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Related Experiment Videos

[Mutants of subtilisin E].

Y H Yang1, Y J Wu, L Jiang

  • 1Shanghai Research Center of Biotechnology.

Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology
|September 8, 2000
PubMed
Summary

Directed mutagenesis of subtilisin E at Ser236Cys (BP-1) significantly enhanced protease activity and enzyme stability. Further mutations negatively impacted these properties.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Context:

  • Subtilisin E is a widely used serine protease.
  • The active site residues (Asp32, His64, Ser221) are crucial for catalytic activity.
  • Ser236 is located on the enzyme surface, distant from the active site.

Purpose:

  • To investigate the effect of mutating surface-exposed Ser236 to Cysteine in subtilisin E.
  • To assess the impact of this mutation on enzyme activity and stability.
  • To explore the consequences of further mutations on the engineered enzyme.

Summary:

  • Directed mutagenesis replaced Ser236 with Cysteine in subtilisin E, creating mutant BP-1.
  • BP-1 exhibited a 150% increase in catalytic efficiency (Kcat/Km) and a 3-fold improvement in stability at pH 7.4 and 50°C.
  • Subsequent mutations in BP-1 (BU-1 and BW-1) resulted in decreased activity and stability.

Impact:

  • The Ser236Cys mutation in subtilisin E enhances both enzyme activity and stability.
  • This finding provides insights into protein engineering strategies for improving enzyme performance.
  • Further modifications can diminish the beneficial effects of initial mutations, highlighting the complexity of enzyme engineering.

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