title: Novel GH family 3 enzymes from the thermophilic organism Rhodothermus marinus
Creators: name: Kandra, Kulwant
Publisher: name: TU Wien
Year: 2014
Subjects: subject: Extremophiles
subject: Thermostable Enzymes
subject: Enzyme Characterization
subject: Design of Experiments

description: The genes encoding a ß-glucosidase (BGL) and a ß-N-acetylglucosaminidase (NAG) from the thermohalophilic bacterium Rhodothermus marinus were successfully expressed in Escherichia coli. These recombinant enzymes, which belong to the glycoside hydrolase family 3 (GH3), were characterized regarding various parameters. Multiple sequence alignment of these two GH3 enzymes with GH3 members from other organisms allowed prediction of the catalytic nucleophiles, which are positioned at D283 for NAG and D306 for BGL. Identifying catalytic acid/base residues proved to be difficult for BGL, whereas for NAG, D207 together with H209 were predicted. Application of LC-MS provided the molecular mass for BGL and NAG with 87.2 kDa, respectively 104.5 kDa. Both enzymes acted very differently regarding substrate specificity. BGL was classified as a broad-specificity ß-glucosidase revealing activity towards many substrates and among the various tested it showed highest activity towards pNPX and xylobiose, followed by pNPG and cellooligosaccharides (cellotriose and -tetraose). NAG on the other hand turned out to be an enzyme with narrow substrate specificity, only exhibiting activity for pNPGlcNAc and chitooligosaccharides (CH2 to CH5). Furthermore, kinetic parameters were determined for NAG, where a catalytic efficiency towards pNPGlcNAc of kcat/Km = 162.30 s-1/mM was obtained. A low Km (0.07 mM) was measured, leading to conclude a high affinity towards the applied substrate. The primarily assay of optimum temperature measurements allowed to measure an activation energy for NAG of 26.9 kJ/mol, between 50°C to 90°C and revealing a maximum activity at 90°C. After 30 minutes, total inactivation was detected at 90°C and a residual activity of 50% was left after 3 hours of incubation at 80°C, with an apparent half-life of 96 minutes for NAG. Higher residual activity was measured at 70°C compared to 80°C for BGL, with a residual activity of 80% remaining after 180 minutes of incubation and an apparent half-life of 79 minutes (at 70°C) was determined. Another approach than the classical one-parameter-at-a-time investigation was conducted (Design of Experiments), where the effect of temperature and pH was studied in regard of enzyme activity, leading to determine a range of both parameters, in which the enzymes show optimum values. The simultaneous variation of temperature and pH on applied aryl substrates, revealed an optimum enzyme activity at pH 5.4, at all specific investigated temperatures, for both enzymes. Analyzing the effects on xylobiose showed a shift with temperature of BGL's optimal range. Response Surface Modeling allowed to build a model for NAG, where the consumption of chitobiose was used as a depended variable. NAG exhibited optimal values with increasing pH and decreasing temperature (pH = 6.2 and 65°C-70°C). The evaluated model provided evidence of an influence by both investigated parameters. This kind of study leads to a better understanding of the enzyme's stability in a more effective way as well as it forms the basis for faster optimum activity determination, which can be easily applied to any enzyme.