From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase

Abstract

Naturally-occurring phytases having the required level of thermostability for application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequence comparisons. A consensus enzyme based on 13 fungal phytase sequences had normal catalytic properties, but showed an unexpected 15-22 degrees C increase in unfolding temperature compared with each of its parents. As a first step towards understanding the molecular basis of increased heat resistance, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed expected, based on comparisons of both three-dimensional structures, to contribute more to phytase stabilization than non-consensus amino acids. For some consensus amino acids, predicted by structural comparisons to destabilize the protein, mutational analysis was performed. Interestingly, these consensus residues in fact increased the unfolding temperature of the consensus phytase. In summary, for fungal phytases apparently an unexpected direct link between protein sequence conservation and protein stability exists.

Designed to be stable: Crystal structure of a consensus ankyrin repeat protein

“…The canonical sequence approximation has previously been used to create molecules with improved stabilities (9,(11)(12)(13)(14). We have successfully applied this consensus design to ARs.…”

“…The collection of nearly 8,000 AR sequences in the SMART database (10) facilitates such an approach. This consensus design concept has been used to generate enzymes with improved thermostability (11,12) and to improve antibody stability (9,13,14). We used this concept to design a consensus AR consisting of fixed framework residues that are responsible for repeat structure maintenance (fold conservation) and of randomized interacting residues, i.e., residues, which, in a repeat domain, create a randomized surface for interaction with target proteins.…”

Current Progresses in Phytase Research: Three‐Dimensional Structure and Protein Engineering

“…Using homologous sequence alignment to improve the EcAppA performance had been more challenging due to the lack of highly identical thermo-tolerant bacterial HAP. AB345F7 showed the highest catalytic efficiency; A234567 showed the highest thermostability [52] EcAppA Homologous sequence alignment Mutating residues near active site and modifying glycosylation status according to Citrobacter phytases V89T mutant showed 17.5 % higher specific activity; triple N-glycosylation mutant showed more than 27 % higher residual activity at 85°C [46] EcAppA Deleting unstable C-terminal region Deleting seven C-terminal residues 39.07 % higher residual activity at 80°C Consensus fungal phytase showed optimal temperature at 71°C and a T m of 78°C [65] Bacillus phytases Consensus sequence Consensus sequence from 15 Bacillus phytases Improved specific activity and higher optimal temperature [66] (CaAppA) and Citrobacter braakii (CbAppA) are good templates for this purpose (identity with EcAppA, 57.1 % and 60.6 %) [21,53,54]. Both Citrobacter phytases showed higher specific activity than EcAppA (>3000 U mg -1 vs.~2000 U mg -1 ), and the CbAppA retains 66 % residual activity after 70°C treatment for 30 min when expressed in Saccharomyces cerevisiae [53,54].…”

Site-Directed Mutagenesis as Applied to Biocatalysts

“…There are numerous examples in which this approach has been used to increase the thermostability of proteins. Some proteins reconstruct the complete consensus sequence (Lehmann et al, 2000;Sullivan et al, 2011). In others, point mutations were used to identify the residues that increased stability.…”