Phage display selects for amylases with improved low pH starch-binding.
Directed evolution of secreted industrial enzymes is hampered by the lack of powerful selection techniques. We have explored surface display to select for enzyme variants with improved binding performance on complex polymeric substrates. By a combination of saturation mutagenesis and phage display we selected alpha-amylase variants, which have the ability to bind starch substrate at industrially preferred low pH conditions. First we displayed active alpha-amylase on the surface of phage fd. Secondly we developed a selection system that is based on the ability of alpha-amylase displaying phages to bind to cross-linked starch. This system was used to probe the involvement of specific beta-strands in substrate interaction. Finally, a saturated library of alpha-amylase mutants with one or more amino acid residues changed in their Cbeta4 starch-binding domain was subjected to phage display selection. Mutant molecules with good starch-binding and hydrolytic capacity could be isolated from the phage library by repeated binding and elution of phage particles at lowered pH value. Apart from the wild type alpha-amylase a specific subset of variants, with only changes in three out of the seven possible positions, was selected. All selected variants could hydrolyse starch and heptamaltose at low pH. Interestingly, variants were found with a starch hydrolysis ratio at pH 4.5/7.5 that is improved relative to the wild type alpha-amylase. These data demonstrate that useful alpha-amylase mutants can be selected via surface display on the basis of their binding properties to starch at lowered pH values.
Pharmaceutical Biology, University Centre for Pharmacy, A. Deusinglaan 1, 9713, AV Groningen, The Netherlands.
Raymond M D
RM
Verhaert
Jules
J
Beekwilder
René
R
Olsthoorn
Jan
J
van Duin
Wim J
WJ
Quax
D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding; D013213:Starch / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D013213:Starch; D000681:Amylases