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Serration and its use in preparation of chiral precurser for dielzepin

A technology for Serratia and strains, which is applied in the field of preparing diltiazem chiral precursors, can solve the problems of unstable enzyme reactor, complicated technology and equipment, large resistance to transmembrane mass transfer, etc., and achieves low cost and technological process. Simple, catalytically active and highly stereoselective effect

Inactive Publication Date: 2007-08-29
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the enzyme still needs to be extracted and then uploaded to the membrane reactor. The technology and equipment are relatively complicated, and the enzyme is not very stable in the reactor. Production efficiency gradually decreases with reactor operation
In addition, this process requires the use of a special hollow fiber membrane that can tolerate organic solvents, and due to the large transmembrane mass transfer resistance of the substrate and product during the reaction, the catalytic efficiency of the enzyme is limited to a certain extent.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Slant culture: peptone 0.5wt%, yeast extract 0.5wt%, K 2 HPO 4 0.2wt%, NaCl 0.05wt%, MgSO 4 ·7H 2 O 0.05wt%, FeSO 4 ·7H 2O 0.001wt%, agar 2wt%, pH6.5. The cryopreserved glycerol suspension was inoculated on the slant (plate) medium, and cultured at a constant temperature of 30° C. for about 1.5 days.

[0030] Enzyme production culture: Tween-80 1.0wt%, peptone 3.0wt%, K 2 HPO 4 0.2wt%, NaCl 0.05wt%, MgSO 4 ·7H 2 O 0.05wt%, FeSO 4 ·7H 2 O 0.001wt%, pH7.0, 250ml shake flask liquid volume 30ml, inoculum size 2.5v / v%, culture temperature 25°C, shaker speed 200rpm, shake flask culture under the above conditions for 2 days, the enzyme production can reach 4780U / L.

[0031] Enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze p-nitrophenol acetate (1.0 mmol / L) per minute at 25°C and pH 7.0 to produce 1 μmol of p-nitrophenol.

Embodiment 2

[0033] The slant culture was the same as in Example 1. Fill a 250ml shake flask with 30ml of culture medium, the composition of which is as follows:

[0034] Tween-80 1.0wt%, Dextrin 2.0wt%, Corn Steep Steep 3.0wt%, Ammonium Sulfate 0.5wt%, K 2 HPO 4 0.2wt%, NaCl 0.05wt%, MgSO 4 ·7H 2 O 0.05wt%, FeSO 4 ·7H 2 O 0.001wt%, pH6.5. The culture solution pre-cultivated at 30°C and 150rpm for 12 hours was used as the seed, and transferred to four 1L shake flasks containing 150ml of the same medium at an inoculum size of 5v / v%. The enzyme production was 9480U / L after culturing for 24 hours under the condition of rotating speed 150rpm.

Embodiment 3

[0036] Seed culture is identical with embodiment 2. Load 3L medium in a 5L fermenter, and its composition is as follows: dextrin 2wt%, corn steep liquor 1.5wt%, ammonium sulfate 0.5wt%, K 2 HPO 4 0.2wt%, NaCl 0.05wt%, MgSO 4 ·7H 2 O0.05wt%, CaCl 2 0.05 wt%, pH 6.5. After the culture medium is sterilized, insert 200ml of the seed solution pre-cultivated for 12 hours, and ferment with aeration under the conditions of 30°C, 400rpm and 0.5vvm. Foam enemy (1wt / v% aqueous solution) was added dropwise for defoaming. After 15 hours of fermentation, the tank was put, and the enzyme production was 5600U / L.

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Abstract

A Serratia bacillus able to generate the stereoselective lipohydrolase features that after it is fermented and cultured, it can generate the lyolipase, which can be used to split the racemate (+ / -)-methylester of p-methoxyphenyl glycilic acid to obtain the (-)-methylester of p-methoxyphenyl glycilic acid, which is the key chiral precursor of diltiazem for treating cardiovascular diseases.

Description

technical field [0001] The present invention relates to a Serratia strain and its application, in particular to a Serratia strain producing stereoselective ester hydrolase and its application method in the preparation of diltiazem chiral precursor. technical background [0002] Diltiazem (diltiazem) is a benzothiapine calcium ion channel blocker synthesized in the 1970s. Due to its definite curative effect and low toxic and side effects, it has been widely used in the long-term treatment of angina pectoris and hypertension for a long time. Synthesis of diltiazem by chemical-enzymatic method can simplify the synthetic route, increase the yield and reduce the cost. In the chemical-enzymatic production process, lipase is generally used to catalyze the resolution of (±)-methyl p-methoxyphenyl glycidate, in which the unwanted (+)-isomer is selectively hydrolyzed by the enzyme, while The useful (-)-methyl p-methoxyphenylglycidate was preserved intact and used as a starting materi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P41/00C12R1/425A01G7/00
Inventor 许建和郜莉潘江龙章德
Owner EAST CHINA UNIV OF SCI & TECH
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