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 -