Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases

Abstract: Two putative cyclodextrinases (CDases) were amplified from two moderate thermophiles isolated in Iceland: Anoxybacillus flavithermus and Laceyella sacchari. They were cloned, expressed and characterized. The expression of the former was optimized in E. coli by using a solubility-enhancing tag, NusA, and tuning of inducer concentration in the expression strain Tuner(DE3). The CDases were optimally active at around 57 ºC and had a poor thermal stability. AfCda13 had significantly higher activity for cyclodextrins and formed a dimer in solution, whereas LsCda13 existed as a monomer, which likely lowered the activity for cyclodextrins. Two beta-glucosidases, members of glycoside hydrolase families 1 and 3 and originating from the extreme thermophile Thermotoga neapolitana were cloned and expressed in E. coli. The apparent thermal unfolding temperatures were around 90 ºC and 102 ºC for TnBgl3B and TnBgl1A, respectively. TnBgl3B hydrolyzed para-nitrophenyl-glucoside with a Vmax at 90 ºC of 93 ± 13 U/mg and a Km of 0.11 ± 0.03 mM. TnBgl3B was tested for its transglycosylating ability by forming hexyl- and octyl-beta-D-glucoside. The selectivity of alcoholysis compared to hydrolysis was higher than for other reported enzymes. Both beta-glucosidases were also tested in reactions cleaving off glucose from quercetin-glycosides extracted from onion by subcritical water. They both performed better than the commercially available almond beta-glucosidase. Predicted catalytic amino acids of TnBgl3B were replaced with non-catalytic glycines. The aspartate nucleophile is highly conserved in GH3 and the D242G mutant lost all activity. The acid/base residue is less conserved and two residues were mutated, Glu458 and Asp461. Mutant E458G had low but significant activity, while D461G lost almost all activity, which points to the latter residue as the catalytic acid/base of TnBgl3B. However, further analyses including structural information should confirm this. TnBgl3B was crystallized and the structure was solved by multiple wavelength anomalous diffraction using selenomethionyl crystals in addition to native crystals. X-ray diffraction data was collected to 2.4 Å resolution and the structure has been solved to 2.7 Å. Manual model building and refinement is in progress.