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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Volontè, Federica Pisanelli, Ines D'Arrigo, Paola Viani, Fiorenza Molla, Gianluca Servi, Stefano Pollegioni, Loredano |
| Description | Country affiliation: Italy Author Affiliation: Volontè F ( Dipartimento di Biotecnologie e Scienze Molecolari, Università degli Studi dell'Insubria, Varese, Italy.) |
| Abstract | 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. |
| File Format | HTM / HTML |
| ISSN | 01410229 |
| Issue Number | 6-7 |
| Volume Number | 49 |
| e-ISSN | 18790909 |
| Journal | Enzyme and Microbial Technology |
| Language | English |
| Publisher | Elsevier |
| Publisher Date | 2011-12-10 |
| Publisher Place | United States |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Discipline Microbiology Discipline Biochemistry Discipline Biotechnology Amino Acids Metabolism Chymotrypsin Amino Acid Sequence Chemistry Base Sequence Genetics Isolation & Purification Dna, Fungal Escherichia Coli Enzymology Esterification Fungal Proteins Hydrolysis Kinetics Metarhizium Molecular Sequence Data Recombinant Proteins Stereoisomerism Journal Article Research Support, Non-u.s. Gov't |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry Bioengineering Applied Microbiology and Biotechnology Biotechnology |
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