Preparation method for cell catalyst with high regioselective acylation activity and microbiologic method for synthesizing cytosine arabinoside monoester

A cytarabine monoester and whole-cell catalyst technology, which is applied in the field of cell catalysts, can solve the problem of low selectivity by-products, etc., and achieve the effects of easy control of product structure, low preparation cost, and no need for group protection and deprotection

Inactive Publication Date: 2013-03-06
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The regioselectivity of the microbial whole-cell catalyst synthesized by the invention reaches more than 96% and the catalytic conversion rate is more than 70%, which overcomes the shortcomings of traditional chemical methods such as low selectivity and easy generation of by-products.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] will be cultivated Pseudomonas fluorescens GIM1.209 seed solution was transferred to the liquid medium containing Tween 80 enzyme-producing inducer for culture. The composition of the liquid medium is: 20 g / L Tween 80, 0.5 g / L yeast extract, 0.2 g / L MgSO 4 ·7H 2 O , 2 g / L (NH 4 ) 2 SO 4 , 1 g / L K 2 HPO 4 , the pH was 7.0. The wet cells were collected by centrifugation after 24 hours of shake flask culture at 20°C, washed twice with citrate buffer at pH 5.5, and the whole cell catalyst was obtained after vacuum freeze-drying for 12 hours. Add the prepared cell catalyst (amount of 5 g / L) into hexane pyridine with a cytarabine content of 25 mmol / L and a molar ratio of carboxylate to cytarabine of 15:1, and the synthesis reaction temperature When the temperature is 60 ℃, the shaking speed of the reaction process is 60 rpm, and the reaction time is 24 h, cytarabine monoester can be obtained. The regioselectivity of the catalyzed cytarabine acylation reaction of the ...

Embodiment 2

[0020] will be cultivated Pseudomonas fluorescens GIM1.209 seed liquid was transferred to liquid medium containing Tween 85 enzyme-producing inducer for culture. The composition of the liquid medium is: 30 g / L Tween 85, 40 g / L yeast extract, 4 g / L MgSO 4 ·7H 2 O , 15 g / L (NH 4 ) 2 SO 4 , 1 g / L K 2 HPO 4 , the pH was 7.0. The wet cells were collected by centrifugation after 72 hours of shake flask culture at 40°C, washed twice with citrate buffer at pH 5.5, and vacuum freeze-dried for 24 hours to obtain the whole cell catalyst. The prepared cell catalyst (amount of 50 g / L) was added to hexane-pyridine with a cytarabine content of 50 mmol / L and a molar ratio of carboxylate to cytarabine of 18:1, and the synthesis reaction The temperature is 60 ℃, the shaking speed of the reaction process is 60 rpm, and the reaction time is 24 h, and cytarabine monoester can be obtained. The regioselectivity of the catalyst for catalyzing the cytarabine acylation reaction is 97%, and the...

Embodiment 3

[0022] will be cultivated Pseudomonas fluorescens GIM1.209 seed liquid was transferred to liquid medium containing Span 60 enzyme-producing inducer for culture. The composition of the liquid medium is: 25 g / L Span 60, 30 g / L yeast extract, 2 g / L MgSO 4 ·7H 2 O , 10g / L (NH 4 ) 2 SO 4 , 1 g / L K 2 HPO 4 , the pH was 7.0. The wet cells were collected by centrifugation after cultured in shake flasks at 30°C for 48 hours, washed twice with pH 5.5 phosphate buffer, and vacuum freeze-dried for 24 hours to obtain the whole-cell catalyst. The prepared cell catalyst (amount of 100 g / L) was added to hexane-isopropyl ether with a cytarabine content of 2 mmol / L and a molar ratio of carboxylate to cytarabine of 50:1. The synthesis reaction temperature is 20°C, the oscillation speed of the reaction process is 100 rpm, and the reaction time is 12 hours, and cytarabine monoester can be obtained. The regioselectivity of the catalyst for catalyzing the cytarabine acylation reaction is 97...

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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 [0001] The present invention is the field of bio -catalysts, involving a method of cell catalyst with a high -region -selective alumation reaction. Background technique [0002] At present, the synthesis of glycotoside derivatives adopts chemical synthesis or ion enzyme method.In order to overcome the disadvantages of chemical preparation methods, poor environmental, poor selectivity, and many by -products, since the end of the last century, people have studied using enzyme method for the synthesis of nucleoside monolitan.However, although the enzyme synthesis has the advantages of high regional selectivity, mild response conditions, and environmentally friendly environment, it has encountered many bottlenecks such as high cost and poor catalyst stability.The whole cell is a type of biocuk. As a natural carrier of enzymes, the steps can be eliminated to separate the steps and other steps, thereby greatly reducing production costs.In addition, the whole cells can p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/38C12R1/39
Inventor 李晓凤吴晖赵光磊余以刚卢志洪
Owner SOUTH CHINA UNIV OF TECH
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