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Heterologous expression of milk-clotting protease pruduced by Thrmomucor indicae-seudaticae N-31 in Escherichia coli and Pichia Pastoris

Grant number: 17/14629-9
Support type:Scholarships in Brazil - Master
Effective date (Start): March 01, 2018
Effective date (End): February 28, 2019
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
Principal researcher:Roberto da Silva
Grantee:Waldir Eduardo Simioni Pereira
Home Institution: Instituto de Biociências, Letras e Ciências Exatas (IBILCE). Universidade Estadual Paulista (UNESP). Campus de São José do Rio Preto. São José do Rio Preto , SP, Brazil

Abstract

The main enzymatic coagulants substituents of conventional rennet, used in the dairy industry, are like proteases. These enzymes act on proteolytic activity, cleaving peptides in proteins and peptides. Aspartic proteases (EC 3.4.23) are enzymes which have the function of specifically hydrolyzing the º-casein peptide bond between the amino acids Phe105-Met106 releasing para-º-casein and glycomacropeptide, which promotes the destabilization of casein micelles, resulting in milk coagulation. The substitution of traditional rennet with enzymatic coagulants is extremely necessary, aiming at reducing the slaughter of animals for these purposes. Even though they are produced by a range of microorganisms, there are still restrictions that hinder the process of obtaining these biocatalysts. To minimize such difficulties, the use of Molecular Biology tools represents an efficient strategy to improve enzymatic production. Cloning and expression of genes encoding proteases in bacteria and yeast have become an interesting alternative for the use of enzymes in consolidated bioprocesses for cheese production. In this way, Escherichia coli and Pichia pastoris are configured as viable systems for heterologous expression. Taking into account the need for a substitute for conventional rennet and its production in significant quantities, the objective of this work will be to clone and express a protease produced by Thermomucor indicae-seudaticae N31 in E. coli and P. pastoris, as well as the biochemical characterization of this enzyme. (AU)

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
PEREIRA, WALDIR EDUARDO SIMIONI; DA SILVA, RONIVALDO RODRIGUES; DE AMO, GABRIELA SALVADOR; RULLER, ROBERTO; KISHI, LUCIANO TAKESHI; BOSCOLO, MAURICIO; GOMES, ELENI; DA SILVA, ROBERTO. A Collagenolytic Aspartic Protease from Thermomucor indicae-seudaticae Expressed in Escherichia coli and Pichia pastoris. Applied Biochemistry and Biotechnology, v. 191, n. 3 FEB 2020. Web of Science Citations: 1.
Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
PEREIRA, Waldir Eduardo Simioni. Expressão heteróloga de uma protease coagulante produzida por Thermomucor indicae-seudaticae N31 em Escherichia coli e Pichia pastoris. 2019. 62 f. Master's Dissertation - Universidade Estadual Paulista "Júlio de Mesquita Filho" Instituto de Biociências, Letras e Ciências Exatas..

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