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Saccharomycete with stereoselectivity lipase liveness and application in producing S- type betaxolol hydrochloride with biological split method thereof

A technology of stereoselectivity and lipase, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of no cell or enzyme immobilization, high catalyst cost, and low flash point of the reaction system

Inactive Publication Date: 2008-07-16
ZHENGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the best effect is Pseudomonas lipase AK, which reacts in tert-butyl methyl ether medium, and the reaction system has a low flash point, is flammable, and has potential safety hazards; the weight ratio of enzyme and substrate is 1:7, and the catalyst cost Very high; their optical purity can only reach 80% (resolution yield 50%), and the ee value reaches 90% by recrystallization of its hydrochloride
The reaction stereoselectivity catalyzed by other microbial enzymes is much worse, and the enzyme-catalyst weight ratio is 1:1, and the catalyst cost is higher
In addition, they did not report on the stability of the enzyme, nor on the immobilization of cells or enzymes, which is far from the requirements of industrialization

Method used

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  • Saccharomycete with stereoselectivity lipase liveness and application in producing S- type betaxolol hydrochloride with biological split method thereof
  • Saccharomycete with stereoselectivity lipase liveness and application in producing S- type betaxolol hydrochloride with biological split method thereof
  • Saccharomycete with stereoselectivity lipase liveness and application in producing S- type betaxolol hydrochloride with biological split method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] 15 soil samples contaminated by oil were collected from hotels and oil plants in downtown Zhengzhou, weighed 10g of various soil samples, suspended in 0.9% NaCl solution, and filtered with 8 layers of gauze to remove larger impurity particles. Inoculate enrichment medium ((NH 4 ) 2 SO 4 0.2%; K 2 HPO 4 0.2%; NaCl 0.05%; MgSO 4 ·7H 2 O 0.05%; substrate 3%; pH natural), after 71 hours of culture, take 10 mL of the culture solution and use the same method to enrich it twice. Spread a selective plate (the composition is the same as the enrichment culture containing 2% agar). After preliminary screening, 52 strains that can grow on the plate with the substrate as the sole carbon source and produce transparent circles were picked out and transferred to LB medium (yeast extract 0.5%, peptone 1%, NaCl 1%, The slant made of agar 2%, pH 7.5) was preserved and used for the transformation test.

[0061] The 52 strains obtained from the above preliminary screening were respectively inoc...

Embodiment 2

[0082] Preliminary identification of bacterial species was carried out by observing the colony morphology, the shape and size of the bacteria, the presence or absence of spores and hyphae, and the mode of reproduction. The results are as follows:

[0083] The cells are round (5μm) and elliptical (5×6μm), without spores and pseudohyphae. They are mainly fission, and a few are budding.

[0084] The morphology of the strain on different media is different, the specific morphology is as follows:

[0085] YEPD (1) Medium: The moss is small, pink, the surface is smooth, shiny, relatively moist, the edges are transparent, neat, no wrinkles, easy to pick, viscous, tasteless, and the cells are round (5μm) and elliptical (5×6μm).

[0086] Wort culture medium: the moss is small, water-red, the surface is wrinkled, dull, relatively dry, the edges are not neat due to the influence of wrinkles, no spores, no pseudohyphae, easy to pick, sticky, tasteless , The cells are more round (3.5-4μm).

[...

Embodiment 3

[0091] Connect the preserved strain CGMCC.No.2257 to the culture medium (corn steep liquor 2%, peptone 0.5%, glycerol 2%, initial pH 6), and 110 mL (ie 45%) in a 250 mL Erlenmeyer flask. After culturing at 32°C for 16 hours Add 1g of substrate (1) and transform at 28°C for 12h. The reaction solution was extracted three times with an equal volume of ethyl acetate, and the ethyl acetate phase was saturated with NaHCO 3 Wash three times, saturated NaCl solution three times, and then wash once with distilled water, saturated NaHCO 3 The two phases and saturated NaCl phase were back-extracted with ethyl acetate once, the organic phases were combined, and anhydrous NaCl was used. 2 SO 4 After drying, 0.42 g of product (2) was obtained, the yield was 47.5%, and the ee value of the product was 95%. The product (3) was 0.58 g, and the yield was 68%. Among them, the product (2) is our target product. From the product (2), S-betalol hydrochloride is prepared according to the method described...

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Abstract

The invention discloses a yeast which has the stereoselective lipase activity and the application of the yeast in the preparation of an S-type betaxolol hydrochloride by using a biological separation method. The method selects one yeast with the stereoselective lipase activity by screening from the soil, utilizes an immobilized cell which is obtained by immobilizing the wet bacteria or sodium alginate-activated carbon-polyethylenimine as an enzyme preparation, carries out an enantiomer separation to a substrate which contains acyl group and prepares the S-type betaxolol hydrochloride. The conversion method is simple, the cost is low and the stereoselectivity is better. The usage of the bacterial strain can carry out the chiral separation of Beta-receptor blocker of betaxolol etc., ephedrine hydrochloride, epinephrine, levodropropizine, salbutamol sulfate, captopril, zofenopril and other compounds which contain hydroxy group or acyl group at the chiral center, thereby having important application value for promoting the development process of the chiral drugs of China.

Description

Technical field [0001] The invention relates to a yeast strain and its application, in particular to a yeast strain with stereoselective lipase activity and its application in the preparation of S-betaolol hydrochloride by a biological resolution method. Background technique [0002] Glaucoma is a common and difficult eye disease with rapid onset, great harm, and blindness at any time. The incidence rate has increased in recent years. According to the statistics of the World Health Organization, there are currently about 67 million glaucoma patients worldwide, and about 4.5 million people have lost their vision due to glaucoma. In my country, the incidence of primary glaucoma among people over 40 years old The rate is 1% to 2%. Based on the country's 1.3 billion population, there are more than 7 million patients with primary glaucoma. Glaucoma has become the second most common ophthalmological disease in my country, accounting for approximately 14.36% of ophthalmic diseases. Stat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12N9/20C12P41/00C12P7/22C12P13/00C12R1/85
CPCY02P20/52
Inventor 刘宏民李永红王瑞于梅艳
Owner ZHENGZHOU UNIV
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