Characterization Of Novel Thermophilic Lipases From Fervidobacterium Nodosum Rt17-B1 And Crystal Structure Determination Of Thermophilic Phosphotriesterase

The researches on thermophilic microorganisms and enzymes which have great impact on theoretical study and industrial applications have always been hot objects since 1990s'. This paper focuses on the characterization of the lipolytic hydrolyses from the thermophilic Fervidobacterium nodosum Rt17-B1 and the crystal structure of the phosphotriesterase from Geobacillus kaustophilus HTA426.In the first part of my thesis, a gene coding for a putative lipase composed of 302 amino acids from Fervidobacterium nodosum Rt17-B1 was cloned and characterized. Phylogenetic analysis suggests that this novel lipase represents a new subfamily of family I of bacterial lipases, annotated as family I.8. The recombinant protein can catalyze the hydrolysis of p-nitrophenyl esters and shows substrate preference for p-nitrophenyl caprate with a k_cat/K_m of 22500 s^-1μM^-1. Most importantly, it can hydrolyze triacylglycerols with long acyl chains. In the test conditions applied here it has a maximum activity at 70°C and pH 9.0 and displays extreme thermal stability. Interestingly, it was not only stable, but was also activated by treatment with polar organic solvents including propanol, acetone, dimethyl sulfoxide (DMSO) and N, N-dimethylformamide (DMF). Structural modeling showed that it is composed of anα/β-hydrolase fold and a lid domain comprised of fourα-helices. A canonical catalytic triad consisting of Ser119, Asp206 and His282 was verified by site-directed mutagenesis. Moreover based on the constructed model, the substrate specificity was explained by analyses in the aspects of the binding free energy, spatial obstruction, and interactions between enzyme and substrate in the active site. Moreover, a putative bacterial esterase encoded by Fond₁301 with typical GDSL family motifs from Fervidobacterium nodosum Rt17-B1 was cloned and characterized. Recombinant FNE exhibited the highest esterase activity (14,000 U/mg) with p-nitrophenyl acetate (pNPC₂) as substrate. The catalytic efficiency (k_cat/K_m) toward p-nitrophenyl acetate (C2₎ was approximately 120-fold higher than toward p-nitrophenyl butyrate (C₄). No significant esterase activity was observed for the substrates with a chain length≥C₈. The monomeric enzyme has a molecular mass of 27.5 kDa and optimal activity around 75℃and at pH 8.5. Its thermostability is relatively high with a half-life of 80 min at 70℃, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the acetylesterase from Aspergillus aculeatus as a template. The model covered most of the FNE. The structure showed anα/β-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis. Sequence analysis showed that FNE is only distantly related to other esterases. A comparison of the conserved motifs shared with GDSL proteins revealed that FNE should be grouped into GDSL family and was further classified as SGNH hydrolase.In the second part of my thesis, the recombinant thermophilic phosphotriesterase GK1506 from Geobacillus kaustophilus HTA426 has been crystallized and the crystal structure of GK1506 has been determined by Singlewavelenth Anamolous Dispersion and molecular replacement.to 2.5 ?. The tertiary structure of this enzyme was anα/βbarrel in which the active site is located at the C-terminal ends of theβ-strands and in the loop regions connecting these strands to their subsequent helices. The active site of GK1506 contains the three substrate binding pockets found in other OPH enzymes: the small, large and leaving group pockets. The two metal ions was concluded in the active site of GK1506. site-directed mutagenesis lead us to make sure that His²³, His²⁵, His¹⁷⁸, His²⁰⁶, Lys¹⁴⁵ and Asp²⁶⁶ are important residues for catalytic function and substrate binding. The structure determination will illuminate the structure base of catalytic mechanisum. Moreover this knowledge can lead to the development of new and more efficient protein engineering strategies and a wide range of biotechnological applications.