Development of Catcher/Tag-based purification method for Panus rudis laccase
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School of Chemical Engineering |
Master's thesis
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en
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59
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Abstract
Recombinant enzymes are a safe and environmentally friendly alternative for chemical catalysis in industrial processes and one such alternative are laccases. Laccases (EC 1.10.3.2) are oxidoreductases and applicable in many industry applications since they oxidize a wide range of substrates with water as the byproduct. There is a demand for laccases in industrial biotechnological applications, but they often require characterization of the enzyme. Extensive characterization requires efficient purification of the enzyme, but the purification of laccases is expensive and produces low yields. Conventional affinity purification with polyhistidine-tag (HisTag) is especially challenging. Potential solution is to use Catcher/Tag-based purification method. Catcher/Tag is a protein/peptide conjugation pair which has shown great efficiency in protein purifications. This thesis aimed to evaluate the purification of white-rot fungal Panus rudis laccase with Catcher/Tag-based purification method. The second aim was to use Catcher/Tag version called SilkCatcher/SilkTag for purification for the first time and assess its suitability as a purification method. SilkCatcher/SilkTag is engineered from a surface protein domain of Lactobacillus plantarum. Three different P. rudis laccase variants with SilkCatcher, SilkTag or SpyTag, were produced in Pichia pastoris. SpySwitch/SpyTag is another Catcher/Tag version originating from the surface protein of Streptococcus pyogenes. The two Catcher variants, SilkCatcher and SpySwitch, were produced in Escherichia coli. SilkCatcher, SilkTag or SpyTag attached to laccases functioned as purification tags while SilkCatcher, SpySwitch and SilkTag were coupled to purification resin to purify laccases. As SpySwitch could not be produced in soluble form, purification was performed with only SilkCatcher and SilkTag. SilkCatcher/Tag showed good degree of purification though SilkCatcher and SilkTag binding was weak. The effectiveness of laccase purification with Catcher/Tag method could not be concluded decisively due to the weak binding and SpySwitch being produced in insoluble form. SilkCatcher/Tag has potential as a purification method, and P. rudis laccase was successfully produced in P. pastoris. This thesis provides the groundwork for future research of laccase purification with Catcher/Tag.Description
Supervisor
Aranko, SesiljaThesis advisor
Wan, XingMöttönen, Nea