CN102952770A - Preparation method for cell catalyst with high regioselective acylation activity and microbiologic method for synthesizing cytosine arabinoside monoester - Google Patents
Preparation method for cell catalyst with high regioselective acylation activity and microbiologic method for synthesizing cytosine arabinoside monoester Download PDFInfo
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- CN102952770A CN102952770A CN2012104481919A CN201210448191A CN102952770A CN 102952770 A CN102952770 A CN 102952770A CN 2012104481919 A CN2012104481919 A CN 2012104481919A CN 201210448191 A CN201210448191 A CN 201210448191A CN 102952770 A CN102952770 A CN 102952770A
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- cytosine arabinoside
- cell catalyst
- monoesters
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- reaction
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Abstract
The invention relates to a preparation method for a cell catalyst with high regioselective acylation activity and a microbiologic method for synthesizing cytosine arabinoside monoester. The preparation method comprises the steps that an initial bacterial strain pseudomonas fluorescens GIM1.209 seed solution is inoculated into a fluid nutrient medium containing 20-40g/L of enzyme producing inducer, thalli in a flask are obtained through centrifugation after 24-72h of shake-flask culture at the temperature of 20-40 DEG C, and distilled water or buffer solution is frozen and dried for more than 12h in vacuum after two times of washing to prepare a whole-cell catalyst. The prepared cell catalyst has high regioselective acylation activity, the regioselectivity is higher than 93%, and the method can be adaptive to the preparation of the cytosine arabinoside monoester and has the advantages that the cost of the cell catalyst is lower than that of an enzyme catalyst, and the efficiency is high.
Description
Technical field
The invention belongs to the biological catalyst field, relate to a kind of method with cell catalyst of high regioselective acylation reaction.
Background technology
Synthetic employing chemical synthesis or the stripped enzyme process of present cytosine arabinoside ester derivative.The shortcomings such as the chemical preparation process environment is unfriendly in order to overcome, poor selectivity, by product are many begin one's study from people at the end of last century and to utilize enzyme process to carry out the synthetic of nucleosides monoesters.Yet, having regioselectivity height, gentle a, advantages of environment protection of reaction conditions although enzyme process is synthetic, it meets with many bottleneck problems such as cost height, poor catalyst stability on industrial application.Full cell is a kind of of biological catalyst, as the natural carrier of enzyme, can save the steps such as enzyme purification and immobilization, thereby reduce production costs widely.In addition, whole cell can be enzyme good environment is provided, and makes enzyme be unlikely to rapid deactivation in non-aqueous media.Yet, up to now, utilize the synthetic research of high regioselectivity microorganism cells catalysis cytosine arabinoside monoesters to there is not yet report.The microbe whole-cell single stage method prepares the cytosine arabinoside monoesters, is green biological respinse approach, for the suitability for industrialized production of cytosine arabinoside monoesters provides new approaches.
Summary of the invention
The present invention is directed to the existing deficiency that is used for the synthetic catalyzer of nucleoside analog ester, purpose is to provide the preparation method of the cell catalyst with high regioselective acylation activity and the microbial process of synthetic cytosine arabinoside monoesters.Described microbe whole-cell method for preparing catalyst is simple to operation, and the inexpensive easy purchase of starting material does not relate to separation and purification of enzyme.The microbe whole-cell catalyst zone field selectivity that the present invention synthesizes reach more than 96% and catalytic conversion more than 70%, the selectivity that has overcome the traditional chemical method is low, easily generate the shortcomings such as by product.Compare with zymin, that the microorganism cells catalyzer has is inexpensive, easily obtain, the high advantage of stability in non-aqueous media.The present invention is achieved through the following technical solutions.
A kind of cell catalyst preparation method with high regioselective acylation activity, the method is: with the initial strains Pseudomonas fluorescens
Pseudomonas fluorescensThe GIM1.209 seed liquor is seeded to and contains in the liquid nutrient medium that produces enzyme inducer, inoculum size is 1% ~ 12%, 20 ~ 40 ℃ of shake-flask culture 24 ~ 72 h, centrifugal results wet thallus, after distilled water or damping fluid washed twice, vacuum lyophilization 12-18 h makes the microbe whole-cell catalyzer.Prepared microbe whole-cell catalyst zone field selectivity more than 96% and catalytic conversion more than 70%, have high zone and select acidylate active.
Further optimize, described product enzyme inducer comprises one or both among Tween 80, Tween 85, Span 60, the Span 80.
Further optimize, take the liquid nutrient medium cumulative volume as benchmark, the composition of described liquid nutrient medium is: 20 ~ 40g/L produces enzyme inducer, 0.5 ~ 50 g/L yeast extract paste, 0.2 ~ 4 g/L MgSO
47H
2O, 2 ~ 15 g/L (NH
4)
2SO
4, 1 g/L K
2HPO
4, liquid nutrient medium pH is 6.8-7.5.
Further optimize, described damping fluid is Citrate trianion or the phosphate buffered saline buffer of pH 5.5 ~ 7.5.
The microbial process of synthetic cytosine arabinoside monoesters of the present invention, specifically: take cytosine arabinoside and carboxylicesters as substrate, take the prepared microbe whole-cell of claim 1 as catalyzer, take pyridine-hexane, pyridine-isopropyl ether as reaction medium generation ester synthesis reaction, obtain cytosine arabinoside monoesters product.
Further optimize, ara content is 10 ~ 50 mmol/L in the substrate, the mol ratio of carboxylicesters and cytosine arabinoside is 15:1 ~ 50:1, the consumption of whole-cell catalyst is 20 ~ 100 g/L reaction systems, synthesis reaction temperature is 20 ~ 60 ℃, reaction process hunting speed 60 rpm ~ 240 rpm, the reaction times can obtain the cytosine arabinoside monoesters at 12-72 h.
The present invention has following advantage compared with prior art:
1. adopt the microbe whole-cell catalyzer come efficient catalytic synthesize cytosine arabinoside 5 '-monoesters synthesizes, have than before the higher regioselectivity of technology, reach the advantages such as product structure is easily controlled, byproduct of reaction is few, and it is unfriendly to have overcome the chemical catalyst environment, product purity is low, easily generates the shortcomings such as by product.
2. the microbe whole-cell catalyst preparation process is easy to operate, preparation cost is low.
3. one-step catalytic cytosine arabinoside monoesters is synthetic, need not radical protection and go to protect, reaction process is simple and easy to control, product separates easily.
4, under the processing condition that limit prepared microbe whole-cell catalyst zone field selectivity more than 96% and catalytic conversion more than 70%, have best catalysis cytosine arabinoside acylation reaction effect.
Embodiment
For better understanding the present invention, below in conjunction with embodiment the present invention is done further detailed description, need to prove that these embodiment do not consist of limiting the scope of the invention.
Embodiment 1
Will be through cultivating
Pseudomonas fluorescensThe GIM1.209 seed liquor is forwarded to contain in the Tween 80 product enzyme inducer liquid nutrient mediums and cultivates.The liquid culture based component is: 20 g/L Tween, 80,0.5 g/L yeast extract pastes, 0.2 g/L MgSO
47H
2O, 2 g/L (NH
4)
2SO
4, 1 g/L K
2HPO
4, pH is 7.0.Centrifugal collection wet thallus behind 20 ℃ of shake-flask culture 24h, pH5.5 citrate buffer washed twice gets whole-cell catalyst behind vacuum lyophilization 12 h.It is 25 mmol/L that prepared cell catalyst (consumption is 5 g/L) is added ara content, the mol ratio of carboxylicesters and cytosine arabinoside is in the hexane pyridine of 15:1, synthesis reaction temperature is 60 ℃, reaction process hunting speed 60 rpm, reaction times can obtain the cytosine arabinoside monoesters at 24 h.The regioselectivity of the catalysis cytosine arabinoside acylation reaction of this catalyzer is 96%, and catalytic conversion is 75%.
Embodiment 2
Will be through cultivating
Pseudomonas fluorescensThe GIM1.209 seed liquor is forwarded to contain in the Tween 85 product enzyme inducer liquid nutrient mediums and cultivates.The liquid culture based component is: 30 g/L Tween, 85,40 g/L yeast extract pastes, 4 g/L MgSO
47H
2O, 15 g/L (NH
4)
2SO
4, 1 g/L K
2HPO
4, pH is 7.0.Centrifugal collection wet thallus behind 40 ℃ of shake-flask culture 72h, pH5.5 citrate buffer washed twice gets whole-cell catalyst behind vacuum lyophilization 24 h.It is 50 mmol/L that prepared cell catalyst (consumption is 50 g/L) is added ara content, the mol ratio of carboxylicesters and cytosine arabinoside is in hexane-pyridine of 18:1, synthesis reaction temperature is 60 ℃, reaction process hunting speed 60 rpm, reaction times can obtain the cytosine arabinoside monoesters at 24 h.The regioselectivity of the catalysis cytosine arabinoside acylation reaction of this catalyzer is 97%, and catalytic conversion is 74%.
Embodiment 3
Will be through cultivating
Pseudomonas fluorescensThe GIM1.209 seed liquor is forwarded to contain in the Span 60 product enzyme inducer liquid nutrient mediums and cultivates.The liquid culture based component is: 25 g/L Span, 60,30 g/L yeast extract pastes, 2 g/L MgSO
47H
2O, 10g/L (NH
4)
2SO
4, 1 g/L K
2HPO
4, pH is 7.0.Centrifugal collection wet thallus behind 30 ℃ of shake-flask culture 48 h, pH 5.5 phosphate buffered saline buffer washed twice get whole-cell catalyst behind vacuum lyophilization 24 h.It is 2 mmol/L that prepared cell catalyst (consumption is 100 g/L) is added ara content, the mol ratio of carboxylicesters and cytosine arabinoside is in hexane-isopropyl ether of 50:1, synthesis reaction temperature is 20 ℃, reaction process hunting speed 100 rpm, reaction times can obtain the cytosine arabinoside monoesters at 12h.The regioselectivity of the catalysis cytosine arabinoside acylation reaction of this catalyzer is 97%, and catalytic conversion is 74%.
Embodiment 4
Will be through cultivating
Pseudomonas fluorescensThe GIM1.209 seed liquor is forwarded to contain in the Span 80 product enzyme inducer liquid nutrient mediums and cultivates.The liquid culture based component is: 25 g/L Span, 80,30 g/L yeast extract pastes, 2 g/L MgSO
47H
2O, 10g/L (NH
4)
2SO
4, 1 g/L K
2HPO
4, pH is 7.0.Centrifugal collection wet thallus behind 30 ℃ of shake-flask culture 48 h, pH 5.5 phosphate buffered saline buffer washed twice get whole-cell catalyst behind vacuum lyophilization 24 h.It is 20 mmol/L that prepared cell catalyst (consumption is 55 g/L) is added ara content, the mol ratio of carboxylicesters and cytosine arabinoside is 35:1, and synthesis reaction temperature is 35 ℃, reaction process hunting speed 240 rpm, reaction times can obtain the cytosine arabinoside monoesters at 36 h.The regioselectivity of the catalysis cytosine arabinoside acylation reaction of this catalyzer is 96%, and catalytic conversion is 76%.
Embodiment 5
Will be through cultivating
Pseudomonas fluorescensThe GIM1.209 seed liquor is forwarded to contain in Tween 85 and the Span 80 product enzyme inducer liquid nutrient mediums and cultivates.The liquid culture based component is: 30 g/L Span 80 and Tween 85 mixtures, 30 g/L yeast extract pastes, 2 g/L MgSO
47H
2O, 10g/L (NH
4)
2SO
4, 1 g/L K
2HPO
4, pH is 7.0.Centrifugal collection wet thallus behind 30 ℃ of shake-flask culture 72h, pH 5.5 phosphate buffered saline buffer washed twice get whole-cell catalyst behind vacuum lyophilization 24 h.It is 30 mmol/L that prepared cell catalyst (consumption is 60 g/L) is added ara content, the mol ratio of carboxylicesters and cytosine arabinoside is 40:1, and synthesis reaction temperature is 40 ℃, reaction process hunting speed 200 rpm, reaction times can obtain the cytosine arabinoside monoesters at 72 h.The regioselectivity of the catalysis cytosine arabinoside acylation reaction of this catalyzer is 97.5%, and catalytic conversion is 76%.
Claims (7)
1. the cell catalyst preparation method with high regioselective acylation activity is characterized in that: with the initial strains Pseudomonas fluorescens
Pseudomonas fluorescensThe GIM1.209 seed liquor is seeded to and contains in the liquid nutrient medium that produces enzyme inducer, inoculum size is 1% ~ 12%, 20 ~ 40 ℃ of shake-flask culture 24 ~ 72 h, centrifugal results wet thallus, after distilled water or damping fluid washed twice, vacuum lyophilization 12-18 h makes the microbe whole-cell catalyzer.
2. described cell catalyst preparation method with high regioselective acylation activity according to claim 1 is characterized in that described product enzyme inducer comprises one or both among Tween 80, Tween 85, Span 60, the Span 80.
3. described cell catalyst preparation method with high regioselective acylation activity according to claim 1; it is characterized in that take the liquid nutrient medium cumulative volume as benchmark; the composition of described liquid nutrient medium is: 20 ~ 40g/L produces enzyme inducer, 0.5 ~ 50 g/L yeast extract paste, 0.2 ~ 4 g/L MgSO
47H
2O, 2 ~ 15 g/L (NH
4)
2SO
4, 1 g/L K
2HPO
4, liquid nutrient medium pH is 6.8-7.5.
4. described cell catalyst preparation method with high regioselective acylation activity according to claim 1 is characterized in that described damping fluid is Citrate trianion or the phosphate buffered saline buffer of pH 5.5 ~ 7.5.
5. the cell catalyst preparation method with high regioselective acylation activity according to claim 1; it is characterized in that prepared microbe whole-cell catalyst zone field selectivity more than 96% and catalytic conversion more than 70%, have high zone and select acidylate active.
6. the microbial process of a synthetic cytosine arabinoside monoesters, it is characterized in that take cytosine arabinoside and carboxylicesters as substrate, take the prepared microbe whole-cell of claim 1 as catalyzer, take pyridine-hexane, pyridine-isopropyl ether as reaction medium generation ester synthesis reaction, obtain cytosine arabinoside monoesters product.
7. the microbial process of a kind of synthetic cytosine arabinoside monoesters according to claim 6, it is characterized in that ara content is 10 ~ 50 mmol/L in the substrate, the mol ratio of carboxylicesters and cytosine arabinoside is 15:1 ~ 50:1, the consumption of whole-cell catalyst is 20 ~ 100 g/L reaction systems, synthesis reaction temperature is 20 ~ 60 ℃, reaction process hunting speed 60 rpm ~ 240 rpm, the reaction times can obtain the cytosine arabinoside monoesters at 12-72 h.
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Patent Citations (5)
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| US6423692B2 (en) * | 1997-04-24 | 2002-07-23 | Dana-Farber Cancer Institute, Inc. | Method of enhancing the effectiveness of DCK phosphorylated molecules |
| CN1661030A (en) * | 2004-12-22 | 2005-08-31 | 华南理工大学 | Method of enzyme catalysis for preparing cytosine ester arabinoside |
| CN1876824A (en) * | 2006-04-21 | 2006-12-13 | 华南理工大学 | Method for preparing cytosine arabinoside ester by enzyme catalysis |
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Application publication date: 20130306 |