In the last decade, new trends for enzyme attachment to solid carriers have emerged in an attempt to rationalize the classical methods for enzyme immobilization. In silico analysis is becoming a powerful tool to predict the orientation of the enzyme covalently-attached to the carrier or the protein regions involved in the adsorption to the support. Significantly, an array of algorithms has been established for the Rational Design of Immobilized Derivatives (RDID), which comprises both the protein size and the textural properties of the support. Ordered mesoporous materials open a challenging pathway to tailor immobilized enzymes with high volumetric activity and minimum lixiviation. In addition, fluorescence confocal microscopy is being successfully employed to understand the diffusional restrictions and the distribution of biomolecules within the support.
Unnatural selection: A fungal laccase was tailored by directed evolution to be active at neutral/alkaline pH. After five generations, the final mutant showed a broader pH profile while retaining 50 to 80 % of its activity at neutral pH.
Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from Saccharomyces cerevisiae showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered Clostridium's 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound.
A purified lipase fraction from Rhizomucor miehei (RML) was immobilized and used to selectively hydrolyze hexa-O-acetyl-lactal 1 to penta-O-acetyl-3-hydroxylactal 2. The reaction was performed in low water content tert-butanol (7% USP water), thus allowing the control of by-product formation. This solvent was superior to the established buffer/acetonitrile system in terms of reagent/product solubility, biocatalyst stability and environmental impact. The reaction was scaled up to 28 g L 21 (50 mM) affording the product 2 in very high yield (85%) and purity (98%).
The growing demand for concentrated extracts of PUFA prompted us to undertake the preparation of natural glycerides containing linoleic acid and a-linolenic acid from hempseed oil. Hempseed is an exceptionally rich source of essential FA but is still underutilized. Hempseed oil was thus submitted to a lipase-catalyzed hydrolysis in a homogeneous medium based on oil and t-BuOH/water. Seven commercial lipases were screened in this reaction medium, which resulted to be superior to the routinely used biphasic systems in terms of substrate/product solubility and biocatalyst stability. Lipase from Pseudomonas cepacia resulted to be sufficiently selective versus saturated FA and it was thus used to hydrolyze hempseed oil in order to obtain acylglycerols enriched with PUFA. The enzymatic reaction was scaled up and the acylglycerol components were purified and analyzed by GC-FID. In the fraction of monoacylglycerols (yield 22%) the total amount of PUFA reached 85% (against 77% of the crude oil) whereas the content of saturated FA was negligible. The omega-6/omega-3 ratio of the total glyceride component (purified acylglycerols, yield 29%) was maintained in the optimal range (4.6:1) for human nutrition. Therefore, PUFA concentrates from hempseed oil here obtained in the form of natural glycerides could be considered as valuable dietary supplements.Practical applications: The enzymatic method here described allows to directly achieve pure monoglycerides of PUFA with a potential nutraceutical use. This procedure has the advantage to occur under very mild conditions in a homogeneous medium composed of a water miscible green solvent, to be easily scalable and to rely on a standard chromatography purification step.
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