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Protein Engineering, Design and Selection Advance Access





Published: Fri, 17 Nov 2017 00:00:00 GMT

Last Build Date: Fri, 17 Nov 2017 07:46:36 GMT

 



Designing a mutant Candida uricase with improved polymerization state and enzymatic activity

2017-11-17

Abstract
As human uricase has been silenced during evolution, counterparts from other species become an alternative for the treatment of hyperuricemia. Candida uricase is a promising option among them, but its aggregation propensity remains a major obstacle to clinical use. In this study, we designed two mutations according to homology-modeled 3D structure of Candida uricase: Cys249Ser substitution and C-terminal Leu deletion. The wild-type uricase and three mutants containing either or both of the mutations were expressed in Escherichiacoli BL21 and validated by mass spectrometry. Size-exclusion chromatography and electrophoresis analysis demonstrated that aggregation was induced by interchain disulfide bonds and could be significantly avoided by Cys249Ser substitution. In combination with Cys249Ser substitution, deletion of Leu increased the enzymatic activity by 8%. Taken together, mutant containing both mutations is chosen as our target protein which is comparatively more suitable for therapeutic use. In addition, homology-modeled 3D structure was proved to be an efficient approach for protein engineering.



Rational design of Pleurotus eryngii versatile ligninolytic peroxidase for enhanced pH and thermal stability through structure-based protein engineering

2017-11-07

Abstract
Versatile peroxidase (VP) from Pleurotus eryngii is a high redox potential peroxidase. It has aroused great biotechnological interest due to its ability to oxidize a wide range of substrates, but its application is still limited due to low pH and thermal stability. Since CiP (Coprinopsis cinerea peroxidase) and PNP (peanut peroxidase) exhibited higher pH and thermal stability than VP, several motifs, which might contribute to their pH and thermal stability, were identified through structure and sequence alignment. Six VP variants incorporating the beneficial motifs were designed and constructed. Most variants were nearly completely inactivated except V1 (Variant 1) and V4. V1 showed comparable activity to WT VP against ABTS, while V4 exhibited reduced activity. V1 displayed improved pH stability than WT VP, at pH 3.0 in particular, whereas the pH stability of V4 did not change a lot. The thermal stabilities of V1 and V4 were enhanced with T50 raised by 3°C. The results demonstrated that variants containing the beneficial motifs of CiP and PNP conferred VP with improved pH and thermal stability.