|dc.description.abstract||Halothermothrix orenii, a gram negative strictly anaerobic thermohalophilic bacterium has been isolated from sediments of a Tunisian salt lake (Chott El Guettar). H. orenii grows optimally at 60˚C with 10% NaCl. Phylogenetically, H. orenii is placed at the base of phylum Firmicutes, in the family Haloanaerobiaceae, order Haloanaerobiales, and is the only member of this order whose genome has been completely sequenced. The availability of the genome sequence will not only enhance our understanding of survival and adaptation strategies of microbes living under dual extremities of high salt and temperature, but will also provide information on novel biocatalysts which could have potential application in research, industrial and bioremediation processes. To date, our laboratory has studied a large number of genes that encode Glycosyl Hydrolases and Transferases from H. orenii, including AmyA and AmyB of the α-amylase family 13, a sucrose phosphate synthase (SPS) and a Fructokinase (Frk). Biochemical analysis demonstrated that all these recombinant enzymes are optimally active between 55-65˚C and in addition, the extracellular amylases AmyA and AmyB are most active with NaCl concentration between 5-25%. All the recombinant enzymes have been crystallized and their structures solved, but their molecular basis of thermohalophilicity has yet to be determined.
In the present study, data mining of the genome of H. orenii further identified six potential α-amylase coding genes (designated Amy1, Amy2, Amy5, Amy6, Amy7 and Amy8). Primers were designed for cloning and overexpression of these genes into two vectors, namely flexi pFN6A and pET22b (+). Clones of Amy1, Amy6, Amy7 and Amy8 were discarded from the freezer by a member of the plant laboratory in my school, and therefore only clones Amy2 and Amy5 were available for studies. Amy5 clone has been studied in detail and forms the basis of this thesis. The Amy5 gene, which consists of 1701 nucleotides, was determined by BLAST analysis to code for an oligo-1,6-glucosidase, was cloned in pET22b (+) vector and the recombinant enzyme successfully over-expressed in the E. coli B strain (BL21 (DE3)). The recombinant enzyme (designated rAmy5) was purified to homogeneity using immobilized metal affinity chromatography (IMAC). The molecular weight of rAmy5 was determined by SDS to be 66kDa, which is close to the expected theoretical calculated value of 66.766kDa. The rAmy5 hydrolysed p-nitrophenyl-α-D-glucopyranoside (PNPG). The temperature optimum for enzyme activity was 60˚C (temperature activity range between 45-65˚C) and the pH optimum was between pH 6 and 6.5 (pH activity range between pH 5.3-7). rAmy5 activity was inhibited by metal ions in the order Cd
2+ > Cu2+ > Zn2+ > Fe3+ > Fe2+ > Ni2+ > NH4+, but enhanced by Ca2+ and Mn2+ with Mg2+ and Co2+ having no effect. The recombinant enzyme was tolerant to proteolysis when tested in presence of various concentrations of Trypsin. DTT (Dithiothreitol) had no effect on enzyme activity suggesting that cysteine residues did not play any role in providing thermostability to the enzyme molecule. Activity of the enzyme against PNPG was completely inhibited by 100mM D-glucose or 0.2mM PNP (p-nitrophenol). EDTA was found to have no effect on enzyme activity. The enzyme activity was enhanced in the presence of salts such as 0.5M NaCl and 1M KCl by 22.5% and 45% respectively. Denaturants inhibited enzyme activity in the order SDS > Tris > Urea. Sugars inhibited the enzyme activity in the order D-glucose > D-fructose > Mannitol > Xylose > Cellobiose > Lactose > Cellobiose > Lactose > Mannose > Raffinose > Galactose. The enzyme displayed no activity when tested against the following PNP-based glycosides: 2-Nitrophenyl-β-D-fucopyranoside, p-Nitrophenyl-α-L-arabinopyranoside, p-Nitrophenyl-α-D-xylopyranoside, p-Nitrophenyl-β-D-xylopyranoside, 2-Nitrophenyl-β-D-glucopyranoside, p-Nitrophenyl-β-D-fucopyranoside, 2-Nitrophenyl-1-thio-β-D-galactopyranoside, and p-Nitrophenyl-β-D-glucuronide. The activation energy of enzyme was found to be 81.3kJ/mol. The enzyme was thermostabilized at 60˚C in the presence of salts and combination of metal ions in the following order: 0.5MNaCl > 0.5M NaCl + 25mM CaCl2 > 1M KCl > 1M KCl + 25mM CaCl2. Presence of 25mM CaCl2 alone was found to destabilize the enzyme at 60˚C. The half-life of the enzyme increased four times in presence of 0.5M (10%) NaCl at 60˚C. The enzyme followed Michaelis Menten kinetics for PNPG and was found to have a KM and VMAX of 1.3mM and 333.3mM PNPG/min per mg of enzyme respectively. The enzyme had a high specific activity and was found to be active against a variety of substrates. The probable order of its specificity was PNPG > dextrin > dextran > isomaltotriose > amylose > pullulan > palatinose > soluble starch > trehalose > maltotetraose > maltose > isomaltose > maltooligosaccharide > panose. The enzyme was more active against substrates containing (1→6)-α-D-glucosidic linkages.
In silico analysis revealed that the Amy5 putative protein sequence consisted of a highly conserved ‘QpDln’ signature for oligo-1,6-glucosidase sub-family. The high thermostability of the rAmy5 enzyme could be correlated to an increased presence of prolines and preference for glycine over alanine in the Amy5 sequence. A preference for charged residues over uncharged residues indicated the possible reason for the increased thermostability with NaCl of the rAmy5 enzyme. The fact that the Amy5 gene sequence had a significant identity (63.7%) to the sequence of solved crystal structure of oligo-1,6-glucosidase (1UOK) from Bacillus cereus formed the basis for a comparative homology modelling of Amy5 protein and as result a 3-Dimensional model of the Amy5 protein could be predicted. Comparative analysis of the predicted Amy5 model with 1UOK revealed that the residues of deep catalytic cleft of the latter were found at the same positions (Asp199, His 328, and Asp329), except that the Glu255 residue of 1UOK aligned with position Glu256 of Amy5. These catalytic residues are considered necessary for enzyme function rather than enzyme substrate specificity. Study of crystal structures of various enzymes has revealed that there are various subsites (amino acid residues) which associate with catalytic residues to impart the character to the enzyme to act on specific substrates. In addition to the present study of comparative modelling and biochemical characterization of the rAmy5 enzyme, protein crystallography studies could add to our understanding of structure-function relationship. In our present study, an attempt to crystallize the rAmy5 protein was made using a hanging drop method by screening it against 240 different buffer conditions (using JBScreen Mixed Crystallography kit, Jena Biosciences, Germany), but crystals were not observed during the three-month incubation period.||en_US