Rational design of a new one-step purification strategy for Candida antarctica lipase B by ion-exchange chromatography.

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 degrees C. The broad isoelectric region of C. antarctica lipase B is unique as compared to almost all other alpha/beta-hydrolases which have a well-defined isoelectric point. A search in the lipase engineering database resulted in only one further alpha/beta-hydrolase, the Fusarium solani cutinase, which also has a broad isoelectric region.

High yield expression of lipase A from Candida antarctica in the methylotrophic yeast Pichia pastoris and its purification and characterisation.

The current investigation focuses on shedding further light on the characteristics of lipase A from Candida antarctica (CalA), which has attracted growing attention in its suitability for industrial applications. CalA was functionally expressed in the methylotrophic yeast Pichia pastoris, purified and characterised. A classical fed-batch process and a semi-continuous process were developed and tested with regard to their yield capacity. Lipase concentrations of 0.88 and 0.55 g l(-1) were obtained using the fed-batch and semi-continuous processes, respectively. The semi-continuous process reaches a total activity of 10,233,000 U and so surpasses the fed-batch process reaching 7,530,000 U. The purified enzyme showed highest activity between 50 and 70 degrees C at pH 7.0 and a preference for short-chain triglycerides (C4-C8). Significantly reduced activity was observed in the presence of hydrophilic esters.

The putative lipase, AF1763, from Archaeoglobus fulgidusis is a carboxylesterase with a very high pH optimum.

The open reading frame AF1763, annotated as a putative lipase gene (lipA) of the hyperthermophilic archaeon, Archaeoglobus fulgidus DSM 4304, was cloned and over-expressed in E. coli. A sequence analysis of LipA and the investigation of a truncated enzyme implied a special function of the C-terminal part of LipA. The substrate spectrum of the enzyme suggested that LipA is a carboxylesterase rather than a canonical lipase. The enzyme showed optimal activity at 70 degrees C and between pH 10 and 11, which is among the most alkaline pH range detected for hydrolases.