Difference between revisions of "Part:BBa K2940002"
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Revision as of 18:23, 21 October 2019
Improved Bpul
Improved Bpul (Laccase from Bacillus pumilus
Usage and Biology
Laccases are enzymes with the potential to biodegrade synthetic dyes with different chemical structures. These enzymes are able to oxidize a wide range of phenolic substrates without the presence of additional co-factors. This improved Bpul enzyme has a major bioactivity for degrading azo dyes.
Characterization
Libraries of laccase mutants were generated using error prone Taq PCR (New England BioLabs). The mutant libraries of assembled PelB and Bpul were then cloned into pET28a-GG-RFP-CD via Golden Gate cloning for expression.
Characterization ABTS assay
98 mutant laccase enzymes were assayed for enzymatic activity using ABTS assay. This assay showed four mutants with improved activity (one with a significant increase). This mutant was sequenced and showed eight-point mutations when compared to WT Bpul.
Results
Laccase activity was determine from Bpul.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 726
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 123
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 726
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 726
Illegal NgoMIV site found at 490
Illegal NgoMIV site found at 958
Illegal AgeI site found at 246
Illegal AgeI site found at 1123 - 1000COMPATIBLE WITH RFC[1000]