טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRabinovich Larisa
SubjectCloning and Characterization of Aminopeptidases from
Bacillus subtilis and Streptomyces
griseus
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yuval Shoham


Abstract

Aminopeptidases catalyze the removal of amino-terminal amino acids from proteins.  These enzymes are widely distributed in all organisms, play major roles in various biological processes and have many biotechnological applications.  The aim of this study was to clone and characterize the aminopeptidases from Bacillus subtilis and Streptomyces griseus, as part of a long term research on zinc-containing aminopeptidases.

Based on the published genome sequence of B. subtilis, the ywaD gene exhibits homology to aminopeptidases from family M28 and encodes for a 425 amino acids protein with a calculated molecular weight of 46,415.  The gene was cloned without its putative leader peptide and over-expressed via the T7 polymerase expression system.  The purified enzyme exhibits optimal activity between pH 8 to 9 and is stable for 20 minutes at 80°C.  The highest activity was toward Arginine, Lysine and Leucine p-nitroanilide derivatives with kcat/Km values of 57, 12 and 15 sec-1mM-1, respectively.  The activity of BSAP was not affected by specific inhibitors of serine and aspartic proteases and was completely inhibited in the presence of the metal chelator - 1,10 -phenanthroline.  Full activity was restored by ~2-2.5 mol Zn per mol BSAP, indicating that BSAP is a metallo-aminopeptidase.

The sgap gene encoding for  S. griseus aminopeptidase (SGAP) was cloned and over-expressed using the T7 polymerase expression system.  Based on high resolution crystal structures of SGAP, Glu131 and Tyr246 were proposed to be the catalytic residues.  The replacement Glu131Ala resulted in 109 fold decrease in activity.  The replacements Tyr246Ser, Tyr246Ala and Tyr246Phe lead to the 103, 102 and 10 fold decrease in activity, respectively.  These results demonstrate unequivocally the involvement of Glu131 and Tyr246 in catalysis.