Overexpression of a bacterial chymotrypsin: Application for L-amino acid ester hydrolysis
| Autor: | Loredano Pollegioni, Gianluca Molla, Paola D'Arrigo, Stefano Servi, Federica Volontè, Fiorenza Viani, Ines Pisanelli |
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| Rok vydání: | 2011 |
| Předmět: |
Metarhizium
Proteases Overexpression medicine.medical_treatment Thioredoxin reductase Molecular Sequence Data Bioengineering Phenylalanine Chymotrypsinogen Applied Microbiology and Biotechnology Biochemistry Pichia pastoris Fungal Proteins Escherichia coli medicine Chymotrypsin Amino Acid Sequence Amino Acids DNA Fungal chemistry.chemical_classification Protease Base Sequence Esterification biology Hydrolysis Chymotrypsin Nonanimal protease Overexpression Amino acid ester hydrolysis Stereoisomerism biology.organism_classification Recombinant Proteins Amino acid Kinetics chemistry Amino acid ester hydrolysis biology.protein Biotechnology Nonanimal protease |
| Zdroj: | Enzyme and microbial technology 49 (2011): 560–566. doi:10.1016/j.enzmictec.2011.06.009 info:cnr-pdr/source/autori:Volonte, Federica; Pisanelli, Ines; D'Arrigo, Paola; Viani, Fiorenza; Molla, Gianluca; Servi, Stefano; Pollegioni, Loredano/titolo:Overexpression of a bacterial chymotrypsin: Application for L-amino acid ester hydrolysis/doi:10.1016%2Fj.enzmictec.2011.06.009/rivista:Enzyme and microbial technology/anno:2011/pagina_da:560/pagina_a:566/intervallo_pagine:560–566/volume:49 |
| DOI: | 10.1016/j.enzmictec.2011.06.009 |
| Popis: | In this work, a reliable protocol was designed to rapidly express and purify a microbial chymotrypsin(ogen) as a useful alternative to using animal proteases. The cDNA encoding for chymotrypsinogen from the deuteromycete Metarhizium anisopliae (chy1) was overexpressed in an Origami2(DE3) E. coli strain deficient in thioredoxin reductase and glutathione reductase activities, thus possibly allowing disulfide exchange. By using a quick purification protocol, in which the hexahistidine tag was added at the C-terminal end of the protease, the recombinant CHY1 protein could be purified in a single step on an Ni-NTA column as a mixture of 19.5- and 15-kDa mature active forms and did not require further activation/maturation steps. This expression and purification procedure offers an easier and faster means of producing recombinant CHY1 chymotrypsin than that previously described for Pichia pastoris. The kinetic properties could be characterized and CHY1 chymotrypsin was demonstrated to efficiently catalyze N-acetylated L-phenylalanine and L-tyrosine methyl ester hydrolysis. (C) 2011 Elsevier Inc. All rights reserved. |
| Databáze: | OpenAIRE |
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