2 Enzyme and Microbial Technology Research Center, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, Serdang 43400, Malaysia
3 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang 43400, Malaysia
4 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang 43400, Malaysia
A functional mini protein can be developed by miniaturising its size. The minimisation technique provides an excellent model system for studying native enzymes, especially in creating an alternative novel biocatalyst. Miniaturised proteins may have enhanced stability, a crucial characteristic for large-scale production and industrial applications. In this study, a huge enzyme molecule, known as diamine oxidase (DAO, comprising 700 amino acids), was selected to undergo the process.
By retaining the arrangement of the original functional sites of DAO in the fourth domain, a mini DAO can be designed via homology modelling. After several downsizing processes, a final configuration of 220 amino acids displayed high binding affinity towards histamine, a short-chain substrate that was catalysed by the parental DAO. The configuration also showed enhanced affinity towards a long-chain substrate known as spermidine. The gene for the designed protein was cloned and expressed in pET102/TOPO vector and overexpressed in E. coli BL21 (DE3).
The new mini DAO had similar temperature tolerance and versatile substrates specificity characteristics as its parental protein. An active mini-protein with these characteristics is potentially useful for several applications such as detecting biogenic amines in the biological fluids and the environment that may give rise to health issues.