Description
In the framework of previous projects, we carried out studies on the biochemical and molecular characterization of a sterol esterase from the ascomycete Ophiostoma piceae. The enzyme hydrolyzes efficiently mixtures of triglycerides and sterol esters from wood extractives and its use to reduce pitch problems produced by these compounds during paper pulp production has been reported in a patent. Comparing the activity of several commercial lipases and esterase and the O. piceae enzyme showed that it is better hydrolyzing triglycerides and esters of p-nitrophenol and cholesterol, in the presence of detergents. Furthermore, the recombinant protein, expressed in Pichia pastoris, showed improved catalytic efficiency (~ x10) with respect to the native one for all the assayed substrates (p-nitrophenol, glycerol, and cholesterol esters). This increased effectiveness is explained by the presence of 6-8 additional amino acids at the N-terminus of the recombinant protein, which come from poor processing of the signal peptide in the vector used for cloning, affecting their aggregation state, making it more soluble.
As lipases do, the O. piceae enzymes can perform synthesis reactions in organic solvents. Recent studies showed that they esterify free phytosterols more efficiently than C. rugosa lipase, since lower enzyme doses and reaction times are required. The daily consumption of phytosterol esters helps to reduce cholesterol levels, so these results were collected in a patent proposing the use of the O. piceae enzyme for enzymatic synthesis of esters of phytosterols, as an alternative to the chemical methods currently used. In addition, the O. piceae esterase transforms polyvinyl acetate into polyvinyl alcohol, a more soluble and biodegradable compound used in many applications. This, together with the fact that the enzyme can perform synthesis reactions, suggests that it may be used both in the production and recycling of polyesters, processes currently carried out using chemical catalysts.
All results indicate that we have a robust enzyme catalyst, which could have different biotechnological applications, although there are still bottlenecks in order to verify its effectiveness at the pilot or industrial level. To solve some of them, this project has raised a number of strategies. First, we have addressed the expression of the enzyme in GRAS hosts such as Saccharomyces cerevisiae, facing the potential use of the enzyme in food industry related applications. The recombinant enzymes produced are being characterized biochemically and structurally to ensure they keep their catalytic properties, to get insight into their differences with other lipases and, finally, to try to explain its effectiveness in the hydrolysis and synthesis of sterol esters, triglycerides and polyesters, all compounds of industrial interest. Similarly, the immobilization of crudes containing the native or the recombinant enzyme expressed in Pichia pastoris is being assayed, since recycling of the catalyst is very interesting from an industrial point of view.
Another aspect of this project relies on "in silico" searching of new sterol esterases, similar to that of O. piceae, from different microbial origins. To do so, we are using public databases to select genes of fungal enzymes and bacterial metagenomic libraries for prokaryotes. The ultimate goal of these experiments is the expression of functional proteins on the heterologous host appropriate for each one of the selected proteins, for comparison of their catalytic properties with those from the Ophiostoma sterol esterase.
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