Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 65, Issue 34, Pages 7337-7344Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.7b02116
Keywords
phytases; pepsin resistance; trypsin resistance; thermostability; stability at low pH; hydrolysis of corn meal phytate; optimizing the polarity and charge of the amino acids
Funding
- National Natural Science Foundation of China [31402110]
- Special Fund for Agro-Scientific Research in Public Health of China [201403047]
- National Science Fund for Distinguished Young Scholars of China [31225026]
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Susceptibility to proteases usually limits the application of phytase. We sought to improve the pepsin and trypsin resistance of YeAPPA from Yersinia enterocolitica and YkAPPA from Y. kristensenii by optimizing amino acid polarity and charge. The predicted pepsin/trypsin cleavage sites F89/K226 in pepsin/trypsin-sensitive YeAPPA and the corresponding sites (F89/E226) in pepsin-sensitive but trypsin-resistant YkAPPA were substituted with S and H, respectively. Six variants were produced in Pichia pastoris for catalytic and biochemical characterization. F89S, E226H, and F89S/E226H elevated pepsin resistance and thermostability and K226H and F89S/K226H improved pepsin and trypsin resistance and stability at 60 degrees C and low pH. All the variants increased the ability of the proteins to hydrolyze phytate in corn meal by 2.6-14.9-fold in the presence of pepsin at 37 degrees C and low pH. This study developed a genetic manipulation strategy specific for pepsin/trypsin-sensitive phytases that can improve enzyme tolerance against proteases and heat and benefit the food and feed industry in a cost-effective way.
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