Details
| Originalsprache | Englisch |
|---|---|
| Seiten (von - bis) | 1840-1846 |
| Seitenumfang | 7 |
| Fachzeitschrift | Process biochemistry |
| Jahrgang | 46 |
| Ausgabenummer | 9 |
| Publikationsstatus | Veröffentlicht - 24 Juni 2011 |
Abstract
The submerged cultivation of Pleurotus ostreatus var. florida on wheat gluten led to an increased solubility of the substrate. The coding sequence of one of the extracellular peptidases was obtained by PCR with degenerated primers and RACE. The sequence amounted to 1161 bp, translating into 386 aa with a molecular mass of 38.7 kDa. By comparison with homologous proteins the new enzyme was classified as a subtilisin-like peptidase of the proteinase K subfamily. P. ostreatus peptidase 1 (POP1) was expressed as a preproenzyme, whose 19 aa signal sequence was cleaved off during export into the extracellular space. The prosequence acted as an intramolecular chaperone and temporary inhibitor and was degraded in an autocatalytic process. The heterologous expression in Escherichia coli resulted in the formation of inclusion bodies, from which the peptidase was refolded by using β-cyclodextrin and CTAB. POP1 showed optima at pH 7.5 and 37 °C. It was most stable at pH 8 and 30 °C. The peptidase was completely inhibited by PMSF, antipain and Cu 2+, while Ca2+ significantly enhanced its performance. The hydrolysis of N-Suc-AAPF-pNA was almost tripled in the presence of 5 M NaCl.
ASJC Scopus Sachgebiete
- Chemische Verfahrenstechnik (insg.)
- Bioengineering
- Biochemie, Genetik und Molekularbiologie (insg.)
- Biochemie
- Immunologie und Mikrobiologie (insg.)
- Angewandte Mikrobiologie und Biotechnologie
Zitieren
- Standard
- Harvard
- Apa
- Vancouver
- BibTex
- RIS
in: Process biochemistry, Jahrgang 46, Nr. 9, 24.06.2011, S. 1840-1846.
Publikation: Beitrag in Fachzeitschrift › Artikel › Forschung › Peer-Review
}
TY - JOUR
T1 - Heterologous expression, refolding and characterization of a salt activated subtilase from Pleurotus ostreatus
AU - Eisele, Nadine
AU - Linke, Diana
AU - Nimtz, Manfred
AU - Berger, Ralf G.
PY - 2011/6/24
Y1 - 2011/6/24
N2 - The submerged cultivation of Pleurotus ostreatus var. florida on wheat gluten led to an increased solubility of the substrate. The coding sequence of one of the extracellular peptidases was obtained by PCR with degenerated primers and RACE. The sequence amounted to 1161 bp, translating into 386 aa with a molecular mass of 38.7 kDa. By comparison with homologous proteins the new enzyme was classified as a subtilisin-like peptidase of the proteinase K subfamily. P. ostreatus peptidase 1 (POP1) was expressed as a preproenzyme, whose 19 aa signal sequence was cleaved off during export into the extracellular space. The prosequence acted as an intramolecular chaperone and temporary inhibitor and was degraded in an autocatalytic process. The heterologous expression in Escherichia coli resulted in the formation of inclusion bodies, from which the peptidase was refolded by using β-cyclodextrin and CTAB. POP1 showed optima at pH 7.5 and 37 °C. It was most stable at pH 8 and 30 °C. The peptidase was completely inhibited by PMSF, antipain and Cu 2+, while Ca2+ significantly enhanced its performance. The hydrolysis of N-Suc-AAPF-pNA was almost tripled in the presence of 5 M NaCl.
AB - The submerged cultivation of Pleurotus ostreatus var. florida on wheat gluten led to an increased solubility of the substrate. The coding sequence of one of the extracellular peptidases was obtained by PCR with degenerated primers and RACE. The sequence amounted to 1161 bp, translating into 386 aa with a molecular mass of 38.7 kDa. By comparison with homologous proteins the new enzyme was classified as a subtilisin-like peptidase of the proteinase K subfamily. P. ostreatus peptidase 1 (POP1) was expressed as a preproenzyme, whose 19 aa signal sequence was cleaved off during export into the extracellular space. The prosequence acted as an intramolecular chaperone and temporary inhibitor and was degraded in an autocatalytic process. The heterologous expression in Escherichia coli resulted in the formation of inclusion bodies, from which the peptidase was refolded by using β-cyclodextrin and CTAB. POP1 showed optima at pH 7.5 and 37 °C. It was most stable at pH 8 and 30 °C. The peptidase was completely inhibited by PMSF, antipain and Cu 2+, while Ca2+ significantly enhanced its performance. The hydrolysis of N-Suc-AAPF-pNA was almost tripled in the presence of 5 M NaCl.
KW - β-Cyclodextrin
KW - Basidiomycete
KW - Pleurotus ostreatus
KW - Refolding
KW - Salt activation
KW - Subtilase
UR - http://www.scopus.com/inward/record.url?scp=80051543926&partnerID=8YFLogxK
U2 - 10.1016/j.procbio.2011.06.013
DO - 10.1016/j.procbio.2011.06.013
M3 - Article
AN - SCOPUS:80051543926
VL - 46
SP - 1840
EP - 1846
JO - Process biochemistry
JF - Process biochemistry
SN - 1359-5113
IS - 9
ER -