Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design

Wu, Tzu-Hui; Chen, Chun‐Chi; Cheng, Ya‐Shan; Ko, Tzu‐Ping; Lin, Cheng-Yen; Lai, H.L.; Huang, Ting-Yung; Liu, Je-Ruei; Guo, Rey‐Ting

doi:10.1016/j.jbiotec.2014.01.034

Cited by 46 publications

(25 citation statements)

References 19 publications

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“…Thus, N-glycosylation represents a stabilizing factor against proteolytic cleavage by proteases (29). Apart from conferring an increased resistance to proteolysis, N-glycosylation can also enhance enzyme thermostability and alter catalytic activity (30)(31)(32)(33). The improved conformational stability of enzymes derived from the steric interactions between N-linked glycan and protein can decrease the enzyme flexibility or increase the rigidity of the enzyme structure (34,35).…”

mentioning

confidence: 99%

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.A s much as 80% of the total phosphorus in cereal grains, oilseeds, and legumes exists in the form of phytate (myo-inositol hexakisphosphate), one of the most important functional ingredients in animal feed (1). However, phytate usually forms indigestible complexes with mineral cations and proteins, thereby causing reductions in nutrient utilization (2, 3). The dietary phytate undigested by monogastric animals is also released into the environment, where it causes phosphorus pollution (4, 5).The enzyme phytase catalyzes the sequential release of inorganic phosphate and lower myo-inositol phosphates from phytate (6); thus, inclusion of exogenous phytases in animal feeds as enzyme additives can enhance the nutrient uptake, lower the feedstuff production costs, and protect the environment in regions where intensive animal farming is conducted (7,8). Numerous phytases have been identified from microorganisms, animals, and plants (9-11), but only a few of them are commercially produced (12). The extensive application of phytases is limited by enzyme lability and protease sensitivity under high processing temperatures and low physiological pHs. Therefore, improving phytase stability at low pHs and high protease concentrations is desirable.Pepsin, a monomeric protein composed of two ␤-barrel-like domains, represents the main proteolytic enzyme in gastric juice (13). The acidic residues D32 and D215 are mainly responsible for proteoly...

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confidence: 99%

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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“…Notably, a putative N-glycosylation site N204 is located at the edge of the phytate-binding pocket, and the N-linked glycans might cause steric hindrance for phytate entry or product release. More recently, our mutagenesis experiments have validated the N204 glycosylation, and we also demonstrated a significantly increased enzyme catalytic activity by eliminating the glycosylation site [46]. Given the unaltered protein thermostability of the N204 de-glycosylation mutants (data not shown), it is likely that the selection of N204C mutant in the GSSM study is a result of higher initial activity.…”

Section: Directed Evolutionmentioning

confidence: 59%

“…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]. Based on the sequence alignment, the residues near the substrate binding pocket of EcAppA were modified in accordance with the Citrobacter phytases [46]. The V89T mutant showed 17.5 % increase in specific activity.…”

Section: Homologous Alignmentmentioning

confidence: 99%

“…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].…”

Section: Homologous Alignmentmentioning

confidence: 99%

“…On the other hand, as the extensive glycosylation of CbAppA in the yeast host attributing to protein thermostability, the EcAppA was modified to adopt the CbAppA glycosylation status [46]. The resulting mutant harboring additional CbAppA glycosylation sites exhibited significant increase in residual activity at 80°C.…”

Section: Homologous Alignmentmentioning

confidence: 99%

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

Chen

Cheng²,

et al. 2015

ChemBioEng Reviews

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Phytase is one of the most important feed additive enzymes for monogastric animal, because it hydrolyzes the indigestible phytate in the cereal-based feedstock to release phosphate as an essential nutrient. To understand its molecular machinery, the three-dimensional structures of various types of phytases and complexes have been studied extensively. For commercial applications, important properties such as higher catalytic efficiency and higher thermostability are desired. Since a phytase with both beneficial characteristics is hardly found in nature, various protein engineering strategies are popular in modifying the existing enzymes with enhanced performance. In this review, the up-todate status of phytase structural and engineering studies is summarized. In addition, structural perspectives of some engineered phytases with improved properties are also provided. These results broaden the understanding of phytases and will be important for phytase applications in the future.

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Enhancing thermal stability and lytic activity of phage lysin PlyAB1 from Acinetobacter baumannii

Yuan

Zhang

et al. 2022

Biotech & Bioengineering

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With the increasingly serious drug resistance of Acinetobacter baumannii, there is an increasingly urgent need for new antibacterial drugs. Phage lysin PlyAB1 has a bactericidal effect on drug‐resistant A. baumannii, which has the potential to replace antibiotics to fight infection caused by A. baumannii. However, its application is limited by its thermal stability and lytic activity. To solve these problems, molecular dynamics (MD) simulations combined with Hotspot wizard 3.0 were used to identify key residue sites affecting thermal stability, and evolutionary analysis combined with multiple sequence alignment was used to identify key residue sites affecting lytic activity. Four single‐point variants with significantly increased thermal stability and four single‐point variants with significantly lytic activity were obtained, respectively. Furthermore, by superimposing mutations, we obtained three double‐point variants, G100Q/K69R, G100R/K69R, and G100K/K69R, with significantly improved thermal stability and improved lytic activity. At 45°C, the lytic activity and half‐life of the optimal variant G100Q/K69R were 1.51‐ and 24‐fold higher than those of the wild PlyAB1, respectively. These results deepen our understanding of the structure and function of phage lysin and contribute to the application of phage lysin in antibiotic substitution.

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Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design

Cited by 46 publications

References 19 publications

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

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

Enhancing thermal stability and lytic activity of phage lysin PlyAB1 from Acinetobacter baumannii

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