Details
| Originalsprache | Englisch |
|---|---|
| Aufsatznummer | e2935 |
| Seiten (von - bis) | e2935 |
| Fachzeitschrift | Biotechnology progress |
| Jahrgang | 36 |
| Ausgabenummer | 2 |
| Frühes Online-Datum | 23 Okt. 2019 |
| Publikationsstatus | Veröffentlicht - 9 Apr. 2020 |
Abstract
The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.
ASJC Scopus Sachgebiete
- Biochemie, Genetik und Molekularbiologie (insg.)
- Biotechnologie
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in: Biotechnology progress, Jahrgang 36, Nr. 2, e2935, 09.04.2020, S. e2935.
Publikation: Beitrag in Fachzeitschrift › Artikel › Forschung › Peer-Review
}
TY - JOUR
T1 - Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase
AU - Ekramzadeh, Kimia
AU - Brämer, Chantal
AU - Frister, Thore
AU - Fohrer, Jörg
AU - Kirschning, Andreas
AU - Scheper, Thomas
AU - Beutel, Sascha
N1 - Funding information: We would like to thank Prof. Ralf G. Berger and Dr. Ulrich Krings (both from Institute of Food Chemistry, Leibniz University Hannover, Germany) for the performance of GC–MS measurements.
PY - 2020/4/9
Y1 - 2020/4/9
N2 - The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.
AB - The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism.
KW - biocatalysis
KW - biocatalytic mechanism
KW - patchouli oil
KW - patchoulol synthase
KW - sesquiterpenes
UR - http://www.scopus.com/inward/record.url?scp=85074864792&partnerID=8YFLogxK
U2 - 10.1002/btpr.2935
DO - 10.1002/btpr.2935
M3 - Article
C2 - 31643144
AN - SCOPUS:85074864792
VL - 36
SP - e2935
JO - Biotechnology progress
JF - Biotechnology progress
SN - 8756-7938
IS - 2
M1 - e2935
ER -