Periodic Reporting for period 1 - PATTENZYME (Sequential and selective patterning of enzymes in modular electrochemical based biorreactor for continuos production of pharmaceutical materials)
Reporting period: 2022-03-17 to 2024-03-16
PATTENZYME addresses a gap in the state of the art in the preparation of pharmaceutical materials by using electrochemical approaches for the targeted and selective immobilization of bio/catalysts in modular 3D printed reactors.
The pharmaceutical industry faces a significant challenge in its reliance on batch processes with synthesis of active pharmaceutical ingredients occurring via individual reactions, methods that are not well suited to modern, flexible, manufacturing processes that need to be agile and responsive to changing needs.
PATTENZYME prepares a modular bioreactor for the controlled delivery of H2O2 in 3D-printed flow reactors for the selective synthesis of pharmaceutical materials in a stand-alone environment.
PATTENZYME is an original and innovative project which 3D printed reactors with catalysts incorporated in a patterned manner in the channels that utilizes continuous flow technology for the controlled delivery of the oxidant as the first step in a cascade reaction. In the subsequent step, bio/catalysts will be utilized for the catalytic oxidation of substrates for the production of pharmaceutical materials.
Overall objectives:
Preparation and characterization of NPG
Optimization of immobilization of UPO and of Mn salen on modified NPG.
Reaction/flow modelling and modelling of patterning and loading of catalysts.
Assembly of 3D printed flow cells that incorporate immobilized bio/catalysts in aqueous and non-aqueous solutions.
The pharmaceutical industry faces a significant challenge in its reliance on batch processes with synthesis of active pharmaceutical ingredients occurring via individual reactions, methods that are not well suited to modern, flexible, manufacturing processes that need to be agile and responsive to changing needs.
PATTENZYME prepares a modular bioreactor for the controlled delivery of H2O2 in 3D-printed flow reactors for the selective synthesis of pharmaceutical materials in a stand-alone environment.
PATTENZYME is an original and innovative project which 3D printed reactors with catalysts incorporated in a patterned manner in the channels that utilizes continuous flow technology for the controlled delivery of the oxidant as the first step in a cascade reaction. In the subsequent step, bio/catalysts will be utilized for the catalytic oxidation of substrates for the production of pharmaceutical materials.
Overall objectives:
Preparation and characterization of NPG
Optimization of immobilization of UPO and of Mn salen on modified NPG.
Reaction/flow modelling and modelling of patterning and loading of catalysts.
Assembly of 3D printed flow cells that incorporate immobilized bio/catalysts in aqueous and non-aqueous solutions.
PATTENZYME project started on 16/03/22 and due to the resignation of the candidate finished on 14/10/22.. During this period the ER focused on WP1 consisting on the preparation and characterization of NPG electrodes with pore size distributions and surface areas. Some initial work on WP2 commenced.
For WP1 the ER has elaborated the following protocols for this propose.
1.Protocol for preparation of nanoporous gold electrodes (NPG)
Gold is an attractive material for biocatalytical applications.1 In comparison to polycrystalline gold (PG), nanoporous gold (NPG), possesses a much higher surface-to-volume ratio that can accommodate higher enzyme loadings. The presence of pores protects the enzyme from the bulk solution, limiting interference effects and protecting the enzyme from denaturation. Nanostructured gold can be prepared using a wide variety of methods.2
Following the procedure described by Siepenkoetter et al1, the protocol for preparation and characterization a library of nanoporous gold electrodes (NPG) with varying pore size is described below.
NPG were fabricated on commercial glass slides at room temperature. Firstly, the glass sheets were cleaned in Ar plasma under vacuum. Then a 10 nm Ti were sputtering followed by a pure Au layer with one third of the thickness of the following alloy layer. After that an Ag70/Au30 alloy layer (100 nm) was deposited on top of this layer. The resultant sheet was cut with a circular saw into squares of 0.5–0.7 cm2. The electrodes were de-alloyed with 70% HNO3 at varying temperatures and time periods to obtain different pore sizes.1 A silver wire was soldered to the result NPG electrode using an indium wire. The soldering point was supported by epoxy glue to ensure adhesion to the surface. Finally, after drying overnight, the electrode components were insulated with dielectric paste, defining the electroactive area.
2.Protocol for characterizatrion of nanoporous gold electrodes (NPG)
2.1 Electrochemical characterization
Firstly, the electrodes were cleaned and characterized by cycling in H2SO4 in the potential range between 0.2 and 1.65 V vs Ag/AgCl at a scan rate of 100 mV/s for two cycles. The electrodes were raised with deionized water and dried under vacuum for 10 minutes. The electro active area of the electrodes was calculated, resulting in an average roughness of 1.1 ± 0.3.
2.2 Morphology Characterization
The morphology of the NPG electrode was determined by scanning electron microscope (SEM) measurements. SEM images were taken at three different magnifications: 25 k, 80 k and 200k magnification at a working distance of 2 mm and 10 kV and then converted into a black and white image with the ISODATA function of ImageJ software, were used to determine the pore size distribution of NPG. Finally, the pore sizes were determined measuring the distances between the pores.
WP2 the ER initially used chloroperoxidase (CPO) as a model protein. The ER immobilized CPO on an agarose matrix following the protocol previously described1 for the immobilization of amine transaminase. The catalytic activity was examined using thioanisole as substrate.
Briefly, agarose (10 g) was suspended in deionized water at 4°C under mechanical stirring. A 1.7 M solution of NaOH containing NaBH4.
After that 50 µl of CPO was incubated with glyoxyl-agarose (1 g) in NaHCO3 buffer (pH 10, 50 mM, 10 mL) at 4°C for 3 h under mechanical stirring. Chemical reduction of imines was carried out over 30 min by adding NaCNBH3 (10 mg) to the mixture. The immobilized enzyme was then filtered under vacuum and washed thoroughly with deionized water and K2HPO4 buffer (pH 8, 50 mM) and stored at 4°C. The immobilization procedure was monitored by evaluating the amounts of residual protein and activity of the supernatant by Bradford and activity assay, respectively.
Finally the catalytic activity in in 0.1 M KPi pH 5 was evaluated employed thioanizol as a model substrate. The conversion the storage and the recycling of the system was studied with success results.
Dissemination of the results
the results obtained in PATTENZYME have been disseminated to both industrial and academic researchers in accordance with the European Charter for Researchers:
a) Publication of articles: with the obtained results two papers are in preparation to be publish in high-level peer-reviewed journal articles
b) Participation in conferences: oral presentations to disseminate the results of PATTENZYME
a. XLII Meeting of the RSEQ Electrochemical Specialized Group (42 GERSEQ 2022). Santander, Spain. 6-8th July 2022.
b. ISE Regional Meeting in Prague, Czech Republic. August 15-19th
c) Dissemination to industry: The ER has participated in quarterly technical meetings of SSPC which include industry partner
For WP1 the ER has elaborated the following protocols for this propose.
1.Protocol for preparation of nanoporous gold electrodes (NPG)
Gold is an attractive material for biocatalytical applications.1 In comparison to polycrystalline gold (PG), nanoporous gold (NPG), possesses a much higher surface-to-volume ratio that can accommodate higher enzyme loadings. The presence of pores protects the enzyme from the bulk solution, limiting interference effects and protecting the enzyme from denaturation. Nanostructured gold can be prepared using a wide variety of methods.2
Following the procedure described by Siepenkoetter et al1, the protocol for preparation and characterization a library of nanoporous gold electrodes (NPG) with varying pore size is described below.
NPG were fabricated on commercial glass slides at room temperature. Firstly, the glass sheets were cleaned in Ar plasma under vacuum. Then a 10 nm Ti were sputtering followed by a pure Au layer with one third of the thickness of the following alloy layer. After that an Ag70/Au30 alloy layer (100 nm) was deposited on top of this layer. The resultant sheet was cut with a circular saw into squares of 0.5–0.7 cm2. The electrodes were de-alloyed with 70% HNO3 at varying temperatures and time periods to obtain different pore sizes.1 A silver wire was soldered to the result NPG electrode using an indium wire. The soldering point was supported by epoxy glue to ensure adhesion to the surface. Finally, after drying overnight, the electrode components were insulated with dielectric paste, defining the electroactive area.
2.Protocol for characterizatrion of nanoporous gold electrodes (NPG)
2.1 Electrochemical characterization
Firstly, the electrodes were cleaned and characterized by cycling in H2SO4 in the potential range between 0.2 and 1.65 V vs Ag/AgCl at a scan rate of 100 mV/s for two cycles. The electrodes were raised with deionized water and dried under vacuum for 10 minutes. The electro active area of the electrodes was calculated, resulting in an average roughness of 1.1 ± 0.3.
2.2 Morphology Characterization
The morphology of the NPG electrode was determined by scanning electron microscope (SEM) measurements. SEM images were taken at three different magnifications: 25 k, 80 k and 200k magnification at a working distance of 2 mm and 10 kV and then converted into a black and white image with the ISODATA function of ImageJ software, were used to determine the pore size distribution of NPG. Finally, the pore sizes were determined measuring the distances between the pores.
WP2 the ER initially used chloroperoxidase (CPO) as a model protein. The ER immobilized CPO on an agarose matrix following the protocol previously described1 for the immobilization of amine transaminase. The catalytic activity was examined using thioanisole as substrate.
Briefly, agarose (10 g) was suspended in deionized water at 4°C under mechanical stirring. A 1.7 M solution of NaOH containing NaBH4.
After that 50 µl of CPO was incubated with glyoxyl-agarose (1 g) in NaHCO3 buffer (pH 10, 50 mM, 10 mL) at 4°C for 3 h under mechanical stirring. Chemical reduction of imines was carried out over 30 min by adding NaCNBH3 (10 mg) to the mixture. The immobilized enzyme was then filtered under vacuum and washed thoroughly with deionized water and K2HPO4 buffer (pH 8, 50 mM) and stored at 4°C. The immobilization procedure was monitored by evaluating the amounts of residual protein and activity of the supernatant by Bradford and activity assay, respectively.
Finally the catalytic activity in in 0.1 M KPi pH 5 was evaluated employed thioanizol as a model substrate. The conversion the storage and the recycling of the system was studied with success results.
Dissemination of the results
the results obtained in PATTENZYME have been disseminated to both industrial and academic researchers in accordance with the European Charter for Researchers:
a) Publication of articles: with the obtained results two papers are in preparation to be publish in high-level peer-reviewed journal articles
b) Participation in conferences: oral presentations to disseminate the results of PATTENZYME
a. XLII Meeting of the RSEQ Electrochemical Specialized Group (42 GERSEQ 2022). Santander, Spain. 6-8th July 2022.
b. ISE Regional Meeting in Prague, Czech Republic. August 15-19th
c) Dissemination to industry: The ER has participated in quarterly technical meetings of SSPC which include industry partner
The ER has obtained a position in the University of Seville in Spain which forced her to resign from the project on 14th of October.