Anal Biochem
January 2025
In this study, we propose a continuous assay that provides a high-throughput, efficient method for screening the regioselectivity of lipases at the sn-1,3 and sn-2 positions on triacylglycerols (TAGs). This assay measures the specific hydrolysis rates at the primary and secondary positions of TAGs derivates containing oleic (O) and punicic (P) acids. The method is based on the absorbance ratio of released punicic acid from the hydrolysis of sn-POP (sn-1,3 regiospecific lipases) and sn-OPO (sn-2 regiospecific lipases).
View Article and Find Full Text PDFPolyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), hold notable significance due to their pharmaceutical relevance. Obtaining PUFAs from diverse sources like vegetables, fish oils, and algae poses challenges due to the mixed fatty acid (FA) composition. Therefore, focusing on particular FAs necessitates purification and resolution processes.
View Article and Find Full Text PDFBiocatalytic processes play a crucial role in the valorization of lignin; therefore, methods enabling the monitoring of enzymes such as β-etherases, capable of breaking β-O-4 aryl-ether bonds, are of significant biotechnological interest. A novel method for quantifying β-etherase activity was developed based on the β-ester bond formation between a chromophore and acetovainillone. The chromogenic substrate β-(ρ-nitrophenoxy)-α-acetovanillone (PNPAV), was chemically synthesized.
View Article and Find Full Text PDFLignin, a complex heteropolymer present in plant cell walls, is now recognized as a valuable renewable resource with potential applications in various industries. The lignin biorefinery concept, which aims to convert lignin into value-added products, has gained significant attention in recent years. β-etherases, enzymes that selectively cleave β--4 aryl ether bonds in lignin, have shown promise in lignin depolymerization.
View Article and Find Full Text PDFA continuous spectrophotometric assay for the screening of PHB depolymerase activity in microtiter plates was developed. We evaluated crystalline PHB in the suspension and coated it with the addition of a pH indicator to detect the breakage of the ester bond by proton titration. The reaction rate and the concentration of the recombinant PhaZ1 from Paucimonas lemoignei PHB depolymerase presented a linear correlation.
View Article and Find Full Text PDFGastrointestinal lipase inhibitors are molecules of pharmaceutical interest due to their use as anti-obesity drugs. In this study, forty strains isolated from soil and sediments were identified with the ability to produce inhibition of gastrointestinal lipase activity. The biomass extract of these strains showed at least 50% inhibition in the hydrolysis of tributyrin by recombinant human pancreatic lipase (rHPL) or rabbit gastric lipase (RGL) by in vitro assays.
View Article and Find Full Text PDFObjectives: Ustilago maydis lipase A (UMLA) expressed in Pichia pastoris was compared with Candida antarctica lipase A (CALA) to study its biochemical properties such as thermostability and selectivity.
Results: UMLA had similar behavior to its homologue CALA regarding the effect of pH and temperature on enzymatic activity, substrate preference and selectivity. Both lipases were active on insoluble triglycerides as well as natural oils and hydrolyzed preferably esters with short and medium acyl and alkyl chains.
Novel microbial phospholipases A (PLAs) can be found in actinomycetes which have been poorly explored as producers of this activity. To investigate microbial PLA production, efficient methods are necessary such as high-throughput screening (HTS) assays for direct search of PLAs in microbial cultures and cultivation conditions to promote this activity. About 200 strains isolated with selected media for actinomycetes and mostly belonging to (73%) and (10%) genus were first screened on agar-plates containing the fluorophore rhodamine 6G and egg yolk phosphatidylcholine (PC) to detect strains producing phospholipase activity.
View Article and Find Full Text PDFA continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method.
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