Biochemical and biological characterisation of GH-J clan enzymes from haloarchaea

Abstract

Fructans are fructose-based oligo- and polysaccharides that are exploited in a variety of industrial, agricultural, food and healthcare applications, most notably as prebiotic dietary supplements. Fructans are synthesised from sucrose by enzymes with fructosyltransferase activity from the glycoside hydrolase 32 and 68 (GH32 and GH68) families. Recently, the existence of GH32 and GH68 enzymes in archaea, specifically haloarchaea, has been demonstrated. The work presented in this thesis focuses on characterizing the structure, function and activity of GH32 and GH68 enzymes from two model haloarchaeal species: Haloarcula hispanica and Haloferax gibbonsii. GH32 and GH68 enzymes from these organisms were expressed in Haloferax volcanii and purified to apparent homogeneity. The three-dimensional structure of GH32 from H. gibbonsii was determined by X-ray crystallography, with analysis revealing the existence of a novel N-terminal domain that is postulated to be involved in levan binding. Extensive functional characterisation of the GH32 and GH68 enzymes was performed, which provided valuable insights into the range of activities and substrate specificities of the enzymes. To complement this biochemical and structural analysis, the biological function of the GH32 and GH68 proteins was addressed by creating single and double knockouts in H. hispanica, revealing that the genes are essential for cell survival when sucrose is present as the sole carbon source. Characterisation of the mechanisms of fructan biosynthesis by haloarchaea could shed light into underlying catalytic mechanisms, as well as the biological significance of fructan biosynthesis in haloarchaea. In addition, it could provide the potential to establish a haloarchaeal platform that could be deployed by industrial biotechnology to produce fructans of novel physiological properties, which could also infer advantages over traditional industrial processes.

Date of Award	2 Jul 2025
Original language	English
Awarding Institution	University of St Andrews
Supervisor	Stuart MacNeill (Supervisor) & Tracey Gloster (Supervisor)