View source for Part:BBa K2940002


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'''Improvement of K863001'''

Bpul (Laccase from <I>Bacillus pumilus</I>) with improved enzymatic activity. Generated by error prone PCR mediated directed evolution. 

===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'''

After mutant library generation vie error prone PCR. 92 mutant laccase enzymes were prescreened via a plate based dye degradation assay. This assay showed 23 which produced functional Bpul laccase enzymes. Enzyme activity for the mutants was assessed by ABTS assay, four of which displayed higher activity when compared to wild type Bpul (Shown in the graph).  One (M26) was shown to have a 0.8 fold increase in activity when compared to wild type Bpul. Sequencing showed eight point mutations present in M26 when compared to wild type Bpul. 


[[Image:T--Edinburgh OG--FigureScreening_Nathan.png|thumb|92 randomly selected mutants were prescreened on agarplates containing 25 μM trypan blue. Transformed colonies were picked and placed onto dye plate followed by incubation at 37 C for 48 h. Yellow squaresindicate selected colonies based on dye degradation. The blue squareindicates WT Bpul. The red square indicates pET218a-GG-RFP-CD.]]


[[Image:T--Edinburgh OG--FigureBpul2 Nathan.png|thumb|Mutants with activity higher than WT Bpul as determined byABTS assay are shown. The results shown are mean ̆standard error forthree replicates. Significant differences between wild type (WT) Bpul and mutants P<0.05 (**)]]


<partinfo>BBa_K2940002 SequenceAndFeatures</partinfo>

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===Functional Parameters===
<partinfo>BBa_K2940002 parameters</partinfo>
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