A rational design to enhance the resistance of Escherichia coli phytase appA to trypsin.


Abstract

Escherichia coli phytase appA, which hydrolyzes phytate, has been widely applied as an important feed supplement, but its resistance to trypsin needs to be improved. Six putative solvent-accessible amino acid residues (K74, K75, K180, R181, K183, and K363), which could be easily attacked by trypsin, were selected to improve trypsin tolerance of Escherichia coli phytase appA. Inspection of the three-dimensional structure and computational design via hydrogen bond analysis, six optimal mutation sites of K74D/K75Q/K180N/R181N/K183S/K363N, which strengthened the hydrogen bonding, were performed to generate three mutants. Results showed that the most beneficial mutant appA-M6 had a specific activity of 3262 U/mg with molecular weight of approximately 52-55 kDa. Similar to appA-WT, the optimal pH (4.5) and temperature (60 °C) of appA-M6 were unchanged. Compared with appA-WT, appA-M6 showed a significant enhancement (p < 0.05) in resistance to trypsin and a 3.8 °C increase in melting temperature (Tm). We concluded that introduction of hydrogen bonds and N-glycosylation modification resulted in decreased enzyme flexibility and increased the enzyme stability against proteolysis and thermal denaturation. The mutant appA-M6 generated in this study could be applied for the large-scale commercial production of phytase and thus could benefit the food and feed industry.

Submission Details

ID: MHVcY6vM4

Submitter: Shu-Ching Ou

Submission Date: March 29, 2019, 11:07 a.m.

Version: 1

Publication Details
Wang X;Du J;Zhang ZY;Fu YJ;Wang WM;Liang AH,Appl Microbiol Biotechnol (2018) A rational design to enhance the resistance of Escherichia coli phytase appA to trypsin. PMID:30178201
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1DKQ 2000-08-03 2.05 CRYSTAL STRUCTURE OF PHYTATE COMPLEX ESCHERICHIA COLI PHYTASE AT PH 5.0. PHYTATE IS BOUND WITH ITS 3-PHOSPHATE IN THE ACTIVE SITE. HG2+ CATION ACTS AS AN INTERMOLECULAR BRIDGE
4TSR 2015-05-06 2.07 The Complex Structure of Mutant Phytase with IHS
1DKM 2000-08-02 2.25 CRYSTAL STRUCTURE OF ESCHERICHIA COLI PHYTASE AT PH 6.6 WITH HG2+ CATION ACTING AS AN INTERMOLECULAR BRIDGE
1DKP 2000-08-03 2.28 CRYSTAL STRUCTURE OF PHYTATE COMPLEX OF ESCHERICHIA COLI PHYTASE AT PH 6.6. PHYTATE IS BOUND WITH ITS 3-PHOSPHATE IN THE ACTIVE SITE. HG2+ CATION ACTS AS AN INTERMOLECULAR BRIDGE
1DKL 2000-08-03 2.3 CRYSTAL STRUCTURE OF ESCHERICHIA COLI PHYTASE AT PH 4.5 (NO LIGAND BOUND)
1DKO 2000-08-03 2.38 CRYSTAL STRUCTURE OF TUNGSTATE COMPLEX OF ESCHERICHIA COLI PHYTASE AT PH 6.6 WITH TUNGSTATE BOUND AT THE ACTIVE SITE AND WITH HG2+ CATION ACTING AS AN INTERMOLECULAR BRIDGE
1DKN 2000-08-03 2.4 CRYSTAL STRUCTURE OF ESCHERICHIA COLI PHYTASE AT PH 5.0 WITH HG2+ CATION ACTING AS AN INTERMOLECULAR BRIDGE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.5 Periplasmic AppA protein P07102 PPA_ECOLI