Abstract
A fast and efficient one-step method for purification of lipase B from Candida antarctica by ion-exchange chromatography was developed by rational design. The electrostatic properties of the enzyme were calculated and validated by isoelectric focusing and measurement of the titration curve. C. antarctica lipase B shows an unusual pH profile with a broad isoelectric region from pH 4 to 8. At pH 3 C. antarctica lipase B can be bound to a cation-exchange chromatography column and was purified to homogeneity with a purification factor of 2.4. It was stable at pH 3, the residual activity was still 80% after 6 days incubation at 20 °C. The broad isoelectric region of C. antarctica lipase B is unique as compared to almost all other α/β-hydrolases which have a well-defined isoelectric point. A search in the lipase engineering database resulted in only one further α/β-hydrolase, the Fusarium solani cutinase, which also has a broad isoelectric region.
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