Improvement of BsAPA Aspartic Protease Thermostability via Autocatalysis-resistant Mutation.

An aspartic protease gene (Bsapa) was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris. The recombinant BsAPA showed maximal activity at pH 3.0 and 75 °C and remained stable at 70 °C and below, indicating the thermostable nature of BsAPA. However, heat inactivation still limits the application of BsAPA. To further improve its thermostability, an autocatalysis site (L205-F206) in BsAPA was identified and three mutants (F193W, K204P, and A371V) were generated based on structure analysis neighboring the autocatalysis site. These mutants have improved thermostability, and their half-life at 75 °C increased by 0.5-, 0.2- and 0.3-fold, respectively. A triple-site mutant (F193W/K204P/A371V) was generated, with 1.5-fold-increased half-life at 80 °C, and a 10.7 °C-increased Tm, compared with the wild-type. These results indicate that autocatalysis of aspartic protease reduces enzyme thermostability. Further, site-directed mutagenesis at regions near the autocatalysis site is an efficient approach to improve aspartic protease thermostability.

• Publication year

  2019

• Venue

  Journal of Agricultural and Food Chemistry

• Publication date

  2019-08-29

• Fields of study

  Medicine, Chemistry

• Identifiers
  DOI 10.1021/acs.jafc.9b03959PMID 31462045
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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