Rhodococcus sp.D12 and its culture method and application

A culture method and technology for Rhodococcus, applied in the directions of bacteria, hydrolase, fermentation, etc., can solve the problem of no cyanohydrolase for sale, and achieve the effect of optimizing culture conditions

Inactive Publication Date: 2003-11-26
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, there are currently no commercial cyanohydrolases available on the market

Method used

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  • Rhodococcus sp.D12 and its culture method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Culture of Rhodococcus sp.D12

[0024] Use an inoculation needle to pick the screened bacteria or their spores and connect them to the slant, grow in the culture medium for 1-3 days, pH3-8, the composition of the culture medium is glucose 8-15g / L, beef extract 2-8g / L, K 2 HPO 4 or K 2 HPO 4 ·3H 2 O0-2g / L, KH 2 PO 4 0-3g / L, NaCl0-0.5g / L, trace element solution 0-2g / L, agar 12-35g / L. Pour a small amount of sterile water into the slope where the bacteria grow well, pick it up with an inoculation loop and pour it directly into the culture medium without inducer, pH3-8, the composition of the culture medium is glucose 8-15g / L, beef extract 2-8g / L, methacrylamide 1-5g / L, K 2 HPO 4 or K 2 HPO 4 ·3H 2 O0-2g / L, KH 2 PO 4 0-3g / L, NaCl0-0.5g / L, trace element solution 0-2g / L, the above culture medium and culture solution are all sterilized at 107-127°C for 10-40 minutes. Cultivate in a reciprocating shaker at 30°C for 24 hours (or 32 hours)...

Embodiment 2

[0026] Effects of Various Inducers on Enzyme Activity and Stereoselectivity

[0027] The effects of various nitriles, amides and acids on the enzyme activity were investigated by fixed culture conditions, reaction conditions, and other components in the culture solution formula except the inducer.

[0028] Pick the well-grown Rhodococcus sp.D12 from the slope and put it into the culture medium, and cultivate it on a reciprocating shaker at room temperature for 48 hours. The formula of the culture medium is trace element solution 1.0g / L, K 2 HPO 4 1.03g / L, KH 2 PO 4 0.75g / L, NaCl0.1g / L, beef extract 5g / L, glucose 10g / L, inducer 10g / L, pH7.0, 30℃.

[0029] Take the newly cultured Rhodococcus sp.D12 cells, suspend in K 2 HPO 4 -KH 2 PO 4 In the buffer solution, the concentration is 10g wet bacteria / 40mlK 2 HPO 4 -KH 2 PO 4 Buffer solution, add substrate 100mg 2-phenylpropionitrile to react, pH7.0, 30°C. TLC followed the progress of the reaction. A...

Embodiment 3

[0038] Effects of Various Nitrogen Sources on Enzyme Activity and Stereoselectivity

[0039] Fixed culture conditions, reaction conditions, other components in the culture solution formula except the nitrogen source, and other experimental conditions were the same as in Example 2, and the effects of various nitrogen sources on the enzyme activity were investigated respectively. The results are shown in Table 2.

[0040] Table 2 Nitrogen source Growth situation Reaction time 2-phenylpropanamide 2-phenylpropionic acid% Total activity Specific activity (unit / g

[0041] (dry weight, g / l) (h) % (yield / ee.) (yield / ee.) (unit / l) dry weight) no 0.84 ND yeast extract 5.47 20 33 / 85 60 / 66 29.3 5.36 beef extract 5.89 18 12 / 99 80 / 60 39.5 6.71 Ammonium nitrate 5.65 19 44 / 63 50 / 59 28.1 4.98 Soybean powder extract 5.47 19 17 / 52 76 / 11 b 34.1 6.23 peptone a 5.26 19 23 / 40 b 27 / 43 15.0 2.84 L-glutamate 4.21 19...

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Abstract

A Rhodococcus sp.D12 is obtained by screening from soil, separating and purifying. Its optimized culture condition including kind of inductor, nitrogen source, carbon source, inducing period and dosage of inductor is also disclosed. Said strain can be used to catalytically hydrolyzing the nitrile compound into carboxylic acid and amide or their mixture.

Description

technical field [0001] The invention relates to a biocatalyst, specifically Rhodococcus sp. D12 (Rhodococcus sp.), its culture conditions and its use for cyanohydrolysis. Background technique [0002] Nitriles are important substances used in organic synthesis, but are also some of the worst pollutants. Therefore, the biodegradation of nitriles is particularly important. Industrially using acrylonitrile as a raw material to synthesize the compound acrylamide with a wide range of uses by microbial catalysis is the most typical and successful example. In the study of asymmetric synthesis catalyzed by cyanohydrolases, the chiral resolution of α-arylpropionitriles has attracted much attention, because their hydrolyzed products α-arylpropionic acids are a very important class of non-steroidal antibacterial and anti-inflammatory agents. Drugs, some varieties have achieved great success in the market [Faber, K.2000.Biotransformations inorganic chemistry: a textbook, 4th ed.Spring...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/14C12P7/40C12P13/02
Inventor 李祖义吴中柳
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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