Improvement of cold adaptation of Bacillus alcalophilus alkaline protease by directed evolution

• Protein engineering of alkaline protease was performed using error-prone PCR.
• A cold-active and alkali-resistant protease E110A/E134A was successfully generated.
• Single mutants E110A and E134A were obtained via site-directed mutagenesis.
• The activity and kcat/Km value were 3.67 and 2.72 times that of wild-type at 10 °C.

Protein engineering of the Bacillus alcalophilus PB92 ATCC 31408 alkaline protease (SBA) was performed to obtain enzymes with improved cold adaptation. The activity of SBA at low temperature was enhanced through direct evolution using error-prone polymerase chain reaction. Two mutation sites, Glu110Ala and Glu134Ala, were obtained in SBA. To identify the mutation of amino acids in E110A/E134A related to its activity at low temperature, single mutants E110A and E134A were obtained via site-directed mutagenesis. The kcat/Km values of the mutants E110A, E134A and E110A/E134A at 10 °C were 1.5-, 2.2- and 2.7-fold higher, respectively, than that of the wild-type. Through the three-dimensional structure analysis, it was indicated that E110A/E134A showed an improved activity at low-temperature condition as a result of the disrupted hydrogen bond, increased protein hydrophobicity, and decreased calcium affinity. The findings of this study provides the theoretical basis and background data for improvement of the cold adaptation in SBA by protein engineering.

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