Δημήτριος Χατζηνικολάου, Αναπληρωτής Καθηγητής Μικροβιακής Βιοτεχνολογίας, Τμήμα Βιολογίας, ΕΚΠΑ
Γεώργιος Διαλλινάς , Καθηγητής Μοριακής Μικροβιολογίας, Τμήμα Βιολογίας, ΕΚΠΑ
Κωνσταντίνος E. Βοργιάς, Καθηγητής Bιοχημείας, Τμήμα Βιολογίας, ΕΚΠΑ
In the framework of this thesis, we performed an analysis regarding a member of the glycoside hydrolase family 48 (GH48) derived from the bacterial strain Bacillus safensis. More specifically, we performed cloning, production, and biochemical characterization of the enzyme.
The microorganism was isolated from soil in 1999 by Christakopoulos and his colleagues (Christakopoulos et al., 1999) and was characterized as B. safensis based on the full sequence of the 16S rRNA gene, in the present study. Detection of the Cel48_B.s. gene followed, based on alignment of respective genes derived from B. safensis and B. pumilus. The gene was transformed into the plasmid vector pET-15b and expressed in modified bacterial cells of Escherichia coli BL21. Purification of the enzyme was performed using nickel affinity chromatography after sonication of the cells.
Subsequently, we performed enzymatic characterization of glycoside hydrolase Cel48_B.s. Initially, the molecular weight of the enzyme was determined as 79 kDa, using SDS-PAGE electrophoresis, while its quaternary structure was determined using molecular filtration chromatography. Its hydrolytic activity was estimated on various substrates. Maximum enzyme activity was observed in phosphoric acid swollen cellulose (PASC), while it was also detected against carboxylmethyl cellulose (CMC). In addition, enzymatic activity was observed against crystalline cellulose (avicel) and filter paper. The maximum enzyme activity was detected at 60 °C and at pH6. The thermostability of the enzyme was tested at 60 °C, 65 °C, 70 °C and 80 °C and we ended up that the enzyme is thermostable at 60 °C and 65 °C. Following incubation of the enzyme on different substrates, it was observed that the largest percentage of the products of enzyme hydrolysis, is cellobiose, while there was also production of glucose and oligosaccharides. Finally, we performed Michaelis - Menten kinetic analysis regarding the substrates PASC and CMC.
Simultaneously, a bioinformatics analysis was conducted for the enzyme. Initially, it was observed that the gene encoding Cel48_B.s. belongs to a gene cluster which includes genes encoding hydrolases that degrade (hemi)cellulose. We also studied the protein organization of GH48 family enzymes regarding the protein domains they carry. Finally, phylogenetic trees were constructed based on both the GH48 domain of these enzymes as well as the full amino acid protein sequence, but also the 16S rRNA gene of the microorganisms carrying the specific enzymes. Higher identity rates of the enzyme Cel48_B.s. appear to be detected with the enzymes of the bacteria Paenibacillus barcinonensis and Myxobacter sp. AL-1, while the latter shares a common, unique protein organization.
cellulose, cellulase, enzyme, Bacillus safensis, glycoside hydrolase GH48