Expression and Characterization of an Extremely Thermostable Beta Glycosidase (Mannosidase) from the Hyperthermophilic Archaeon Pyrococcus furiosus DSM3638 and Mutation Studies in the Active Site.

摘要

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF0356) similar to the enzymes in glycoside hydrolase family 1. This beta-glycosidase, designated PFTG (Pyrococcus furiosus thermostable glycosidase), was cloned and expressed in Escherichia coli. The expressed enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The gene was composed of 1452 bp encoding 483 amino acids for a protein with a predicted molecular mass of 56,326 Da. The temperature and pH optima were 100°C and 5.0 in sodium citrate buffer, respectively. The substrate specificity studies by conventional assays showed characteristics of both beta-galactosidase and beta-mannosidase activities. However, through kinetic studies by ITC (Isothermal Titration Colarimetry), this enzyme showed the highest catalytic activity in p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) with kcat/Km (3.02). The enzyme showed transglycosylation and transgalactosylation activities toward cellobiose, lactose and mannooligosaccharides, that could produce GOS (galactooligosaccharides) and MOS (mannooligosaccharides) which are the important prebiotic (bifidogenic) ingredients.;By substituting Gln150 to Trp and Gln77/150Trp, the catalytic efficiency (kcat/Km) of the mutants by ITC 200 on both synthetic and natural substrates were slightly changed. As compared with the wild-type enzyme, substrate specificities on the mutant enzymes were similar but with more affinity (Km) to substrates and low turnover number (kcat).;To confirm the category of PFTG, the protein structural studies were performed. After the wild-type enzyme was purified, the enzyme was set up for protein crystal screening under 200 different conditions. With several hit conditions, X-ray diffraction study was performed, but during the X-ray analysis, the data was too high to obtain the reasonable data. Through protein sequence analysis of PFTG, two Lys-Lys sequences which interfere the protein crystallization were revealed. After Lys-Lys amino acids were mutated to Ala-Ala by site-directed mutagenesis, the mutant enzymes were set up and incubated for crystallization. Clear protein crystals were not obtained, but the computer 3D model structure indicated that this enzyme was similar to that of beta-glycosidase from Thermosphaera aggregans.;Keywords: beta-glycosidase; beta-galactosidase; beta-mannosidase; hyperthermophile; Pyrococcus furiosus; hyperthermostable enzyme; ITC (Isothermal Titration Calorimetry); glycoside hydrolase; protein crystallography;Sequence alignments and homology modeling of PFTG showed that the residue 150 is conserved as tryptophan in beta--glycosidase and in other related enzymes such as beta-mannosidase and beta-galactosidase. To elucidate the relationship between the substrate size and geometric shape of the catalytic site of thermophilic beta-glycosidase and category of PFTG, the Q77, the Q150 and the D206 located at the interface of the dimer were replaced with Trp and Asn. Also, to confirm the role of active sites of PFTG, the Q77/150W double mutant was created through subcloning. The mutant enzymes were successfully cloned, expressed in E. coli and showed the same temperature and pH optima at 100 °C and pH 5.0, respectively.