Exploring the Biotechnological Potential of the Secretome from the Marine Fungus Digitatispora marina Proteomics, Bioinformatics and Heterologous Expression of Novel Proteases

Abstract

Cold-adapted enzymes are of biotechnological value due to their high catalytic powers at low to moderate temperatures and their high thermolability at elevated temperatures. Marine fungi are an understudied group of organisms, which makes them interesting as enzyme producers because of their adaptions to marine habitats. In this thesis study, a bioprospecting pipeline was constructed, and different approaches were assembled to identify the genetic source in the marine fungus Digitatispora marina that is responsible for previously observed protease activity in growth medium. Biochemical characterization in form of temperature optimum, salt tolerance and pH optimum were made to acquire information about the properties of the protease(s). The activity was maintained at a range of different NaCl concentrations and pH values of Tris-HCl. Moreover, the protease(s) were also active at lower temperatures with an activity peak at 50°C. Protein extracts from the fungal culture medium were also analyzed by Mass spectrometry for possible identification of proteases contributing to the protease activity. Through bioinformatic analysis, a selection of seven serine proteases (SP1, SP2, SP3, SP4, SP5, SP6 and SP7) and one metalloprotease (MP) was chosen for recombinant protein expression in E. coli BL21(DE3) cells. There was a successful expression of SP1, however the activity was not recovered in any of the samples. Further studies are needed to identify the genetic source that is responsible for the protease activity from the secretome of D. marina.