Penicillin acylase, as well as high-yield strain and application thereof
A high-yield penicillin acylase and penicillin acylase technology, applied in the fields of application, bacteria, hydrolase, etc., can solve the problems of bacterial drug resistance, etc., and achieve high activity, high substrate specificity, and organic solvent tolerance strong effect
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Embodiment 1
[0030] This example illustrates the screening procedure for natural strains producing penicillin acylase.
[0031] The initial screening adopts the following method: use high-concentration penicillin G as the screening pressure to obtain high-concentration penicillin-resistant microorganisms from soil samples around Nanjing and Suzhou pharmaceutical factories. The formula of specific screening medium is: yeast extract 5 g / L, peptone 5 g / L, NaCl 2 g / L, phenylacetic acid 2 g / L, penicillin 10 5 ~10 7 U / ml , pH 7.0, solvent is water. The culture temperature was 30°C, the culture time was 24-48 h, and the shaker speed was 180 rpm. This method can screen a large number of high-concentration penicillin-resistant microorganisms.
[0032]The strains obtained from the primary screening were re-screened, and the specific method was as follows: 3-phenylacetamide-6-nitrobenzoic acid (abbreviated as NIPAB, the same below) with a concentration of 2mg / ml was prepared with 50mM phosphate bu...
Embodiment 2
[0035] This example illustrates the biological properties and identification of penicillin acylase producing strain K18.
[0036] Biological properties of the strain K18: the strain is a Gram-negative strain without spores, the colony is light yellow, slightly raised, round, with neat edges, moist, translucent, and smooth; the microscopic observation shows that the bacteria are bacilli, 0.5-1.2 ×0.5~2.6μm, obligate aerobic, the optimum growth temperature is 28℃~37℃. Its physiological and biochemical characteristics are manifested in: the results of catalase reaction, oxidase reaction and nitric acid reduction reaction are positive, the results of gelatin reaction are negative, it has power, oxidizes xylose to produce acid, and can use xylose, fructose, mannose, etc. Trehalose, mannitol, and arabinose are not available.
[0037] After being identified by the BIOLOG automatic bacterial identification instrument, the strain K18 and Achromobacter The Sim value of the genus was ...
Embodiment 3
[0040] This example illustrates Achromobacter xylosoxidans ( Achromobacter xylosoxidans ) K18 fermentation enzyme production method.
[0041] Plate medium formula: Yeast powder 5 g / L, peptone 10 g / L, NaCl 10 g / L, agar 1.8-2.0 g / L, pH 7.0, solvent is water.
[0042] Seed medium formula: yeast powder 5 g / L, peptone 10 g / L, NaCl 10 g / L, pH 7.0, solvent is water.
[0043] Preparation of seed solution: The volume of seed medium in a 250 mL Erlenmeyer flask was 30 mL. The bacterial strain K18 cultured on the plate for 24 hours was scraped and inserted into the seed medium, the rotation speed of the shaker was 180 rpm, the cultivation temperature was 30°C, and the cultivation time was 8-10 hours to obtain the seed liquid.
[0044] method 1
[0045] Enzyme production fermentation medium formula: glycerol 5 g / L, yeast powder 7.5 g / L, NaCl 2 g / L, Triton X-100 0.25 mL / L, phenylacetic acid 1 g / L, pH 6.0, solvent is water.
[0046] The filling volume of the enzyme-producing fermentatio...
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