Wataru Kugimiya

Improvement of the optimum temperature of lipase activity for Rhizopus niveus by random mutagenesis and its structural interpretation.

Random mutagenesis was used to improve the optimum temperature for Rhizopus niveus lipase (RNL) activity. The lipase gene was mutated using the error-prone PCR technique. One desirable mutant was isolated, and three amino acids were substituted in this mutant (P18H, A36T and E218V). The wild-type and this randomly mutated lipase were both purified and characterized. The specific activity of the mutant lipase was 80% that of the wild-type. The optimum temperature of the mutant lipase was higher by 15 degrees C than that of the wild-type. To confirm which substitution contributed to enhancing the optimum temperature for enzymic activity, two chimeric lipases from the wild-type and randomly mutated gene were constructed: chimeric lipase 1 (CL-1; P18H and A36T) and chimeric lipase 2 (CL-2; E218V). Each of the chimeric enzymes was purified, and the optimum temperature for lipase activity was measured. CL-1 had a similar optimum temperature to that of the wild-type, and CL-2 had a higher temperature like the randomly mutated lipase. The mutational effect is interpreted in terms of a three-dimensional structure for the wild-type lipase.

Chitosan-Soyprotein Interaction as Determined by Thermal Unfolding Experiments

Chitosan interaction with soybean β-conglycinin β3 was investigated by thermal unfolding experiments using CD spectroscopy. The negative ellipticity of the protein was enhanced with rising solution temperature. The transition temperature of thermal unfolding of the protein (T m) was 63.4 °C at pH 3.0 (0.15 M KCl). When chitosan was added to the protein solution, the T m value was elevated by 7.7 °C, whereas the T m elevation upon addition of chitosan hexamer (GlcN)6 was 2.2 °C. These carbohydrates appear to interact with the protein stabilizing the protein structure, and the interaction ability could be evaluated from the T m elevation. Similar experiments were conducted at various pHs from 2.0 to 3.5, and the T m elevation was found to be enhanced in the higher pH region. We conclude that chitosan interacts with β-conglycinin through electrostatic interactions between the positive charges of the chitosan polysaccharide and the negative charges of the protein surface.

Thermal stability of Rhizopus niveus lipase expressed in a kex2 mutant yeast.

Lipase from Rhizopus niveus (RNL) has a complex structure, and recombinant RNL, has even more complex structural properties in the yeast, Saccharomyces cerevisiae. These properties are due to the processing and to the size of the glycosylated sugar chain. The processing site was presumed to be that for the proteinase product of the KEX2 gene in yeast. We therefore, constructed an expression system in which the KEX2 gene was disrupted to produce a non-processed type of lipase with high thermal stability. This type of lipase was thermally stable to a temperature 15 degrees C higher than that of each processed type of lipase. This non-processed lipase had 50% residual activity after 2 h at 50 degrees C, while the residual activity of the processed lipases was only 10% after 30-45 min of incubation at 50 degrees C. The CD spectrum of the non-processed type of lipase at 222 nm was almost unchanged by heating, suggesting that this group of lipases had a very rigid structure and that the peptide bond between the A- and B-chain contributed to maintain this rigid structure. On the other hand, the length of the sugar chain bound to the lipase had no effect on the thermal stability.