d'Errico, Clotilde

Et vigtigt redskab for bioteknologiske virksomheder, i enzymudvikling til biomasse delignificering er adgang til veldefinerede model substrater. En dybere forståelse af enzymers substratspecificitet kan anvendes til at evaluere og optimere enzymblandinger til naturlige, komplekse substrater. Derfor udkonkurrerer kemisk syntetiserede substrater ofte tilsvarende isoleret fra naturlige kilder i form af reproducerbarhed, homogenitet og renhed.Den første del af dette arbejde var syntesen af to glucuronoxylanfragmenter udformet som modelsubstrater for xylanaser. Syntesen involverede anvendelsen af thioxylosider som byggeklods i en iterativ lineær glycosylerings strategi. To sidekæder af glucuronat byggeklodser blev syntetiseret via en divergerende strategi fra samme thioethylglucose derivat.I anden del af projektet blev tre alkylaromatiske og aromatiske estere forberedt som efterligninger af de lignin-kulhydrat komplekser, der findes i lignocellulose biomasse, som modelsubstrater til glucuronoyl esteraser (GE). Disse estere er blevet anvendt til at karakterisere en hidtil ukendt GE fra Cerrena unicolor (CuGE), produceret af Novozymes, for at opnå indsigt i substratspecificiteten af enzymet. HPLC-analyse af enzymatiske reaktioner førte til bestemmelsen af kinetiskeparametre, der gav oplysninger om både limning affinitet og katalytisk effektivitet.An essential tool for biotechnology companies in enzyme development for biomass delignification is the access to well-defined model substrates. A deeper understanding of the enzymes substrate specificity can be used to address and optimize enzyme mixtures towards natural, complex substrates. Hence, the chemically synthesized substrates often outcompete those isolated from natural sources in terms of reproducibility, homogeneity and purity.The first part of this work was the synthesis of two glucuronoxylan fragments designed as model substrates for xylanases. The synthesis involved the use of thioxyloside building blocks in an iterative, linear glycosylation strategy. Two sidechain glucuronate building blocks were synthesized via a divergentstrategy from the same thioethylglucose derivative.In the second part of the project three alkylaromatic and aromatic esters have been prepared as mimics of lignin-carbohydrate complexes found in lignocellulosic biomass, as model substrates for glucuronoyl esterases (GEs). These esters have been used to characterize a novel GE from Cerrena unicolor (CuGE), produced by Novozymes, to obtain insights into the substrate specificity of the enzymes. HPLC analysis of the enzymatic reactions led to the determination of kinetic parameters that gave information about both bonding affinity and catalytic efficiency

Online Research Database In Technology

Glucuronic Acid Derivatives in Enzymatic Biomass Degradation: Synthesis and Evaluation of Enzymatic Activity

http://orbit.dtu.dk/ws/files/127495495/Glucuronic_Acid_Derivatives.pdf

Glucuronic Acid Derivatives in Enzymatic Biomass Degradation: Synthesis and Evaluation of Enzymatic Activity

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