Document Type: Research Paper
Department of Reproductive Genetics, Royan Reproductive Biomedicine Research Center, ACECR, Tehran, Iran.
Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Rd., Ratchathewi, Bangkok 10400, Thailand.
Background: D-Phenylglycine aminotransferase (D-PhgAT) is highly beneficial in pharmaceutical biotechnology. Like many other enzymes, D-PhgAT suffers from low stability under harsh processing conditions, poor solubility of substrate, products and occasional microbial contamination. Incorporation of miscible organic solvents into the enzyme’s reaction is considered as a solution for these problems; however, native D-PhgAT is not significantly stable in such solvents.
Objective: Halophiles are known to survive and withstand unsavory habitats owing to their proteome bios. In the current study, with an eye on further industrial applications, we examined the performance and thermostability of four halophilic peptides fused D-PhgAT variants in reaction mixtures of various proportions of different miscible organic solvents and various temperatures as well as desiccation.
Materials and Methods: Plasmid constructs from the previous study (Two alpha helixes and loops between them from Halobacterium salinarum ferredoxin enzyme fused at N-terminus domain of D-PhgAT) expressed in Escherichia coli and then D-PhgAT purified. Purified proteins were subjected to various proportions of miscible organic solvents, different temperatures, and desiccation and then performance and thermostability monitored.
Results: Study confirmed increased C50 of all halophilic fused D-PhgAT variants, where the highest C50 observed for ALAL-D-PhgAT (30.20±2.84 %V/V). Additionally, all halophilic fused variants showed higher thermostability than the wild-type D-PhgAT in the presence of different fractions of acetone, N,N-Dimethylformamide and isopropanol in aqueous binary media, while zero activity observed at the presence of methanol.
Conclusion: Our results suggest that applying this new technique could be invaluable for making enzymes durable in discordant industrial conditions.