Journal of Microbiology and Biotechnology

  • 제34권8호
  • /
  • Pages.1660-1670
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  • 2024
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Engineering of the Phytase YiAPPA to Improve Thermostability and Activity and Its Application Potential in Dephytinization of Food Ingredients

  • Jing Zeng (Institute of Microbiology, Jiangxi Academy of Sciences) ;
  • Jianjun Guo (Institute of Microbiology, Jiangxi Academy of Sciences) ;
  • Lin Yuan (Institute of Microbiology, Jiangxi Academy of Sciences)
  • 투고 : 2024.03.14
  • 심사 : 2024.06.14
  • 발행 : 2024.08.28
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초록

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80℃ was extended from 14.81 min to 23.35 min, half-inactivation temperature (T5030) was increased from 55.12℃ to 62.44℃, and Tm value was increased from 48.36℃ to 53.18℃. Meanwhile, the specific activity of K189R/K216R at 37℃ and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzyme-substrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

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과제정보

This work was financially supported by grants from the National Natural Science Foundation of China (grant number 32160579) and Jiangxi Natural Science Foundation (grant numbers 20212BCJ23033, S2021GDQN2403, and 2022YJC2006).

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