0～10 ℃ of concentration is the crosslinked reinforcement of 1.5% (wt) glutaraldehyde water solution 20～25 minutes, takes out the back and cleans with deionized water, obtains rough fixed yeast cell;
(4) (3) described rough fixed yeast cell is used the solution washing that contains 0.1～1% (wt) Methionin earlier, again with the solution washing that contains 0.2～2% (wt) sodium bisulfite, after cleaning with deionized water at last, place-25～-40 ℃ of cryogenic refrigerator quick-frozens, obtain the said fixed yeast cell of the present invention.
2, the method for claim 1, wherein carrier concn is with 15%(Wt) be good.
3, method as claimed in claim 1 or 2, it is characterized in that carrier and zymic ratio of mixture be preferably 1: 1.0～1.5.
4, as the method for one of claim 1～3, the suitable constant temperature to 34 of carrier when it is characterized in that immobilization (gelatin solution)～36 ℃, the suitable constant temperature to 18 of yeast～25 ℃.
5,, it is characterized in that using 1.5%(Wt as the method for one of claim 1-4) the glutaraldehyde cross-linking temperature of strengthening fixed yeast cell is 4 ℃, crosslinking time is 22 minutes.
When 6, being used for the biosynthesizing adenosine triphosphate as the prepared fixed yeast cell of the method for one of claim 1-5, being characterized as of its processing condition:
(1) reaction solution (substrate) mol ratio is:
A adenosine or adenylic acid (AMP): 30～70 mMol/L,
B glucose: 100～300 mMol/L,
C sal epsom: 4～20 mMol/L,
The d nadide: 0～1 mMol/L,
The e phosphoric acid buffer: 150～350 mMol/L,
(PH=6.5～7.5)
F water: all the other;
(2) fixed yeast cell (Wt) is 1: 1～2 with the ratio of reaction solution (V);
(3) temperature range of enzymatic reaction is 26～38 ℃;
(4) time of enzymatic reacting is 1.5～3 hours.
7, technology as claimed in claim 6 is characterized in that the mol ratio of reaction solution is:
A adenosine or adenylic acid (AMP): 35～45 mMol/L,
B glucose: 150～200 mMol/L,
C sal epsom: 6～10 mMol/L,
The d nadide: 0.1～0.3 mMol/L,
E phosphoric acid buffer PH=7:200～300 mMol/L,
F water: all the other.
8,, it is characterized in that the enzymatic reaction temperature is 34～36 ℃ as claim 6 or 7 described technologies.
9, as the described technology of one of claim 6-8, it is characterized in that the ratio of fixed yeast cell (Wt) and reaction solution (V) is preferably 1: 1～1.5.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 92113813 CN1038257C (en) | 1992-12-18 | 1992-12-18 | Fixed yeast cell and its application to production of adenosine triphosphate |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 92113813 CN1038257C (en) | 1992-12-18 | 1992-12-18 | Fixed yeast cell and its application to production of adenosine triphosphate |
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| Publication Number | Publication Date |
|---|---|
| CN1072957A true CN1072957A (en) | 1993-06-09 |
| CN1038257C CN1038257C (en) | 1998-05-06 |
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|---|---|---|---|
| CN 92113813 Expired - Fee Related CN1038257C (en) | 1992-12-18 | 1992-12-18 | Fixed yeast cell and its application to production of adenosine triphosphate |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1062603C (en) * | 1996-01-31 | 2001-02-28 | 中国科学院大连化学物理研究所 | Film reaction technology for producing D-p-hydroxy-phenyl glycine by enzyme method |
| CN1086736C (en) * | 1999-05-17 | 2002-06-26 | 华东理工大学 | Microorganism carrier fixed with glue compounded with carragheen and konjak polysaccharide and use thereof |
| CN108120833A (en) * | 2018-02-24 | 2018-06-05 | 江南大学 | A kind of method of quick detection yeast activity |
| CN108753808A (en) * | 2018-05-30 | 2018-11-06 | 浙江海正药业股份有限公司 | A kind of recombinant expression carrier, recombinant expression host and its method for synthesizing adenosine triphosphoric acid |
| CN111218441A (en) * | 2020-02-25 | 2020-06-02 | 浙江工业大学 | Magnetic immobilized yeast cell and application thereof in preparation of (R) -2-hydroxy-4-phenylbutyrate ethyl ester |
-
1992
- 1992-12-18 CN CN 92113813 patent/CN1038257C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1062603C (en) * | 1996-01-31 | 2001-02-28 | 中国科学院大连化学物理研究所 | Film reaction technology for producing D-p-hydroxy-phenyl glycine by enzyme method |
| CN1086736C (en) * | 1999-05-17 | 2002-06-26 | 华东理工大学 | Microorganism carrier fixed with glue compounded with carragheen and konjak polysaccharide and use thereof |
| CN108120833A (en) * | 2018-02-24 | 2018-06-05 | 江南大学 | A kind of method of quick detection yeast activity |
| CN108753808A (en) * | 2018-05-30 | 2018-11-06 | 浙江海正药业股份有限公司 | A kind of recombinant expression carrier, recombinant expression host and its method for synthesizing adenosine triphosphoric acid |
| CN108753808B (en) * | 2018-05-30 | 2021-02-23 | 浙江海正药业股份有限公司 | Recombinant expression vector, recombinant expression host and method for synthesizing adenosine triphosphate by using recombinant expression vector |
| CN111218441A (en) * | 2020-02-25 | 2020-06-02 | 浙江工业大学 | Magnetic immobilized yeast cell and application thereof in preparation of (R) -2-hydroxy-4-phenylbutyrate ethyl ester |
| CN111218441B (en) * | 2020-02-25 | 2022-03-18 | 浙江工业大学 | Magnetic immobilized yeast cell and application thereof in preparation of (R) -2-hydroxy-4-phenylbutyrate ethyl ester |
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| Publication number | Publication date |
|---|---|
| CN1038257C (en) | 1998-05-06 |
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