Expression and immobilization of trypsin-like domain of serine protease from Pseudomonas aeruginosa for improved stability and catalytic activity

Protein engineering and enzyme immobilization strategies have produced numerous biocatalysts for modern industrial applications. In this study, we have also used these two strategies for improving the operational stability and catalytic efficiency of serine protease from Pseudomonas aeruginosa. The enzyme serine protease was truncated to separate its trypsin-like domain from the PDZ1 and PDZ2 domains. The truncated trypsin-like domain was expressed in Escherichia coli BL21, and its catalytic activity and thermostability were estimated. Later this trypsin-like domain was immobilized with 2% Na-alginate. The immobilized domain showed 10 degrees C increase in optimum temperature compared to its free counterpart. Kinetic studies showed two-folds increased V-max of the immobilized domain. Likewise, the K-m value of this domain was 11.5 folds lower compared to the free trypsin-like domain. The catalytic efficiency (K-cat/K-m) of the immobilized enzyme also elevated to 311 folds. Additionally, the immobilized trypsin-like domain remained active in the presence of surfactants (Triton-X 100, SDS, and Tween-40) and metal ions (Mg2+, Ca2+, Na+, and Zn2+). It also efficiently removes gelatin layer from X-ray film and hair from sheepskin. Thus, the immobilized trypsin-like domain of serine protease, with increased thermostability and catalytic efficiency, is operationally more stable than the soluble truncated trypsin-like domain.