Biocatalysis is considered a green and environmentally friendly technology. Therefore, novel enzymes and enzymatic systems, together with cascades and protein engineering approaches, are in high demand. Here, three very different biocatalytic approaches have been studied. First, the richness of enzymes in the Red Sea brine pools has been assessed, and the discovery and characterization of a novel halophilic γ-carbonic anhydrase is described, together with the protein engineering approach, which boosted the initial catalytic activity of the γ- carbonic anhydrase. The understanding of polyextremophilicity principles from enzymes from the Red Sea brine pool, contributes to the bioengineering effort of turning mesophilic enzymes into more stable variants. Next, focus is given to the use of amine-transaminases in cascades for chiral amine synthesis. This resulted in the development of a self-sufficient sustainable cascade for chiral and non-chiral amine synthesis. This cascade was achieved by combining a lysine decarboxylase with an amine-transaminase to generate a cheap amino donor source for a more sustainable reaction economy. Finally, gas vesicle nanoparticles are functionalized by various engineering principles to create floating platforms for the immobilization of enzymes. The proof-of-concept was achieved by anchoring a phytase via anchoring peptides on the gas vesicle nanoparticles surface. These bioengineering approaches contributed to the effort of generating first principles for protein engineering.

Amine transaminases (ATA) catalyse the transfer of an amino group from one molecule and replaces a ketone or aldehyde with the amino group, the amino group on the amino-donor is replaced with a ketone or aldehyde. This enzyme, ATA from Chromobacterium violaceum, has previously been used to catalyse the reaction involving amphetamine, therefore, it might be possible to use this enzyme to convert amphetamine and the product absorbs in the UV spectrum and can therefore be measured spectrophotometrically. The aim of the project was to explore the possibility of using ATA in a portable biosensor for the detection of amphetamine. A literature study of commercially available portable biosensors was performed, activity of the free enzyme was tested against two substrates, methylbenzylamine (MBA) and amphetamine. Research on immobilization techniques, materials, and surface functionalization was done to chose suitable methods for immobilizing ATA. Two immobilization methods were suggested and one of the methods, ionic immobilization through His-tag towards Ni2+ on the surface, was tested for enzyme activity toward MBA. The enzyme activity of the free enzyme in solution towards MBA was comparable to previously reported enzyme activity, however, no enzyme activity towards amphetamine was observed. No activity was observed for the immobilized enzyme, but it might be due to the experimental design, more experiments need to be performed to draw conclusions. / Amintransaminaser (ATA) katalyserar överförandet av en amingrupp från en molekyl och ersätter en keton eller aldehyd med den amingruppen, amingruppen på amin-donatorn ersätts med en keton eller aldehyd. Det här enzymet, ATA från Chromobacterium violaceum (CvATA), har tidigare använts för att katalysera en reaktion som involverar amfetamin, därför skulle detta enzym kunna användas på amfetamin. Produkten av reaktionen absorberar i UV spektrumet och kan mätas med en spektrofotometer. Målet med projektet var att utforska möjligheten av att använda CvATA i en biosensor för att detektera amfetamin. En litteraturstudie på kommersiellt tillgängliga bärbara biosensorer genomfördes, aktiviteten av det fria enzymet testades mot två substrat, metylbenzylamin (MBA) och amfetamin. Information samlades om immobiliseringstekniker, material, och ytfunktionalisering gjordes för att välja ut lämpliga metoder för immobilisering av CvATA. Två immobiliseringsmetoder föreslogs och en av metoderna, immobilisering via enzymets His6-tagg och Ni2+ joner på ytan, testades för enzymaktivitet mot MBA. Enzymaktiviteten av det fria enzymet i lösning mot MBA var i samma storleksordning som tidigare rapporterad enzymaktivitet, men ingen enzymaktivitet mot amfetamin kunde observeras. Ingen aktivitet kunde observeras för det immobiliserade enzymet, men det kan vara på grund av designen på experimentet, fler experiment behöver göras för att kunna dra några fler slutsatser.