Evaluation of different enzyme immobilisation methods for multi-step synthesis in microreactors
Lead Research Organisation:
University College London
Department Name: Biochemical Engineering
Abstract
The aim of this project is to establish a framework for systematic comparison of different immobilisation methods (entrapment, affinity, and covalent binding) for three different enzyme-catalysed processes (inulinase, transketolase and transaminase) in batch and continuous flow systems under defined reaction conditions. To achieve this, the performance of the different immobilisation methods will be compared in terms of enzyme activity, productivity, and stability. Initially, reactions will be performed in batch mode with inulinase. Then the most promising immobilisation methods based on enzyme stability and productivity will be applied to the second enzyme, transketolase, in order to determine if the methods can be transferred directly. Based on data from the batch reactions, two methods will be chosen to immobilise first transketolase and then transaminase and carry out reactions under continuous flow in microreactors. The aim is to determine if the immobilised enzymes perform similar in batch and continuous flow systems. The findings of this project will provide a framework for the selection of suitable immobilisation methods for rapid testing of enzymes at the microscale thereby reducing time and cost of biocatalyst development.
Planned Impact
The IDC has a proven track record of delivering impact from its research and training activities and this will continue in the new Centre. The main types of impact relate to: (i) provision of highly skilled EngD graduates; (ii) generation of intellectual property (IP) in support of collaborating companies or for new venture creation; (iii) knowledge exchange to the wider bioprocess-using industries; (iv) benefits to patients in terms of new and more cost effective medicines, and (v) benefits to wider society via involvement in public engagement activities and encouraging future generations of researchers.
With regard to training, the provision of future bioindustry leaders is the primary mission of the IDC and some 97% of previous EngD graduates have progressed to relevant bioindustry careers. These highly skilled individuals help catalyse the development and expansion of private sector innovation and biomanufacturing activity. This is of enormous importance to capitalise on emerging markets and to create new jobs and a skilled labour force to underpin the UK economy.
In terms of IP generation each industry-collaborative EngD project will have direct impact on the industry sponsor in terms of new technology generation and improvements to existing processes or procedures. Where substantial IP is generated this has the potential to lead to spin-out company creation and job creation with wider UK economic benefit. IDC research has already led to creation of two UCL spin-out companies focussed on the emerging field of Synthetic Biology (Synthace) and novel nanofibre adsorbents for improved bioseparations (Puridify). Once arising IP is protected the IDC also provides a route for wider dissemination of project outputs and knowledge exchange available to all UK bioprocess-using companies. This occurs via UCL MBI Training Programme modules which have been attended by more than 1000 individuals from over 250 companies to date.
The majority of IDC projects address production of new medicines or process improvements for pharmaceutical or biopharmaceutical manufacture which directly benefit healthcare providers and patients. Examples arising from previous EngD projects have included: engineered enzymes used in the synthesis of a novel pharmaceutical; early stage bioprocess development for a new meningitis vaccine; redevelopment of the bioprocess for manufacture of the UK anthrax vaccine; and establishment of a cGMP process for manufacture of a tissue-engineered trachea (this was subsequently transplanted into a child with airway disease and the EngD researcher was featured preparing the trachea in the BBC's Great Ormond Street series). Each of these examples demonstrates IDC impact on the development of cost-effective new medicines and therapies. These will benefit society and provide new tools for the NHS to meet the changing requirements for 21st Century healthcare provision.
Finally, in terms of wider public engagement and society, the IDC has achieved substantial impact via involvement of staff and researchers in activities with schools (STEMnet, HeadStart courses), presentations at science fairs (Big Bang, Cheltenham), delivery of high profile public lectures (Wellcome Trust, Royal Institution) as well as TV and radio presentations. The next generation of IDC researchers will be increasingly involved in such outreach activities to explain how the potential economic and environmental benefits of Synthetic Biology can be delivered safely and responsibly.
With regard to training, the provision of future bioindustry leaders is the primary mission of the IDC and some 97% of previous EngD graduates have progressed to relevant bioindustry careers. These highly skilled individuals help catalyse the development and expansion of private sector innovation and biomanufacturing activity. This is of enormous importance to capitalise on emerging markets and to create new jobs and a skilled labour force to underpin the UK economy.
In terms of IP generation each industry-collaborative EngD project will have direct impact on the industry sponsor in terms of new technology generation and improvements to existing processes or procedures. Where substantial IP is generated this has the potential to lead to spin-out company creation and job creation with wider UK economic benefit. IDC research has already led to creation of two UCL spin-out companies focussed on the emerging field of Synthetic Biology (Synthace) and novel nanofibre adsorbents for improved bioseparations (Puridify). Once arising IP is protected the IDC also provides a route for wider dissemination of project outputs and knowledge exchange available to all UK bioprocess-using companies. This occurs via UCL MBI Training Programme modules which have been attended by more than 1000 individuals from over 250 companies to date.
The majority of IDC projects address production of new medicines or process improvements for pharmaceutical or biopharmaceutical manufacture which directly benefit healthcare providers and patients. Examples arising from previous EngD projects have included: engineered enzymes used in the synthesis of a novel pharmaceutical; early stage bioprocess development for a new meningitis vaccine; redevelopment of the bioprocess for manufacture of the UK anthrax vaccine; and establishment of a cGMP process for manufacture of a tissue-engineered trachea (this was subsequently transplanted into a child with airway disease and the EngD researcher was featured preparing the trachea in the BBC's Great Ormond Street series). Each of these examples demonstrates IDC impact on the development of cost-effective new medicines and therapies. These will benefit society and provide new tools for the NHS to meet the changing requirements for 21st Century healthcare provision.
Finally, in terms of wider public engagement and society, the IDC has achieved substantial impact via involvement of staff and researchers in activities with schools (STEMnet, HeadStart courses), presentations at science fairs (Big Bang, Cheltenham), delivery of high profile public lectures (Wellcome Trust, Royal Institution) as well as TV and radio presentations. The next generation of IDC researchers will be increasingly involved in such outreach activities to explain how the potential economic and environmental benefits of Synthetic Biology can be delivered safely and responsibly.
Organisations
| Description | The project has shifted some focus to compare the performance of different immobilization methods by using three model enzymes and microreactors. The comparisons are essential and help to build a guide for immobilization method selection. In the first year, the experiments conducted were based on one prototype of a packed bed microreactor with immobilised transaminase and transketolase with the concept of characterising de novo metabolism. Enzymes are immobilised with His6-tag on Ni-NTA agarose beads and packed into tubes to provide the place for multi-step enzyme reactions. Enzyme activity of TAm and TK expressed from E.coli strain BL21gold (DE3) were characterised in the form of free enzyme, which contains three different plasmids CV2025 ?-TAm (16.9 ± 0.68 U/mL), RSPH17029-3177 ?-TAm 1(7.3 U/mL), WT-TK with pQR 791 (12.0 ± 1.21 U/mL) based on the investigated conditions. Also, 31.4% of TK was expressed in one 50 mL scale fermentation, while for CV2025 ?-TAm and RSPH17029-3177 ?-TAm were (42 ± 1.2)% and (17.0±4.8)% respectively. For the immobilised enzyme, there were around 55% and 51% (CV2025 ?-TAm and RSPH17029-3177 ?-TAm respectively) of the feed was immobilised inside the microreactor estimated through Bradford assay. A model reaction of inulin hydrolysis catalysed by inulinase was applied to compare and characterise the immobilised particles, including enzyme entrapment in PVA hydrogel, Sol-gel, and alginate beads, covalent bonding in Chitosan. Inulin hydrolysis, as a typical Michaelis-Menten reaction with well-established detection method DNS assay, is a suitable model reaction. The preferred immobilised particles can benefit the food industry, sugar production and potentially be applied in cascade reactions. Four different particles showed different properties when comparing the immobilisation efficiency, storage and operational stability, optimal pH and temperature. In general, all methods stabilized enzyme activity in both storage and operational stability compared to free enzyme. Among them, chitosan immobilised inulinase showed the highest activity recovery while lowest storage stability; PVA hydrogel retained the highest initial enzyme activity though has worse operational stability; Sol-gel performed well when evaluating, though due to it is powder and micrometre size, it's difficult to recover from the reaction mixture. Alginate beads showed the best storage stability due to the well-formed bead structure, though with the lowest activity recovery and therefore lowest initial enzyme activity. Alginate beads are considered to be preferred with cell immobilisation and hence will not be evaluated in future work. Another model enzyme categorized as transferase with higher molar weight will be applied to verify the immobilisation methods' applicability. So far, four different immobilisation material have been applied on inulinase and transketolase, characterisation of each material immobilised enzyme were compared in between of each method and in batch and continuous form. Currently, the continuous packed bed reactors are testing and characterising. |
| Exploitation Route | Microreactor technology has been applied as a powerful platform for biocatalytic cascade reactions with a tiny reaction volume and rapid analysis. There are difficulties with its implementation due to the operational control for the least volume of substrate, product, solvent, cofactors, but the small dimensions and physical advantages of microreactors allow a cost-effective optimization of cascade reactions. The integration of sensors adds insight into the reaction progress and eases the reaction optimization process. One of the common applications of microreactor technology is the enzyme-catalyzed reaction and cascade reactions. Thorough researches have been done for this related field for various reactions, some researchers looked into the enzyme immobilised in or on the carrier with some model reaction or cascade reactions, though there is a lack of research to characterise immobilisation methods systematically. One of the commonly studied enzymes is transketolase, which can help produce erythrose. This single-step reaction will be focused in the later work. Therefore, the project aims to compare the immobilisation methods to help build a guidebook for helping choose the suitable immobilisation method for different groups of enzymes or reaction(s). |
| Sectors | Manufacturing, including Industrial Biotechology |
| Description | Enzymes are proteins that accelerate many biochemical and chemical reactions that exist in different organisms. Compared to the free enzyme, immobilized enzymes are of increasing interest due to their enhanced stability and reusability. Immobilized enzymes simplify the downstream process and decrease the contamination of products. However, although there exists a large number of methods or supports for enzymes to be immobilized, there is a lack of research to characterize immobilization methods systematically, i.e. there is no enzyme immobilization method screening. Therefore, the project aims to compare the immobilization methods to build a guidebook for helping choose the suitable immobilization method for different groups of enzymes or reaction(s). The model reactions studied in the project are inulin hydrolysis, which can be applied in food industry for fructose production, and transketolase catalysed HPA and GA reaction, which can be used in a cascade reactions for producing amino alcohol. |
| Sector | Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| Title | Enzyme expression, purification and immobilisation |
| Description | Enzymes include transketolase, transaminase and inulinase. Transketolase and transaminase are expressed through a small scale fermentation process of E.coli (E.coli BL21gold DE3 containing plasmid pQR 801 transaminase; E.coli BL21gold DE3 containing plasmid pQR791 transketolase), Plasmid DNA incl. antibiotic resistance gene, the fermentation process. Cell lysate and purified enzymes are kept in a small volume (1-3ml) in a -20 freezer. Immobilised enzymes are kept in a fridge at 4 degree, room temperature and -20 freezer. |
| Type Of Material | Technology assay or reagent |
| Provided To Others? | No |
| Impact | The fermentation process was stabilized by using different culture media and therefore shortened. |
| Title | Enzyme immobilisation methods comparisons |
| Description | Several different immobilization methods (PVA, Sol-gel, chitosan and alginate) of inulinase were compared for the enzyme immobilization performance under the same characterization place and reaction conditions. Lentikats Bioencapsulation Polyvinyl alcohol (PVA) can be used for the production of a hydrogel carrier since aqueous solutions of this material are able to gel on their own when stored for a long time. Hydrogen bonds between hydroxyl groups of neighboring polymer chains form a non-covalent H-bond network. LentiKat®Liquid (GeniaLab, Germany) was melted in a boiling water bath to a homogenous and clear liquid form and then transferred to a beaker with a magnetic stirrer. A thermometer was used in the process of encapsulation to monitor the temperature. The biocatalysts suspension (0.5 ml, 10-fold diluted in 50mM Acetate buffer pH 5.0) was added and mixed thoroughly with 4.5ml LentiKat liquid when the temperature dropped below 50 degrees. Then the mixture was extruded through a needle into 150 mL of PEG 600 under mild magnetic stirring. After a 2 h period of beads gelation, the hemispheric capsules were harvested and washed with acetate buffer, weighed after removal of the excess buffer with qualitative filter paper. Then the capsules were resuspended in the acetate buffer at 4°C until use. During the process of the biocapsulation, the samples were taken for measuring the immobilization efficiency. Sol-gel Sol-gel stands for a solution, which can form a gel-structure material with cross-linking of polymers. The mixture of 100µL TMOS and 40 µL HCL was sonicated for 10 mins and then added with 160 µL enzyme suspension (10 fold). The 300 µL solution was quickly transferred to 6mL of AOT/Isoctane (150 mM AOT in isooctane) in a 15ml falcon tube, which was stirred in the vortex for a few seconds. The falcon tube was then centrifuged (20min,4000 rpm, 4?) and the pellet was resuspended with 6ml acetate buffer. The centrifugation was repeated at least three times. Then the pellet was then resuspended with 1 ml of acetate buffer and transferred to the Eppendorf tube. After a 5 min centrifugation with 12000 rpm, the supernatant was removed. The Eppendorf tube was then placed with an open lid in a closed box with a saturated solution of NaCl. Then place the box in 4? fridge for one week for drying. The dried sol-gel was weighted and then added with acetate buffer for storage (4? fridge) and further use. Chitosan 3 g of powdered chitosan was added to 150 mL of 3% (v/v) acetic acid solution. When reaching a homogeneous status, the solution was dropped through a syringe needle(syringe pump. 0.5 mL/min ) into a gently stirred 350 mL of 1M NaOH solution and 125 mL of ethanol by a peristaltic pump, and the gelled spheres formed instantaneously. After 3 h of hardening, the beads were filtered and washed with distilled water at least three times to neutrality. The beads were later stirred with 50 mM, pH 5.0 acetate buffer containing 1% (v/v) of glutaraldehyde for 30 min. Then the beads were washed with distilled water to remove the excess of the glutaraldehyde and dried for future usage. 5 ml of inulinase (diluted 10 times) was mixed with 1.0 g of chitosan beads (0.6-1.4 mm particle size) at 150 rpm up to 40h, 4?. The immobilized enzymes were then washed with a corresponded buffer for removing non-bound enzyme and stored in 4? for further use. Alginate 35 ml of 3% (wt.) sodium alginate solution was mixed with 5 ml enzyme solution (10x diluted) then transferred into a 10ml syringe and needle. The alginate beads were formed by dripping the prepared solution from a height of approximately 20 cm into an excess (100 ml) of stirred 0.2M CaCl2 solution at room temperature. The bead size can be controlled by pump pressure and the needle gauge. The flow rate used with the syringe pump was 0.5ml/min. The beads swam in the calcium solution for 3 hours and stirred with a magnetic stir. Then the beads were harvested and washed with 20mM pH 4.5 acetate buffer containing 1%(w/v)calcium chloride at least three times. Dried on absorbent paper, the beads were weighed and resuspended in the corresponded buffer in 4? for further use |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2010 |
| Provided To Others? | No |
| Impact | We compared the parameters of each immobilised inulinase and the results showed the pros and cons of each method. |
| Title | Transketolase 96 well plate screening assay |
| Description | A high throughput screening method for free transketolase activity measurement. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2012 |
| Provided To Others? | Yes |
| Impact | By using this method, with the control of temperature/ph, a fast screening of one transketolase under different conditions were conducted. |
| URL | https://www.researchgate.net/publication/263332776_A_pH-Based_High-Throughput_Assay_for_Transketolas... |
| Description | 18th Annual bioProcessUK Conference 2021- poster presentation |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | I was selected for the bursary and invited to attend the bioProcessUK conference and displayed a poster showcasing my research. In this international conference, I attended several workshop, learned with different projects in several industries, made connections with differnet companies related to the bioprocess industry. There are more than 300 hundred people attended the conference. |
| Year(s) Of Engagement Activity | 2021 |
| Description | Advanced Biocatalysis Workshop |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | This is a virtual workshop talking about advanced biocatalysis with a focus on immobilized enzymes processes, organized by Purolite life sciences. |
| Year(s) Of Engagement Activity | 2021 |
| URL | https://tks.clickmeeting.com/advanced-biocatalysis-workshop |
| Description | Artesc Writing Skills Training course |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | The module was run for over 6 weeks. Each week, I attended at a given time a 2.5hour online teaching session plus a 1-hour video tutorial. I was work with a group of 6 for the 2.5hour online teaching session for each week. As with the face-to-face module delivery, at the end of this module, we have written a 3-4 page document for our projects with each specific part to form one paper. |
| Year(s) Of Engagement Activity | 2020 |
| Description | Design of Experiments for Bioprocess Optimisation (MBI) |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | This is a one-week workshop for the training of the design of experiments. After the workshop, I know how to think like a researcher and start to design my own project experiments based on the different parameters for process optimization. |
| Year(s) Of Engagement Activity | 2018 |
| Description | First-year seminar |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Study participants or study members |
| Results and Impact | Professors, postdocs, and doctoral students attended my first-year seminar, the presentation was 20 minutes to present and 5 minutes for questions. The seminar happened on 16/05/2019. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Friday wrap- Third year seminar |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Third year seminar for the whole department, mainly talk about the research I've done so far and summarize the main data of the research. |
| Year(s) Of Engagement Activity | 2021 |
| Description | Pilot Plant Unit Operations |
| Form Of Engagement Activity | A formal working group, expert panel or dialogue |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | We designed a project for postgraduate students to go through all operations during one week. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Poster and presentation EUROMBR |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | During the EUROMBR training courses, there were more than 50 people, come from different countries and universities. There was much useful advice from the professors, lecturers, and other graduate students. We had a poster and presentation session for our project and I was awarded Top Three Posters At Poster Presentation At the 2nd International EUROMBR training course, TU Braunschweig, 09-13.19.2019 |
| Year(s) Of Engagement Activity | 2019 |
| URL | http://www.eurombr.nu |
| Description | Research Skills |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Data analysis is the key to know how good and how confident the lab data are, in this session, methods of data analysis and the related calculations were discussed. |
| Year(s) Of Engagement Activity | 2018 |
| Description | UCL- Dept. Biochem. Eng. Open day |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Public/other audiences |
| Results and Impact | As a member of Beakers Society, UCL Dept. Biochem. Eng., I took part in the consulting team on the Open day of UCL, June 2019. We introduced the bachelor and graduate degree information to the potential students as well as the parents. We showed one of the main projects of the department, algae fermentation to the public. |
| Year(s) Of Engagement Activity | 2019 |