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Comparative study of transformation techniques of the different hosts for recombinant penicillin G acylase production

Abstract

Penicillin G acylase (PGA) is a very important catalyst for the pharmaceutical industry. Its main application is in the hydrolysis of penicillin G, generating 6-aminopenicillanic acid, a precursor of semi-synthetic antibiotics. The focus of this project is the application of biology molecular techniques to the penicillin G acylase production process by Bacillus megaterium. The project aims at comparing techniques for transformation of B. megaterium, which unlike E. coli is Gram-positive microorganism. The microorganism transformation is a critical step in recombinant DNA technology. Depending on the microorganism, a technique can be more appropriate than another. In this project, different transformation techniques will be compared: treatment with bivalent cations, electroporation and protoplasts, in E. coli for host propagation and in B. megaterium for PGA expression. The proponent has got her PhD degree studying applications of computational techniques to infer variables in the production of PGA by wild B. megaterium, and now intends to specialize in the use of molecular biology techniques to improve the enzyme expression. The application of heterologous expression to bioprocesses is an iterative and interactive process, often demanding going back to benchtop to improve the clone. The existence of chemical engineers with good knowledge of molecular biology techniques is very important to increase the productivity of bioprocesses with recombinant microorganisms. The proponent is part of a research group with access to a well-equipped molecular biology laboratory and that includes researchers in recombinant PGA production. Nowadays, the group has one post-doctoral expert in this area, two PhD students in progress and contributes with different groups in Brazil and in the world. This project will be the first step in the specialization of the proponent in molecular biology. Thus, in addition to the progress in the technology focusing an enzyme of great interest for the pharmaceutical industry, this project will allow the consolidation to the molecular biology laboratory in researches with heterologous protein production in DEQ/UFSCar. (AU)

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