Int J Biochem Mol Biol 2012;3(2):209-218
Original Article
Site-directed mutagenesis of a family 42 β-galactosidase from an antarctic bacterium
Matthew V Shumway, Peter P Sheridan
Department of Biological Sciences, Idaho State University, Pocatello, Idaho USA 83209
Received March 23, 2012; accepted May 11, 2012; Epub May 18, 2012; Published June 15, 2012
Abstract: Site directed mutagenesis was used to modify the active site of a cold active beta-galactosidase taken from an Antarctic
psychrotolerant Planococcus Bacterial isolate. The goal was to modify the active site such that there would be an increase in activity on certain
substrates which showed little to no activity with the wild type enzyme. A total of 5 mutant enzymes were constructed with amino acid changes
based on an analysis done via homology modeling. All 5 modified enzymes were assayed using 14 different nitrophenol substrates. In most
cases there was a loss of activity on substrates that showed activity with the wild type enzymes. None of the expected activity was observed with
any of the mutants, possibly in part due to a decrease in hydrogen bonding between the active site and the substrates. With the substrates p-
nitrophenyl-β-d-galacturonide and p-nitrophenyl-α-d-glucopyranoside we saw increased activity. With one of the mutants we measured a 320%
increase in activity on p-nitrophenyl-β-d-galacturonide. Two other mutants showed activity on p-nitrophenyl-α-d-glucopyranoside, which showed
no activity at all with the wild type enzyme. (IJBMB1203008)
Keywords: Directed evolution, cold active, beta-galactosidase, site directed mutagenesis
Address all correspondence to:
Dr. Peter P Sheridan
Department of Biological Sciences
Idaho State University, Pocatello, ID 83209, USA.
Tel: 208-757-7971
E-mail: sherpete@isu.edu
Original Article
Site-directed mutagenesis of a family 42 β-galactosidase from an antarctic bacterium
Matthew V Shumway, Peter P Sheridan
Department of Biological Sciences, Idaho State University, Pocatello, Idaho USA 83209
Received March 23, 2012; accepted May 11, 2012; Epub May 18, 2012; Published June 15, 2012
Abstract: Site directed mutagenesis was used to modify the active site of a cold active beta-galactosidase taken from an Antarctic
psychrotolerant Planococcus Bacterial isolate. The goal was to modify the active site such that there would be an increase in activity on certain
substrates which showed little to no activity with the wild type enzyme. A total of 5 mutant enzymes were constructed with amino acid changes
based on an analysis done via homology modeling. All 5 modified enzymes were assayed using 14 different nitrophenol substrates. In most
cases there was a loss of activity on substrates that showed activity with the wild type enzymes. None of the expected activity was observed with
any of the mutants, possibly in part due to a decrease in hydrogen bonding between the active site and the substrates. With the substrates p-
nitrophenyl-β-d-galacturonide and p-nitrophenyl-α-d-glucopyranoside we saw increased activity. With one of the mutants we measured a 320%
increase in activity on p-nitrophenyl-β-d-galacturonide. Two other mutants showed activity on p-nitrophenyl-α-d-glucopyranoside, which showed
no activity at all with the wild type enzyme. (IJBMB1203008)
Keywords: Directed evolution, cold active, beta-galactosidase, site directed mutagenesis
Address all correspondence to:
Dr. Peter P Sheridan
Department of Biological Sciences
Idaho State University, Pocatello, ID 83209, USA.
Tel: 208-757-7971
E-mail: sherpete@isu.edu
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