Activity ReportCOOP-CT-2006-032628COMBIO
COOP-CT-2006-032628
COMBIO
Composite Materials for the Immobilisation of Biocatalysts
Instrument
SIXTH FRAMEWORK PROGRAMME, Co-operative Research,
CRAFT
Thematic Priority
Horizontal Research Activities involving SMEs
COMBIO Report
Publishable Final Activity Report
Period covered: from 01.10.2006 to 30.09.2008
Date of preparation: Nov. 2008
Start date of project:01.10.2006 Duration: 24 months
Project coordinator name Dr Wolfgang SKIBAR
Project coordinator organisation name C-Tech Innovation Ltd
Contents
Project Execution
Project Objectives
Project-Coordinator:
Consortium
Work performed
Dissemination and use
Project Execution
Project Objectives
Biocatalytic production processes are established in a number of industrial manufacturing and process sectors (e.g. chemical, pharmaceutical, textile, food). Until recently, biocatalysis in manufacture has been based mainly on use of whole microorganisms or low cost enzymes. The use of immobilised biocatalysts extends the typeof accessible reactor systems, facilitates clean product recovery, and can reduce production costs by making an (expensive) biocatalyst reusable. However the commercial availability of immobilised biocatalysts (enzymes) is limited in the types of carrier commercially available, the classes of enzymes immobilised, and the costs of the immobilised formulations. The aim of this two year project was to enable a substantial increase in the rate and extent of the uptake of biocatalysis processes by industry by developing a range of cost-effective enzyme stabilisation and immobilisation technologies which can be applied to commercial processes.
The project focused on using support materials and novel internal cross-linking methods where the enzyme becomes its own support to develop and maintain microenvironments with enhanced stability and catalytic functionality. Although one major target was enhancement in performance of biocatalysts in the application of the synthesis of fine chemicals, the potential applicability of the technology is very broad. Within this project applications were being developed for the immobilised enzymes in processes as diverse as food manufacture, textile processing and fuel cell components. Development of these immobilisation protocols also has potential commercial applications in the production of novel enzyme-coated materials for other applications.
Extension of the working life of the immobilised catalysts achieved by the combination of stabilisation/immobilisation can also be exploited in the development of new reactor types where process intensification can be achieved, and supported catalysts can be physically retained to ensure pure, enzyme-free product streams. The immobilised biocatalysts developed in COMBIO are designed to be applied in familiar chemical reactor types such as stirred tanks, packed beds and fluidised beds. Incorporation of magnetic particles in the supports has also been demonstrated. This not only assists in the retention/reuse of the biocatalyst, but also allows the development of novel magnetically stabilised fluidised bed reactors (MSFBR) for chemical processes requiring efficient mass transfer. The ultimate aim of COMBIO was to produce biocatalytic enzyme nanocomposites as free flowing dry powders that may be stored at room temperature for many months (possibly years) and used off-the-shelf for the manufacture of fine chemicals etc.
The objectives of this project were:
- Production of low cost immobilisation materials that can be designed to be commercially viable regarding cost to use ratio
- Increasing stability in commercial preparations of supported biocatalysts and understanding the molecular events occurring that cause the improvement
- Exploration of different immobilisation chemistries available to assess the ‘best fit’ for production of immobilised biocatalysts
- Exploration of cross-linking of enzymes to use the enzyme itself as its own support and to add in additives to produce nanocomposite biocatalysts
- Development of magnetic biocatalyst supports for the development of magnetically stabilised reactors and improved separation/recycling
- Evaluation of performance enhancement in fine chemicals synthesis and assessment of scalability
- Limited evaluation of immobilised enzyme opportunities in textile processing and as a novel fuel cell electrode
Project-Coordinator:
Wolfgang Skibar
C-Tech Innovation Ltd
CapenhurstTechnologyPark
Chester, CH1 6EH
UK
Tel. +44 (0)1513472919
E-mail:
Consortium
- C-Tech Innovation Ltd, CapenhurstTechnologyPark, Chester, CH1 6EH, UK
- Namos GmbH, Tatzberg 47, 01307 Dresden, Germany
- DI Dr. Andreas Paar KEG – Qualizyme, Leechgasse 55/8, 8010 Graz, Austria
- Alkomohr Biotech Oy Ltd, Lehtotie 8, 00630 Helsinki, Finland
- CLEA Technologies BV, Julianalaan 136, 2628BL,Delft, Netherlands
- Kylolab S.L., Rio Guadalentin, 13 - Poligono Industrial, 30562 Ceuti (Murcia), Spain
- MacroSynth Limited, 21 Bracken Park, Scarcroft, LS14 3HZ Leeds, UK
- University of Leeds, Woodhouse Lane, LS2 9JTLeeds, UK
- Dublin City University, Glasnevin, Dublin 9, Ireland
- Delft University of Technology, Julianalaan 134, 2600 AA Delft, Netherlands
For more information, please visit the COMBIO website:
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Activity ReportCOOP-CT-2006-032628COMBIO
Work performed
For the duration of the project, the work performed within each workpackage (WP) was as follows:
WP 1 – Enzyme Immobilisation
Screening of free enzymes for solution stabilisation using an array of stabilisers: selection of additives for secondary screens.
Optimal enzyme stabilisers have been identified for subtilisin, subtilisin-nanoparticles conjugate, laccase, laccase- nanoparticles conjugate, lipase-nanoparticles conjugate and PQQ-MDH.Three stabilizer compositions have been selected for stabilisation of enzymes in dry state. Sucrose as a basic stabilizer was selected for comparison with the other stabilizing mixtures.
Immobilisation of enzymes in carrageenan and other porous matrices in presence of polymers, and additives emerging from primary screens.
A range of nanoparticles made from porous materials have been produced. Enzyme immobilized onto nanoparticles can result in low diffusion limitation for substrate molecules, i.e they behave like free enzyme. In the same time such nanoparticles can possess special properties such as magnetic, and the possibility to precipitate at different pH or temperature. Such properties allow the separation of enzyme-loaded nanoparticles from products after reaction. A number of enzymes (subtilisin, laccase, CalB and PQQ-MDH) have been immobilised on the developed nanoparticles.
Mesoporous silicates (MPS) have also been explored as enzyme carriers. Both native enzymes and chemically modified enzymes have been immobilised on them. A range of industrially relevant enzymes were successfully immobilised on four different types of MPs: Qualizyme laccase (from Trametes hirsuta), the subtilisins alcalase and subtilisin Carlsberg, the industrial proteases esperase, everlase and savinase, PQQ-MDH, CalB lipase, horseradish peroxidase, beta-galactosidase and trypsin.
Self-organizing surface proteins and hydroxyapatite coatings have been used to design universal intermediate layers and technologies to establish a fully functional attachment of enzymes on metallic or ceramic surface. Transglutaminase enzyme, which is of interest in processes for cheese hardening has been has thus been immobilised to sintermetallic porous substrates.
Several different support materials: agarose, Eupergit C, and membranes Nytran nylon-66 and Hybond P have been explored for immobilization of PQQ-MDH for fuel cell applications and showed reasonable efficiency.
In order to be tested in a magnetically stabilised fluidised bed reactor magnetic particles for enzyme immobilisation have been developed. One method was based on FeCl3 complexes using BSA and histidine as bio-templates. Particles with a size in the order of magnitude of about 100nm could be produced and used for immobilisation of transglutaminase. Fe-Ag magnetic nanoparticles have also been used for immobilization of enzymes. These nanoparticles can immobilise enzymes with SH groups such as laccase directly without additional modification
Carageenan particles of the low m to single mm dimensions have been produced for testing as enzyme immobilisation matrices. Both magnetic and non-magnetic beads have been made and Candida antarctica lipase B (CalB) has been immobilized on them.
To compare immobilisation methods developed within this project with commercially available products, a number of commercial formulations has been explored. Eupergit CM (Rohm), an acrylate bead material, and covalently bound Cal B lipase have been chosen as a test system.
Development of self-supporting enzymes as cross linked aggregates and cross-linked nano-composites
Cross-linked enzyme aggregates (CLEAs) are a very good method for immobilizing enzymes. It is very simple and robust allowing for easy recovery and reuse of the enzyme. The procedure consists of the precipitation of an enzyme with a salt or an organic solvent and the covalent cross-linking with glutaraldehyde. CLEAs of the enzymes chloroperoxidase, CalB, glucose oxidase, laccase, ß – galactosidase, amidases, esterases and various proteases have been produced. Co-immobilisation of chloroperoxidase and glucose oxidase has also been achieved.
In addition CLEAs with paramagnetic nanoparticles incorporated in their structure were developed. Prototypes of Candida antarctica lipase A and alcalase with paramagnetic properties are available now.
WP 2 – Molecular Interaction Studies
Characterisationof stabilising effects observed at the molecular level.
Laccase, MDH-PQQ, CalB, β-galactosidase and chloroperoxidase and subtilisin A have been studied. Stabilizing chemical modifications have been performed on CaLB, laccase and MDH-PQQ. Three distinct types of chemical modification have been applied and their effects on thermal stability assessed.
Characterization of the stabilising effects of cross-linkers have been carried out by determining the storage stability as well as the mechanical stability of the enzymes.
Investigation of the interactions occurring between enzymes and stabilisers.
Both biochemical and biophysical characterizations of the stabilization effects achieved for the various immobilised enzyme formulations developed by the COMBIO partners have been undertaken.
Give understanding at the nanoscale to the macroscopic effects observed experimentally.
The effectiveness of simple-to-perform chemical modification procedures in stabilising a range of enzymes has been demonstrated. Glutaraldehyde has proven quite successful as a stabilizing covalent modifier of soluble enzymes and in the formation of CLEAs; it is arguably the most versatile, reliable and successful single chemical used so far; it is also quite cheap.
It is possible to perform chemical modification in solution and follow this by immobilization.
It has also been shown that enzyme molecules have occupied the pores in the respective immobilisation matrices by measuring the pore volume decrease for each type of solid particle following enzyme loading.
WP 3 – Immobilised Enzyme Stability and Activity
Stability testing of enzymes in optimised formulation. Effects of drying and re-hydration
The stability at different temperatures (from 25 ºC to 60 ºC) of the soluble enzyme and the immobilized enzymes was compared with the soluble enzymes.
It was demonstrated that nine investigated industrially-relevant enzymes (see WP1) can be stabilized by chemical modification in solution with simple, cost-effective reagents and without prejudice to subsequent MPS immobilisation protocols.
Drying of CLEAs to a dry powder was investigated for a number of enzymes. Several CLEAs were found to be stable in a dried or partial dried form. Most of the CLEAs showed good stability if stored at 4ºC in buffer. Some were extremely stable over the period not loosing any activity at all. The CLEAs could also be recycled several times without any loss of activity. The stability of the several CLEAs was also dramatically improved by changing the formulation.
Assessment of immobilised enzyme activity (small laboratory scale)
Immobilised forms of CalB were evaluated in two lab scale test synthetic reactions– transesterification of phenyl alcohol with vinyl acetate, and synthesis of myristyl myristate.Five principle methods of immobilisation were planned subjects for this investigation- Eupergit CM, CLEA, Mesoporous silicates (MPS), Carrageenan beads, and polymeric nanoparticles.
The activity of transglutaminase immobilised on sintermetallic carriers or silica nanoparticles has been tested by analysing the size change of caseine proteins during milk processing. Immobilisation led to a drop of activity of about 25 % compared to free transglutaminase.
Immobilised laccase was tested on dye effluents and model substrates from the textile industry. Testing of these laccases successfully worked on different model dyes (indigo carmine, lanaset blue, cibacrone marine).
Immobilised protease was tested for the so called scouring process for wool fibre treatment. The scalability of enzyme activity and the hindered penetration into the fibre are useful for wool fibre treatment.
Chemically modified forms of the MDH-PQQ were tested and it was shown that chemical modification either severely reduce its activity or completely eliminates it. Thus, it seems that this particular enzyme is not well suited for chemical modifications without serious activity losses.The MDH-PQQ enzyme was tested together with laccase enzyme in enzymatic fuel cell application. Both enzymes seemed to work well as long as the pH on anode and on cathode is at suitable level for activity of each enzyme.
WP 4 – Design and Construct Scaleable Laboratory Reactors
Design and construct of scaleable laboratory experimental systems incorporating standard reactors types for use in immobilised enzyme applications testing in WP5.
Three reactor designs based on conventional (established) reactor configurations were developed, constructed and trialled for a range of solvent/reactant/catalyst combinations. The designs were stirred tank, fixed bed and fluidised bed reactors. The reactors afford access to both scale-up and improved process control as compared with conventional lab-bench scale systems used for synthetic chemistry research.
A scalable experiment has been developed for the hydrolysis of penicillin G. The obtained data was used to design a continuous reactor setup in a membrane slurry reactor (MSR).
Design and construct of one scaleable laboratory experimental system incorporating an innovative magnetically stabilised fluidised bed (MSFBR) reactor for further development and testing in WP5
A Magnetically Stabilised Fluidised Bed Reactor (MSFBR) has been constructed. It is less conventional than the other reactors built in this workpackage in that it is designed to exploit the features of catalyst formulations which contain magnetically susceptible particles. The controlled application of a magnetic field is used in this reactor to stabilise a fluidised bed of particulate supported biocatalyst.
WP 5 – Fine Chemicals Applications Testing
Evaluation of immobilised enzymes applied to the synthesis of fine chemical products.
A number of chemical reactions (e.g. sulfoxidation of Thioanisole; Hydrolisis of o-nitrophenyl β-D-galactoside) have been used to evaluate CLEAs developed in this project.
Evaluation of comparative performance of standard prototype reactors
Trial runs have been performed with penicillin G amidase CLEAs in a membrane slurry reactor (MSR) for the hydrolysis of penicillin G.
Two test reactions (transesterification and myristyl myristate synthesis) were scaled up for evaluation in the stirred tank, packed column and fluidised bed reactors as appropriate.The scale-up typically represented a 10-100 fold increase in reaction volume over the lab tests. The physical integrity of immobilised biocatalyst during or after process (e.g. microscopy examination of Eupergit from stirred tank reactors) and the degree of enzyme activity loss following reactor operation was assessed.
Sintermetallic supports used for immobilisation of transglutaminase have been tested on a prototype flow-through tube reactor.
β-galactosidase immobilised as CLEAswas tested in order to compare their performance against the free enzymes, which are used on a normal basis for the production of different sugars (by coupling of mono-saccharides). All the reactions were done in stirred-tank vessels of up to 2 liters of capacity. It was shown that substrate selectivity of the immobilised enzyme was within range and that the enzymes can be recovered after one reaction and tested in a subsequent batch.
Optimisation and evaluation of the Magnetically Stabilised Fluidised Bed Reactor (MSFBR)
Two magnetic enzyme support formulations were developed: Carageenan beads and CLEAs with magnetic properties. These formulations were evaluated using the previously developed test reactions (transesterification and myristyl myristate synthesis) in the MFBR and performance compared with non-magnetically stabilised fluidised bed reactors.
WP 6 – Evaluation and Implementation Plan
Update and refining of market intelligence
The state of the art of commercial immobilised biocatalysts has been reviewed. Information in the availability of off-the-shelf immobilisation matrices such as polymer beads and functionalised materials was included.
Measure technical performance against quantified criteria
The commercial benefits to be realised by the use of immobilized/stabilised enzymes developed in this project have been reviewed and compared with commercially available enzyme formulations.
The performance of the four scalable reactor types developed in work package 4 was assessed regarding economic and environmental impact.
Prepare a final plan for technological implementation, further technical development, dissemination and exploitation
The intelligence on the scope of potential applications has been strengthened and detailed information on the market potential considered. Based on this a forward strategy for approaching the market for the specific applications a broader marketing strategy for further applications and industry sectors was developed.
Dissemination and use
List of publishable results:
Result / Market applications / Stage of development / Collaborator details / Contact detailsTest systems to evaluate a portfolio of immobilised enzymes in different reactor designs to enhance properties for industrial processes /
- Fine & Speciality Chemical Synthesis
- Pharmaceutical Manufacture
- Food Processing
- Waste treatment
C-Tech Innovation Ltd
Capenhurst Industrial Park
Chester, CH1 6EH
UK
+44 (0)151 347 2919
Magnetically fluidised bed reactor (MSFBR) /
- Fine & Speciality Chemical Synthesis
- Pharmaceutical Manufacture
- Food Processing
- Waste treatment
C-Tech Innovation Ltd
Capenhurst Industrial Park
Chester, CH1 6EH
UK
+44 (0)151 347 2919
β-Galactosidase CLEA /
- Pharmaceutical
- Food industry
Roger A. Sheldon.
Julianalaan 136, 2628 BL Delft, The Netherlands.
Tel: +31 (0)15 27 82683
Chloroperoxidase CLEA /
- Pharmaceutical
Roger A. Sheldon.
Julianalaan 136, 2628 BL Delft, The Netherlands.
Tel: +31 (0)15 27 82683
Positioning of Nanoparticles on surfaces through protein templates /
- Nanoimprint templates
- Solar panels
- Fuel Cells
Namos GmbH,
Tatzberg 47
01307 Dresden, Germany
+49-351-796-5720
Protocols for chemical modification of enzymes in solution, followed by immobilisation on MPS /
- Fine & Speciality Chemical Synthesis
- Pharmaceutical Manufacture
- Food Processing
- Waste treatment
Dublin City University
School of Biotechnology
Dublin 9
Ireland
+353 (0)1 700 5288
Modified proteases /
- Textile Industry
- Waste treatment
- Pulp & Paper Industry
“Qualizyme Biotechnology”
Leechgasse 55/8
A-8010 Graz, Austria
Methanol dehydrogenase (MDH-PQQ) product from Methylophilus methylotrophus /
- Enzymatic fuel cells
- Alcohol sensors
- Analytics of alcohols
Alkomohr Biotech Ltd
Lehtotie 8
FIN-00630 Helsinki
Finland
+358 (0)50 5626903
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