Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60min at 80°C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants.
Submitter: Shu-Ching Ou
Submission Date: Feb. 1, 2019, 5:44 p.m.
Only values in Table 2 are included.
|Number of data points||60|
|Proteins||Spore coat protein A|
|Assays/Quantities/Protocols||Experimental Assay: kcat/Km: SGZ ; Experimental Assay: kcat: SGZ ; Experimental Assay: Km: SGZ ; Experimental Assay: kcat/Km: ABTS ; Experimental Assay: kcat: ABTS ; Experimental Assay: Km: ABTS ; Derived Quantity: Specificity: SGZ/ABTS ; Derived Quantity: Specificity: ABTS/SGZ ; Derived Quantity: SD of kcat: SGZ ; Derived Quantity: SD of Km: SGZ ; Derived Quantity: SD of kcat: ABTS ; Derived Quantity: SD of Km: ABTS|
|Libraries||Variants for Laccase_SGZ ; Variants for Laccase_ABTS|
|Structure ID||Release Date||Resolution||Structure Title|
|1GSK||2002-01-08T00:00:00+0000||1.7||Crystal structure of CotA, an endospore coat protein from Bacillus subtilis|
|1OF0||2003-04-03T00:00:00+0000||2.45||CRYSTAL STRUCTURE OF BACILLUS SUBTILIS COTA AFTER 1H SOAKING WITH ABTS|
|1W6L||2004-08-19T00:00:00+0000||2.0||3D structure of CotA incubated with CuCl2|
|1W6W||2004-08-24T00:00:00+0000||2.2||3D structure of CotA incubated with sodium azide|
|1W8E||2004-09-21T00:00:00+0000||2.2||3D structure of CotA incubated with hydrogen peroxide|
|2BHF||2005-01-10T00:00:00+0000||2.5||3D structure of the reduced form of CotA|
|2WSD||2009-09-04T00:00:00+0000||1.6||Proximal mutations at the type 1 Cu site of CotA-laccase: I494A mutant|
|2X87||2010-03-06T00:00:00+0000||2.0||Crystal Structure of the reconstituted CotA|
|2X88||2010-03-06T00:00:00+0000||1.8||Crystal Structure of HoloCotA|
|3ZDW||2012-12-01T00:00:00+0000||2.4||Substrate and dioxygen binding to the endospore coat laccase CotA from Bacillus subtilis|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|100.0||Spore coat protein A||P07788||COTA_BACSU|