Details
| Original language | English |
|---|---|
| Article number | e2812 |
| Number of pages | 10 |
| Journal | Biotechnology progress |
| Volume | 35 |
| Issue number | 4 |
| Early online date | 1 Apr 2019 |
| Publication status | Published - 14 Aug 2019 |
Abstract
The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co2+-immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl2 and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm2 each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (-)-patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.
Keywords
- membrane chromatography, patchouli oil, patchoulol synthase, periodic counter-current chromatography, sesquiterpenes, Recombinant Proteins/chemistry, Chromatography, Affinity, Adsorption, Isomerases/chemistry, Escherichia coli/enzymology
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
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In: Biotechnology progress, Vol. 35, No. 4, e2812, 14.08.2019.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Optimization of continuous purification of recombinant patchoulol synthase from Escherichia coli with membrane adsorbers
AU - Brämer, Chantal
AU - Ekramzadeh, Kimia
AU - Lammers, Frank
AU - Scheper, Thomas
AU - Beutel, Sascha
N1 - Funding information: We would like to thank the EU for financial support of the project “Refinement of plant raw materials” within the frame of the EFRE-program.
PY - 2019/8/14
Y1 - 2019/8/14
N2 - The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co2+-immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl2 and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm2 each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (-)-patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.
AB - The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co2+-immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl2 and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm2 each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (-)-patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.
KW - membrane chromatography
KW - patchouli oil
KW - patchoulol synthase
KW - periodic counter-current chromatography
KW - sesquiterpenes
KW - Recombinant Proteins/chemistry
KW - Chromatography, Affinity
KW - Adsorption
KW - Isomerases/chemistry
KW - Escherichia coli/enzymology
UR - http://www.scopus.com/inward/record.url?scp=85064070459&partnerID=8YFLogxK
U2 - 10.1002/btpr.2812
DO - 10.1002/btpr.2812
M3 - Article
C2 - 30932363
AN - SCOPUS:85064070459
VL - 35
JO - Biotechnology progress
JF - Biotechnology progress
SN - 8756-7938
IS - 4
M1 - e2812
ER -