Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity.


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.

Submission Details

ID: vFMPoNsk3

Submitter: Shu-Ching Ou

Submission Date: Feb. 1, 2019, 5:44 p.m.

Version: 1

Publication Details
Khodakarami A;Goodarzi N;Hoseinzadehdehkordi M;Amani F;Khodaverdian S;Khajeh K;Ghazi F;Ranjbar B;Amanlou M;Dabirmanesh B,Int J Biol Macromol (2018) Rational design toward developing a more efficient laccase: Catalytic efficiency and selectivity. PMID:29425870
Additional Information

Only values in Table 2 are included.

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Spore coat protein A P07788 COTA_BACSU