[Show abstract][Hide abstract] 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.
PROCESS BIOCHEMISTRY 12/2012; 47(12):2064–2071. · 2.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas sp. 42A2 LipC is a foldase-dependent lipase show-ing an interesting optimal temperature at 4-20 • C but with a moderate resistance to higher temperatures (Bofill et al., 2010). In order to increase the thermostability of LipC without losing its psychrophilic behaviour, we performed directed evolution based on a rational design mutation schedule. Since highly thermostable enzymes are known as rigid molecules, using the B-FITTER program (Reetz and Carballeira, 2007) on the LipC 3D-homology model, sites displaying the highest degree of flexibility were targeted to intro-duce appropriate mutations. Eight libraries were designed using different codon degeneracy systems, and the minimum number of clones to saturate each aminoacid position was calculated by means of CASTER program (Reetz and Carballeira, 2007). QuickChange PCR (Stratagene, 2003) was performed to mutate plasmid construction pBBlipClipH, bearing lipase and foldase genes in cis, and the result-ing variants were electroporated into PABST7.1, a Pseudomonas lipase and foldase deficient strain (Jaeger et al., 1999). Clones showing activity on tributyrin agar plates were selected and used for highthroughput screening of increased thermostability. Super-natants of above 3000 clones of PABS-pBBlipClipH were analyzed for lipolytic activity on pNP-caprylate before and after exposure to 60 • C for 15 min, and the residual activity was determined in com-parison with that of the wild type enzyme. Only mutants showing more than 20% residual activity after heat-shock were considered as potential hits and further characterized in detail. Seven hits were selected and their activity analyzed individually after standard-izing the protein concentration, and analysis of their nucleotide sequence was performed to check the mutations occurred. When all aminoacidic mutations have been analysed, new rounds of directed evolution through iterative saturation mutagenesis will be per-formed to get a mutant LipC with an even higher thermostability.
Journal of Biotechnology 11/2010; 150:404-404. · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Growth of Pseudomonas sp. 42A2 on oleic acid releases polymerized hydroxy-fatty acids as a result of several enzymatic conversions that could involve one or more lipases. To test this hypothesis, the lipolytic system of strain Pseudomonas sp. 42A2 was analyzed, revealing the presence of at least an intracellular carboxylesterase and a secreted lipase. Consensus primers derived from a conserved region of bacterial lipase subfamilies I.1 and I.2 allowed isolation of two secreted lipase genes, lipA and lipC, highly homologous to those of Pseudomonas aeruginosa PAO1. Homologous cloning of the isolated lipA and lipC genes was performed in Pseudomonas sp. 42A2 for LipA and LipC over-expression. The overproduced lipases were further purified and characterized, both showing preference for medium fatty acid chain-length substrates. However, significant differences could be detected between LipA and LipC in terms of enzyme kinetics and behaviour pattern. Accordingly, LipA showed maximum activity at moderate temperatures, and displayed a typical Michaelis-Menten kinetics. On the contrary, LipC was more active at low temperatures and displayed partial interfacial activation, showing a shift in substrate specificity when assayed at different temperatures, and displaying increased activity in the presence of certain heavy metal ions. The versatile properties shown by LipC suggest that this lipase could be expressed in response to variable environmental conditions.
[Show abstract][Hide abstract] ABSTRACT: Both the secretion and the cell surface display of Bacillus subtilis lipase A (Lip A) in Saccharomyces cerevisiae was investigated using different domains of the cell wall protein Pir4 as translational fusion partners. LipA gene minus its leader peptide was fused inframe in two places of PIR4 to achieve cell wall targeting, or substituting most of the PIR4 sequence, after the signal peptide and the Kex2 processed subunit I of Pir4 to achieve secretion to the growth medium. Expression of the recombinant fusion proteins was investigated in a standard and a glycosylation-deficient strain of S. cerevisiae, grown in selective or rich medium. Fusion proteins intended to be retained at the cell wall were secreted to the growth medium, most likely as result of the degradation of the Pir4 moiety containing the cell wall retention domain, giving low levels of lipase activity. However, the fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 400 IU of lipase activity per milliliter of cell supernatant. This is, to our knowledge, the first report of the efficient production, as a secreted protein, of lipase A of B. subtilis in baker's yeast.
Applied Microbiology and Biotechnology 08/2008; 80(3):437-45. · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Strain Pseudomonas sp. CR-611, previously isolated from a subtropical forest soil on tributyrine-supplemented plates, displays phenotypic and physiological properties consistent with those described for Pseudomonas fluorescens. However, no complete match to this species could be found after 16S rDNA comparison. Zymographic analysis of the strain revealed a complex lipolytic system, showing the presence of at least two enzymes with activity on MUF-butyrate. Alignment of Pseudomonas fluorescens lipase/esterase-coding sequences allowed the design of specific primers for family VI lipases, and the isolation and cloning of the resulting gene estA6. The recombinant clone obtained displayed high activity on fatty acid-derivative substrates, indicating that one of the lipolytic enzymes of the strain had been cloned. The enzyme, named EstA6, was then purified and characterized, showing maximum activity on short chain-length substrates under conditions of high temperature and neutral pH. Amino acid sequence alignment of EstA6 with other family VI esterases allowed identification of a highly conserved beta-/gamma-protobacterial cluster in family VI lipases, to which EstA6 belongs.