Construction of the combinatorial library of Rhizopus oryzae lipase mutated in the lid domain by displaying on yeast cell surface

The lid domain of lipase is an interesting portion which has a large effect on the substrate specificity of the enzyme. To investigate the relationship between the amino acid sequence of the lid domain of Rhizopus oryzae lipase (ROL) and its substrate specificity, six amino acids (Phe88–Arg89–Ser90–Ala91–Ile92–Thr93) consisting the lid domain were combinatorially changed and mutated ROLs were displayed on the yeast cell surface by cell surface engineering. Clones exhibiting halos around colonies on the plates containing tributyrin or soybean oil were screened. As the preliminary results, seven clones among 20,000 clones showed clear halos on tributyrin-containing plates, while no halos were detected on soybean oil-containing plates. Assays using fluorescent substrates (fluorescein dibutyrate and fluorescein dilaurate) indicated that these cells displaying mutated enzymes had a lower activity than the cells displaying the wild-type enzymes, but there were several cells which exhibited a unique substrate specificity. The results obtained from the determination of the DNA sequences of the lid domain of combinatorially mutated enzymes indicated that the sequential alignment of the (basic amino acid)–(polar amino acid)–(non-polar amino acid) might be important for the function of the lid domain.