Ringborg, Rolf H.

Med biokatalyse er det muligt at syntetisere og producere værdifulde kemikalier under mildebetingelser. Ved udvikling af nye processer, bliver enzymer modificeret til at katalysere ønskede produkter med stor succes. Nu om dage introduceres mutationer i enzymer, derved dannes mange nye mutanter og der søges herefter iblandt disse. Med high throughput screening kan man udføre screening af millioner af mutanter pr. dag. Der er derfor høj sandsynlighed for at finde enzym mutanter med egnede egenskaber. Specielt bliver enzymet amin transaminase her evalueret, da disse enzymer giver mulighed foren unik vej til fremstilling af chirale aminer. Chirale aminer er vigtige byggesten både i lægemidler og i kemikalier til landbrug. Et lovende enzym er blevet isoleret, men det har væretvanskeligt at vurdere dets ydeevne samt at give retningslinier for en procesudvikling.Almindeligt kendte begrænsninger i processer er opløselighed af substrat og produkt,ufordelagtig termodynamik¸ inhibering samt stabilitet. Det er vanskeligt at vurdere, hvor flaskehalsen i en given proces vil være. Desuden kan man ikke forvente, at der kun er en enkeltløsningsmodel til afhjælpning af flaskehalse, og man bør i fremtiden have som mål at finde frem til integrerede problemløsninger. Alle de nævnte begrænsninger vedrører reaktoren i en proces, og når ydeevnen af et enzym er ukendt, er det næsten umuligt at opstille forslag til procesudvikling. Et fokuspunkt må derfor i fremtiden være at udvikle kinetiske modeller og metoder til at registrere kinetiske data på en robust og generisk måde. Modeller for mange enzymer eksisterer allerede og findes beskrevet i lærebøger omhandlende enzym kinetik. For at tilpasse modellerne er der brug for kinetisk data der er specifik for den enkelte mutant og skal være opsamlet med reaktionen der har interesse.I denne afhandling beskrives en ny måde hvormed man kan opsamle kinetiske data. Det er blevet opnået ved en kombination af eksisterende teknologier og giver mulighed for onlineanalyse af vandige opløsninger. Endvidere er det muligt at kvantificere enzym og UV/VIS aktivemolekyler ved kombineret af størrelseskromatografi og UV/VIS detektion. Størrelseskromatografi giver ikke basislinie separation, hvilket heller ikke er nødvendigt. Brug af kemometriske metoder gør det nemlig muligt at detektere forbindelser på baggrund af opsamling af retentionstids‐ og bølgelængde data. Et stort fremskridt har været at kunne kvantificere enzymkoncentration, da man herigennem kan anvende specifik aktivitet til modeltilpasning. Dette setup udnytter fordelene ved mikrofluidiske skala og leverer semiautomatisk experimentmuligheder. Alt i alt reducerer dette setup både forbrug af dyrebart materiale og arbejdstid.Biocatalysis offers the ability to carry out important synthesis and production of valuablechemicals at benign conditions. In the development of new processes, enzymes are being engineered towards specific products with great success. Currently, mutations are introduced into enzymes, and mutants are formed thereof and a search among these is conducted. High throughput screening can deliver screening of mutants in the order of millions a day. Enzyme mutants with increased performance are therefore likely to be found. Here, the enzyme amine transaminases is evaluated since it offers a unique way of producing chiral amines. These amines are important as building blocks for pharmaceuticals and agrochemicals. A promising enzyme has been found, but it has been a problem to assess its performance and give process development direction. Common limitations are substrate and product solubility, unfavourable thermodynamics, inhibition and stability. It is a difficult task to assess where the current bottle neck is for a desired process. Moreover, it cannot be expected that a single solution to the limitations can be found and rather an integrated solution of all of the problems should be the future aim. All the limitations surround the reactor of a process,and with the performance of this being unknown, it is almost impossible to direct development. A focal point must therefore lie in the determination of kinetic models and howkinetic data can be obtained in a robust and generic way. Models for many enzymes alreadyexist and can be found in common text books. These models do however require mutant specific data and must be collected with the target reaction. In this thesis a novel way of collecting kinetic data is created, this is carried out by combining existing technology and enables the analysis of aqueous solutions on‐line. Furthermore, the use of a size exclusion column enables the simultaneous detection of enzymes and UV/VISactive compounds. The size exclusion chromatography does not provide baseline separated results, nor is this required. The application of chemometric tools enable detection of compounds in the collected retention time wavelength data. A major improvement over traditional techniques is the quantification of enzyme concentration and this makes it possible to use specific activities for model fitting. The setup takes advantage of microfluidic featuresand delivers semi‐automatic experimentation, overall reducing both consumption of precious materials and costly labor

Online Research Database In Technology

Application of A Microfluidic Tool for the Determination of Enzyme Kinetics

http://orbit.dtu.dk/ws/files/121767170/Application_of_A_Microfluidic_Tool_for_the_Determination_of_Enzyme_Kinetics.pdf

Application of A Microfluidic Tool for the Determination of Enzyme Kinetics

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Online Research Database In Technology