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Cold-adapted lipase
Rational design
Saturation mutagenesis
Enzyme improvement
Pseudomonas
Abstract:
Cold-adapted Pseudomonas aeruginosa LipC is a secreted lipase showing differential properties compared
to its well-known counterpart LipA. LipC is fundamentally a cold-acting lipase, capable of tolerating high
concentrations of ions and heavy metals, and showing a shift in substrate specificity when incubated
at higher temperatures. These properties make LipC an interesting enzyme, well suited for biotechnological
or environmental applications, where activity at low temperatures would be required. However,
a relatively low thermal resistance constitutes the main drawback for using this enzyme in long-term
operational processes. To overcome the lability of LipC, we developed a rational design system to modify
specific sites on the enzyme structure to obtain an improved variant of the lipase bearing higher thermal
stability, but without loss of its cold-adapted properties. Eight mutant libraries plus two point mutations
were constructed affecting those amino acids showing the highest flexibility on the 3D model structure.
After screening more than 3000 mutant clones, a LipC variant bearing two amino acid changes and the
required thermostability and cold-adapted properties was obtained. The new variant D2 H8, with a 7-
fold increased thermal stability in comparison to wild type LipC, will guarantee the use and maintenance
of such a lipase in a number of processes being performed at low (4–20 ◦C) temperatures.
