Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression.
Concomitant activity improvement of an evolved enzyme toward two very different ester substrates was achieved when a unique combination of functional periplasmic enzyme expression in Escherichia coli, random mutagenesis, DNA shuffling and cell-based kinetic screenings was applied. Specifically, we focused on the conversion of subtilisin E into an enzyme with broader esterase activity as opposed to its native amidase activity. Cell-based microtiter assays were performed on N-acetyl-D,L-phenylalanine p-nitrophenyl ester (Phe-NPE) and sucrose 1'-adipate (S1'A), as well as on the tetrapeptide amide substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide. After a single modified cycle of directed molecular evolution, we isolated a number of clones exhibiting increased activity toward Phe-NPE. In the following rounds of screenings, mutants with improved activity on Phe-NPE were also tested on S1'A. Three mutants were identified with increased esterolytic activity on Phe-NPE and S1'A, while having similar amidase activity to that of the parental enzymes. Because the two ester substrates are structurally distinct, we have evolved a more general esterolytic subtilisin and this may have important applications in synthesis.
Sroga
G E
GE
Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180-3590, USA.
Dordick
J S
JS
D000581:Amidohydrolases / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000378:metabolism; D004950:Esterases / Q000737:chemistry; D019143:Evolution, Molecular; D015723:Gene Library; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019897:Periplasm / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020860:Subtilisin / Q000737:chemistry*; D013381:Subtilisins / Q000737:chemistry
D000596:Amino Acids; D004950:Esterases; D013381:Subtilisins; D020860:Subtilisin; D000581:Amidohydrolases; C019665:amidase; D008274:Magnesium