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Expresión heteróloga, caracterización y aplicaciones biotecnológicas de la esterol esterasa-lipasa de Ophiostoma piceae y de otras enzimas seleccionadas de genomas fúngicos
|Authors:||Vaquero, María Eugenia|
|Advisor:||Barriuso, Jorge ; Martínez Hernández, María Jesús|
|Publisher:||CSIC - Centro de Investigaciones Biológicas Margarita Salas (CIB)|
|Abstract:||The ascomycete Ophiostoma piceae secretes a sterol esterase/lipase (OPE)
characterized for its wide substrate specificity. This protein was successfully
expressed in the yeast Pichia pastoris, and preliminary studies showed its
broad biotechnological potential in hydrolysis and synthesis reactions.
Therefore, across this work we tried to deepen the knowledge on this enzyme
and find similar enzymes, being the main objectives:
i. To explore different heterologous expression systems in search of the
most suitable for the production of OPE.
ii. To crystallize the protein to get insight into both its structure and the
molecular mechanisms involved in catalysis.
iii. To select from fungal genomes new enzymes similar to OPE, to be
expressed and characterized.
iv. To evaluate the potential of these new enzymes in various
Search for new heterologous expression systems for OPE.
By using different strains of Escherichia coli as hosts, the sterol esterase
expressed formed inclusion bodies and was solubilized only in the presence of
the detergent Sarkosyl, except the form containing certain extra amino acids
at the N-terminal terminus that yielded a protein fraction soluble but inactive.
The enzyme produced in the prokaryotic host and solubilized with Sarkosyl
presented different folding to the native and recombinant OPE expressed in P.
pastoris, which may explain its lack of activity. In contrast, expression of
OPE in the yeast Saccharomyces cerevisiae, which also holds the status of
GRAS organism, yielded soluble and active enzyme with all constructs and
strains. This recombinant protein showed similar molecular weight,
glycosylation and secondary structure to that expressed in P. pastoris.
However, the amount of enzyme produced in S. cerevisiae and its solubility
were lower, and the kinetic parameters demonstrated its reduced efficiency. In
view of the results, it follows that several post-translational modifications,
may affect enzymes’ folding and be crucial to obtain an active protein. Up to
now, the expression levels of OPE in S. cerevisiae have been low, but the current findings open the way towards improving its production and/or catalytic properties in this yeast by directed evolution techniques, as well as to its use in food-related processes.|
Resolution of the three dimensional structure of OPE: homologies and differences with other lipases C. rugosa-like family. The resolution of the molecular structure of OPE in both its open and closed conformations gave very interesting information. The closed form has an amphiphilic lid that limits the access to the active site. The displacement of this element 30 Å from its original position allows the entry of substrates to the active site. These are housed in a tunnel-shaped substrate’s pocket of 30 Å-length, high in hydrophobic amino acids, ending in an area close to the surface that could serve as a way out of the reaction products. The design of a mutant with a blocked tunnel caused a drastic hydrolysis reduction of long chain fatty acids substrates (16 C atoms), but not of those with short (4 C atoms) or medium acyl chains (12 C atoms). The open form of OPE is organized as a homodimer, leaving a large cavity between the two monomers. Analytical ultracentrifugation experiments showed that the transition from monomer to dimer occurred in the presence of a substrate or inhibitor. The resolution of the three-dimensional structure of OPE in both conformations allowed to deduce its functional dimeric state and will facilitate conducting future structure-function studies to better understand its catalytic mechanisms and the role of the inner tunnel. Properties of novel sterol esterases/lipases selected from fungal genomes. Biotechnological applications. Three enzymes of the C. rugosa-like lipases’ family, belonging to Nectria haematococca, Trichoderma reesei and Aspergillus niger, were selected after in silico search in fungal genomes, expressed and compared to OPE. Common features to all of them, concerning their size, stability and multimeric forms in solution, were extrapolated. However, their kinetic characterization revealed that the A. niger lipase was unable of hydrolyzing cholesterol esters or synthesizing sitostanol esters, showing that this enzyme was not an sterol esterase. However, similar results were obtained in the synthesis of polycaprolactone using either the commercial enzyme of C. rugosa, OPE and A. niger. Compared to the other enzymes tested, OPE showed the highest efficiency in cholesterol ester hydrolysis and in synthesis sitostanyl oleate by transesterification, producing high acylation degrees in very short times. The expression and characterization of these novel lipases belonging to the C. rugosa-like family carried out in this work allowed to find out some of their similarities and differences, and indicated that the versatility in the hydrolysis of triglycerides and sterol esters is not a common feature of all members of this family. Comparing the results for the newly characterized enzymes, OPE, and commercial crude of C. rugosa in several biotechnological applications, OPE appeared as the most promising enzyme.
Finally, the search in fungal genomes of novel enzymes similar to the C. antarctica lipase B (CalB), one of the most used lipases at industrial level, is described. Because of their versatility, most of the proteins showing high homology with CalB are under patent. In this work, the sequence of a lipase from the basidiomycete Plicaturopsis crispa (PlicB), that showed a 44% sequence similarity with CalB, was identified. PlicB successfully expressed in P. pastoris and characterized, appearing as a monomer of molecular mass around ~ 37 kDa, similarly to CalB. Although PlicB hydrolyzed p-nitrophenol esters less efficiently than CalB, the novel enzyme showed greater activity against a triglyceride analog. In view of the results, genome mining appears as an interesting alternative to traditional screening methods.
|Description:||200 p.-40 fig.-6 tab.|
|Appears in Collections:||(CIB) Tesis|
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