ABSTRACT
The aim of this investigation was to synthesize the adipic acid-modified magnetic nanoparticles for the efficient immobilization of C-terminally lysine-tagged α-amylase (BACΔNC-Lys7) from thermophilic Bacillus sp. strain TS-23. The carboxylated magnetic nanoparticles were prepared by the simple co-precipitation of Fe³âº/Fe²âº in aqueous medium and then subsequently modified with adipic acid. Transmission electron microscopy micrographs showed that the carboxylated magnetic nanoparticles remained discrete and had no significant change in size after the binding of BACΔNC-Lys7. Free enzyme was active in the temperature range of 45-70 °C and had an optimum of 60 °C, whereas the thermal stability of BACΔNC-Lys7 was improved as a result of immobilization. The immobilized BACΔNC-Lys7 could be recycled 20 times without a significant loss of the amylase activity and had a better stability during storage with respect to free enzyme. Taken together, the magnetic nanoparticles coated with this functional moiety can be a versatile platform for the effective manipulation of various kinds of engineered proteins.
Subject(s)
Bacterial Proteins/chemistry , Enzymes, Immobilized/chemistry , Magnetite Nanoparticles/chemistry , alpha-Amylases/chemistry , Adipates/chemistry , Bacillus/genetics , Bacterial Proteins/genetics , Enzyme Stability , Enzymes, Immobilized/genetics , Escherichia coli , Ferric Compounds/chemistry , Ferrous Compounds/chemistry , Hot Temperature , Hydrogen-Ion Concentration , Kinetics , Lysine/chemistry , Lysine/genetics , Magnetite Nanoparticles/ultrastructure , Microscopy, Electron, Transmission , Particle Size , Recycling , alpha-Amylases/geneticsABSTRACT
BACDeltaNC/Delta RS is a thermostable variant derived from the truncated alpha-amylase (BAC Delta NC) of alkaliphilic Bacillus sp. strain TS-23. With the aim of enhancing its resistance towards chemical oxidation, Met231 of BAC Delta NC/Delta RS was replaced by leucine to create BAC Delta NC/Delta RS/M231L. The functional significance of the 31 C-terminal residues of BAC Delta NC/Delta RS/M231L was also explored by site-directed mutagenesis of the 483 th codon in the gene to stop codon (TAA), thereon the engineered enzyme was named BAC Delta NC/Delta RS/M231L/Delta C31. BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 were very similar to BAC Delta NC in terms of specific activity, kinetic parameters, pH-activity profile, and the hydrolysis of raw starch; however, the engineered enzymes showed an increased half-life at 70 degrees C. The intrinsic fluorescence and circular dichroism spectra were nearly identical for wild-type and engineered enzymes, but they exhibited a different sensitivity towards GdnHCl-induced denaturation. This implicates that the rigidity of the enzyme has been changed as the consequence of mutations. Performance of the engineered enzymes was evaluated in the presence of commonly used detergent compounds and some detergents from the local markets. A high compatibility and performance of both BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 may be desirable for their practical uses in the detergent industry.
Subject(s)
Bacillus/enzymology , alpha-Amylases/chemistry , Amino Acid Sequence , Bacillus/genetics , Detergents/chemistry , Enzyme Stability , Half-Life , Hydrolysis , Leucine/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxidation-Reduction , Protein Engineering , Sequence Deletion , Starch/metabolism , Temperature , alpha-Amylases/genetics , alpha-Amylases/isolation & purificationABSTRACT
Role of the conserved Asn345 and Asn435 residues of Bacillus kaustophilus leucine aminopeptidase (BkLAP) was investigated by performing computer modeling and site-directed mutagenesis. Replacement of BkLAP Asn345 with Gln or Leu resulted in a dramatic reduction in enzymatic activity. A complete loss of the LAP activity was observed in Asn435 variants. Circular dichroism spectra were nearly identical for wild-type and all mutant enzymes, while measurement of intrinsic tryptophan fluorescence revealed the significant alterations of the microenvironment of aromatic amino acid residues in Asn345 and Asn435 replacements. Except for N435R and N435L, wild-type and other mutant enzymes showed a similar sensitivity towards temperature-induced denaturation. Computer modeling of the active-site structures of wild-type and mutant enzymes exhibits a partial or complete loss of the hydrogen bonding in the variants.