Design of Thermostable Beta-Propeller Phytases with Activity over a Broad Range of pHs and Their Overproduction by Pichia pastoris

Abstract

Thermostable phytases, which are active over broad pH ranges, may be useful as feed additives, since they can resist the temperatures used in the feed-pelleting process. We designed new beta-propeller phytases, using a structure-guided consensus approach, from a set of amino acid sequences from Bacillus phytases and engineered Pichia pastoris strains to overproduce the enzymes. The recombinant phytases were N-glycosylated, had the correct amino-terminal sequence, showed activity over a pH range of 2.5 to 9, showed a high residual activity after 10 min of heat treatment at 80°C and pH 5.5 or 7.5, and were more thermostable at pH 7.5 than a recombinant form of phytase C from Bacillus subtilis (GenBank accession no. AAC31775). A structural analysis suggested that the higher thermostability may be due to a larger number of hydrogen bonds and to the presence of P257 in a surface loop. In addition, D336 likely plays an important role in the thermostability of the phytases at pH 7.5. The recombinant phytases showed higher thermostability at pH 5.5 than at pH 7.5. This difference was likely due to a different protein total charge at pH 5.5 from that at pH 7.5. The recombinant beta-propeller phytases described here may have potential as feed additives and in the pretreatment of vegetable flours used as ingredients in animal diets.

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

“…Encouraged by the results of HAP engineering, attempts to modify the alkaline BPP phytases were also reported recently [58,66]. However, the initial activity is low and the improvements are rather limited.…”

“…In addition, a flexible loop was stabilized in the hybrid enzyme, which is considered as a combined effect of multiple mutations. Similar approach has also been applied to the BPP phytase [66]. The consensus sequence that was generated from an alignment containing 15 Bacillus phytases showed higher specific activity and optimal temperature comparing to the most identical protein (B. amyloliquefaciens phytase; sequence identity, 98 %).…”

“…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].…”

Effect of Beta-propeller Phytase from Pichia pastoris on Energy Partition in Juvenile Litopenaeus vannamei Fed a Plant Protein-Based Diet

“…The β-propeller phytase FTEII from Bacillus species is an alternative to histidine acid phytases because of its high thermostability, calcium phytate-complex substrate specificity, pH profile, and proteolysis resistance (Oh, Choi, Park, Kim, & Oh, 2004). Several β-propeller phytases, one from B. subtilis (Guerrero-Olazarán, Rodríguez-Blanco, Carreon-Treviño, Gallegos-López, Viader-Salvadó, 2010), and others designed by a structure-guided consensus approach, have been produced by the methylotrophic yeast P. pastoris (Viader-Salvadó, Gallegos-López, Carreón-Treviño, Castillo-Galván, Rojo-Domínguez, & Guerrero-Olazarán, 2010). Therefore, during the last two decades, optimizing plant-based diets using phytase as a feed additive has been a way to reduce phytates' antinutritional effect (Fox, Lawrence, Saccardi, Davis, Ricque-Marie, Cruz-Suarez, & Samocha 2006;Lei et al, 2007;Lim & Lee, 2009), thereby improving the absorption and retention of minerals and amino acids (Rebollar and Mateos, 1999;Gómez-Villalva, 2005), and enhancing the activity of proteolytic enzymes, survival rate, and weight gain (Ricque-Marie, Cruz-Suarez, Zavala-Chavez, Nieto-Lopez, Guajardo, Tapia-Salazar, McCallum, & Newkirk, 2004;Cao, Ye, Wang, & Guo, 2010;Wang, Yang, Han, Dong, Yang, & Zou, 2009;Gamboa, Aguilera, Gaxiola, Cuzon, Guerrero, & López, 2011).…”

Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

“…Of the rational design methods, a structureguided consensus approach combines information about the quaternary structure with knowledge of a few sequences that have moderate identity to reduce the number of target residues in a given protein to be mutated. At present, it is considered an effi-cient and reliable method for improving enzymatic properties (16,(22)(23)(24)(25).…”