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

Inactive Publication Date: 2013-07-03
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] β-lactam antibiotics are a very important class of antibiotics in clinical practice, but their large-scale use has also brought some negative effects. The abuse of antibiotics has led to more and more bacteria becoming resistant to them. To overcome this problem An important approach is the development and use of new semi-synthetic antibiotics

Method used

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  • Penicillin acylase, as well as high-yield strain and application thereof
  • Penicillin acylase, as well as high-yield strain and application thereof
  • Penicillin acylase, as well as high-yield strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention provides penicillin acylase, as well as a high-yield strain and an application thereof, belonging to the field of bio-pharmaceuticals. The strain is Achromobacter xylosoxidans K18 with the collection number of CCTCC NO: M2012541. The penicillin acylase is obtained by fermenting the strain K18. The amino acid sequence of the penicillin acylase is as shown in SEQ ID NO: 2, and the gene sequence of the penicillin acylase is as shown in SEQ ID NO: 1. The invention further provides the application of the penicillin acylase in synthesis of beta-lactam type antibiotics. The strain K18 is the high-yield strain of the penicillin acylase, and when fermentation is performed through a culture medium containing an inducing agent, the yield of the penicillin acylase can be above 200U/L. The penicillin acylase produced by the strain K18 has the advantages of high temperature resistance, capability of keeping higher activity under an acidic environment, higher specificity of a substrate, strong organic solvent tolerance and the like.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to penicillin acylase, its high-yield strain and application. Background technique [0002] Penicillin acylase (penicillin acylase, EC3.5.1.11), also known as penicillin amidase or penicillin aminohydrolase, is a subclass of the β-lactam antibiotic acyltransferase family. This kind of enzyme is currently one of the few important enzymes that has been used in large-scale industrialization. [0003] Penicillin acylase plays an important role in the pharmaceutical industry. It is mainly used in the industrial production of semi-synthetic penicillins and cephalosporins. It can catalyze the preparation of new β-lactam antibiotics with high efficiency, broad spectrum and suitable for different purposes. On the one hand, the enzyme can catalyze the hydrolysis of penicillin or cephalosporin to obtain important intermediates of semi-synthetic antibiotics, 6-aminopenici...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/84C12N15/55C12P35/04C12P37/04C12R1/025
Inventor 何冰芳孔佳佳王欣潘鑫柏中中吴斌
Owner NANJING UNIV OF TECH
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