Engineering, expression, purification, and production of recombinant thermolysin.

Thermolysin [EC 3.4.24.27] is a thermostable neutral zinc metalloproteinase originally identified in the culture broth of Bacillus thermoproteolyticus Rokko. Since the discovery in 1962, the enzyme has been extensively studied regarding its structure and catalytic mechanism. Today, thermolysin is a representative of zinc metalloproteinase and an attractive target in protein engineering to understand the catalytic mechanism, thermostability, and halophilicity. Thermolysin is used in industry, especially for the enzymatic synthesis of N-carbobenzoxy L-Asp-L-Phe methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame. Generation of genetically engineered thermolysin with higher activity in the synthesis of ZDFM has been highly desired. In accordance with the expansion of studies on thermolysin, various strategies for its expression and purification have been devised and successfully used. In this review, we aim to outline recombinant thermolysins associated with their engineering, expression, purification, and production.