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Improving Productivity and Enzyme Stability Through Process Design: Lipase-catalysed Synthesis of Epoxides and Esters

Hagström, Anna LU (2010)
Abstract (Swedish)
Popular Abstract in Swedish

Bioteknik är ett mycket brett ämne som handlar om att använda biologiska reaktioner för att tillverka önskvärda produkter. Det innefattar arbete med

mikroorganismer, svampar, enzymer och att få dem att göra som man vill. Enzymer är naturens egna katalysatorer som gör att reaktioner går snabbare utan att själva förbrukas och de är till exempel en viktig del i vår matsmältning för att bryta ner maten till smådelar som kroppen kan använda till energi eller som byggstenar. Inom forskningsprogrammet Greenchem, där mitt arbete ingår, har vi fokuserat på att utnyttja enzymer med en förhoppning om att tillverka kemikalier av olika slag såsom tensider, smörjmedel och komponenter till ytbehandling... (More)
Popular Abstract in Swedish

Bioteknik är ett mycket brett ämne som handlar om att använda biologiska reaktioner för att tillverka önskvärda produkter. Det innefattar arbete med

mikroorganismer, svampar, enzymer och att få dem att göra som man vill. Enzymer är naturens egna katalysatorer som gör att reaktioner går snabbare utan att själva förbrukas och de är till exempel en viktig del i vår matsmältning för att bryta ner maten till smådelar som kroppen kan använda till energi eller som byggstenar. Inom forskningsprogrammet Greenchem, där mitt arbete ingår, har vi fokuserat på att utnyttja enzymer med en förhoppning om att tillverka kemikalier av olika slag såsom tensider, smörjmedel och komponenter till ytbehandling på ett mer miljövänligt

sätt. En annan grundpelare inom programmet har varit att använda förnyelsebara råvaror, som t.ex. vegetabiliska oljor och socker. För länge sedan utgjorde denna typ av ämnen grunden för kemiindustrin, men byttes så småningom ut mot fossil olja. I och med all uppmärksamhet under senare tid på att oljan kommer att ta slut (eller i alla fall stiga i pris) har intresset för förnyelsebara råvaror återigen ökat. Enzymer, som finns till för att omvandla naturliga ämnen, kan därmed vara utmärkta redskap för att kunna tillverka användbara kemikalier.

Vatten är det vanligaste lösningsmedlet i naturen. Det är mycket viktigt för allt liv och naturligtvis också för enzymer som finns i alla levande organismer. En del enzymer är därför vana vid att fungera i vattenlösningar, medan andra bara kräver några få vattenmolekyler för att kunna fungera. I många fall är det viktigt att kunna kontrollera fuktigheten i reaktorn där enzymerna ska användas för att de ska kunna

arbeta så effektivt som möjligt, vilket var målet med ett av delprojekten som beskrivs i denna avhandling. Den relativa fuktigheten i reaktorn mättes och justerades till det önskade värdet genom bubbling av antingen torr eller fuktig luft genom systemet för att sänka respektive höja den relativa fuktigheten i reaktorn.

I mitt arbete har jag använt en typ av enzym som kallas lipas, vars naturliga funktion är att bryta esterbindningar i fett. Genom att ändra reaktionsförhållandena vi runt lipaserna kan man vända på reaktionen och istället få dem att bygga upp esterbindningar, men också att utföra andra reaktioner som inte är lika naturliga för enzymet. Det effektivaste sättet för att kunna bilda esterbindningar är att minska på mängden vatten. Det lipas som jag oftast har använt i mitt arbete klarar av att arbeta vid mycket små vattenmängder, vilket jag utnyttjade i mitt första projekt som gick ut på att tillverka vax för ytbehandling av trä, närmare bestämt s.k. vaxestrar – feta estrar baserade på en fettalkohol och en fettsyra. Reaktionen torkades i detta fall genom att blåsa torr luft genom reaktionsvätskan för att ta bort vattnet som bildades i reaktionen. Vid utvärderingen av vaxesterprodukterna visade det sig att de inte skyddade träet tillräckligt bra, speciellt inte mot fett. Arbetet gick då vidare med att tillverka en lack genom att sätta på akrylgrupper på polyestrar för att de skulle kunna binda ihop sig till ett nätverk och bilda en film på träet. Akrylgrupperna innehåller en reaktiv del (terminal dubbelbindning) som när de utsätts för UV-ljus lätt reagerar med varandra. Om det då finns två eller fler akrylgrupper på varje molekyl kan det bildas ett nätverk. Istället för att bubbla luft genom reaktorn utnyttjades här vakuum för att ta bort en biprodukt, etanol, och på så sätt få reaktionen att gå mot mer av

den önskade produkten. Vakuum användes också för att driva bort vatten från

reaktionen då vi tillverkade smörjmedel, TMP-oleate, som kan användas i

skogsmaskiner som ett miljövänligt alternativ till mineraloljor. Det kan brytas ner i naturen och är det dessutom tillverkat med hjälp av enzymer istället för metalliska katalysatorer kan man undvika utsläpp av tungmetaller i naturen då de kan finnas kvar i produkten.

Det fjärde projektet som ingår i denna avhandling skiljer sig från de tidigare genom att det inte var en esterbindning som skulle bildas utan en så kallad persyra från en fettsyra och väteperoxid. Persyran kan i sin tur reagera vidare med en dubbelbindning (i en fettsyra) och bildar då en s.k. epoxidgrupp. Epoxider används inom en mängd olika områden, t.ex. i epoxylim, lacker, plast, m.m. Problemet med denna process är att enzymet inaktiveras när det kommer i kontakt med väteperoxiden. Mitt arbete inom detta projekt gick ut på att hitta en process för denna reaktion som var så skonsam som möjligt för enzymet så att det kan användas som katalysator och inte förbrukas på samma sätt som en reaktant. (Less)
Abstract
Interest in sustainable development has increased throughout society during the past decades, and the chemical industry is no exception. The appeal for renewable raw materials has consequently increased and has paved the way for the implementation of industrial biotechnology. Since enzymes are used to convert biomass in nature, they may also be a good choice in the chemical industry. However, to identify if the use of enzymes is the best alternative for a particular application they must be evaluated. Investigations can be carried out on the enzyme itself, the reaction, the reactor design or the complete process. Analyses of the environmental impact and the economics of the process are also important. The focus of the interdisciplinary... (More)
Interest in sustainable development has increased throughout society during the past decades, and the chemical industry is no exception. The appeal for renewable raw materials has consequently increased and has paved the way for the implementation of industrial biotechnology. Since enzymes are used to convert biomass in nature, they may also be a good choice in the chemical industry. However, to identify if the use of enzymes is the best alternative for a particular application they must be evaluated. Investigations can be carried out on the enzyme itself, the reaction, the reactor design or the complete process. Analyses of the environmental impact and the economics of the process are also important. The focus of the interdisciplinary research programme Greenchem at Lund University is to develop speciality chemicals from renewable raw materials using enzymes as catalysts. The work presented in this thesis was focused on the reactor and the process, rather than the catalyst itself, or a specific reaction, as those have been covered in other projects within Greenchem.

Lipases are among the most extensively studied enzymes. Candida antarctica lipase B (CalB), which can be used to catalyse several different reactions, is one example. This enzyme was most frequently used in the work described in this thesis, usually in an immobilised preparation, Novozym® 435 (N435).

In the initial study presented in this thesis, four wax esters were produced and tested regarding their applicability as wood coatings. Unfortunately, even the best of these waxes showed too low resistance to grease stains. Towards the end of this work a new study was initiated; acrylation of polymers to be used as the main component in lacquers, with the hope of achieving better resistance than that achieved with the wax esters. These acrylates were produced on litre-scale via lipase-catalysed trans-esterification between ethyl acrylate and polyesters and polyethers, respectively, and were found to be comparable to the commercial alternatives available today. A biolubricant, trimethylolpropane (TMP) oleate, was produced via esterification using the same lipase preparation, as well as epoxidised rapeseed methyl ester (RME) through chemo-enzymatic epoxidation.

Several parameters must be considered when setting up a biocatalytic process. Water activity is one of them, and a system for water activity control was developed using wax ester synthesis as a model reaction. The system consisted of a sensor, a control unit and the facility to bubble air through the reactor, either dry or saturated with water, to decrease or increase the humidity in the reactor. The optimum water activity for esterification using N435 was determined and found to be close to zero. For comparison, an immobilised lipase from Candida rugosa was used, and this preparation was found to work best at higher water activities, i.e. above 0.5. By-product removal is another important parameter, and this was accomplished by evaporation through bubbling air through the reactor in wax ester synthesis, or by vacuum in the synthesis of acrylates and TMP oleate.

The stability of the enzyme and the performance of the processes were investigated by varying different parameters. In the acrylation reaction half-lives of up to 320 h were achieved for two different CalB preparations, which resulted in reasonable catalyst costs for the process. However, when using N435 for TMP oleate synthesis, the process was not viable due to the combination of the long reaction times required at moderate temperatures and the relatively low stability of the enzyme (~90 h half-life). The stability of the biocatalyst was also found to be too low for an economically feasible production of epoxidised RME, although it could be enhanced by appropriate process design. Productivity and enzyme stability were investigated in different reactor designs, and it was found to be crucial to maintain the hydrogen peroxide concentration at a low, controlled level in order to prolong the life-time of the biocatalyst.

The work presented in this thesis shows the direct applicability of biocatalysis to some processes such as the acrylation reaction, which has been proven to be competitive with traditional chemical production methods. In other cases, further development regarding choice of biocatalyst, reaction medium and process design are needed. One of the most important factors in investigations regarding industrial biotechnology is the enzyme stability, as this determines the possibility of implementing the process on an industrial scale. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Nielsen, Per Munk, Senior Science Manager, Novozymes A/S, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
publisher
Department of Chemistry, Lund University
defense location
Lecture Hall B, Center for Chemistry and Chemical Engineering, Lund University Faculty of Engineering, Sölvegatan 39, Lund
defense date
2010-06-04 10:30
ISBN
978-91-89627-65-9
language
English
LU publication?
yes
id
6ece5149-f922-4fc7-854f-edcd50178f76 (old id 1599270)
date added to LUP
2010-05-05 09:00:54
date last changed
2016-09-19 08:45:05
@phdthesis{6ece5149-f922-4fc7-854f-edcd50178f76,
  abstract     = {Interest in sustainable development has increased throughout society during the past decades, and the chemical industry is no exception. The appeal for renewable raw materials has consequently increased and has paved the way for the implementation of industrial biotechnology. Since enzymes are used to convert biomass in nature, they may also be a good choice in the chemical industry. However, to identify if the use of enzymes is the best alternative for a particular application they must be evaluated. Investigations can be carried out on the enzyme itself, the reaction, the reactor design or the complete process. Analyses of the environmental impact and the economics of the process are also important. The focus of the interdisciplinary research programme Greenchem at Lund University is to develop speciality chemicals from renewable raw materials using enzymes as catalysts. The work presented in this thesis was focused on the reactor and the process, rather than the catalyst itself, or a specific reaction, as those have been covered in other projects within Greenchem. <br/><br>
Lipases are among the most extensively studied enzymes. Candida antarctica lipase B (CalB), which can be used to catalyse several different reactions, is one example. This enzyme was most frequently used in the work described in this thesis, usually in an immobilised preparation, Novozym® 435 (N435).<br/><br>
In the initial study presented in this thesis, four wax esters were produced and tested regarding their applicability as wood coatings. Unfortunately, even the best of these waxes showed too low resistance to grease stains. Towards the end of this work a new study was initiated; acrylation of polymers to be used as the main component in lacquers, with the hope of achieving better resistance than that achieved with the wax esters. These acrylates were produced on litre-scale via lipase-catalysed trans-esterification between ethyl acrylate and polyesters and polyethers, respectively, and were found to be comparable to the commercial alternatives available today. A biolubricant, trimethylolpropane (TMP) oleate, was produced via esterification using the same lipase preparation, as well as epoxidised rapeseed methyl ester (RME) through chemo-enzymatic epoxidation. <br/><br>
Several parameters must be considered when setting up a biocatalytic process. Water activity is one of them, and a system for water activity control was developed using wax ester synthesis as a model reaction. The system consisted of a sensor, a control unit and the facility to bubble air through the reactor, either dry or saturated with water, to decrease or increase the humidity in the reactor. The optimum water activity for esterification using N435 was determined and found to be close to zero. For comparison, an immobilised lipase from Candida rugosa was used, and this preparation was found to work best at higher water activities, i.e. above 0.5. By-product removal is another important parameter, and this was accomplished by evaporation through bubbling air through the reactor in wax ester synthesis, or by vacuum in the synthesis of acrylates and TMP oleate. <br/><br>
The stability of the enzyme and the performance of the processes were investigated by varying different parameters. In the acrylation reaction half-lives of up to 320 h were achieved for two different CalB preparations, which resulted in reasonable catalyst costs for the process. However, when using N435 for TMP oleate synthesis, the process was not viable due to the combination of the long reaction times required at moderate temperatures and the relatively low stability of the enzyme (~90 h half-life). The stability of the biocatalyst was also found to be too low for an economically feasible production of epoxidised RME, although it could be enhanced by appropriate process design. Productivity and enzyme stability were investigated in different reactor designs, and it was found to be crucial to maintain the hydrogen peroxide concentration at a low, controlled level in order to prolong the life-time of the biocatalyst. <br/><br>
The work presented in this thesis shows the direct applicability of biocatalysis to some processes such as the acrylation reaction, which has been proven to be competitive with traditional chemical production methods. In other cases, further development regarding choice of biocatalyst, reaction medium and process design are needed. One of the most important factors in investigations regarding industrial biotechnology is the enzyme stability, as this determines the possibility of implementing the process on an industrial scale.},
  author       = {Hagström, Anna},
  isbn         = {978-91-89627-65-9},
  language     = {eng},
  publisher    = {Department of Chemistry, Lund University},
  school       = {Lund University},
  title        = {Improving Productivity and Enzyme Stability Through Process Design: Lipase-catalysed Synthesis of Epoxides and Esters},
  year         = {2010},
}