β-N-Acetylhexosaminidases (CAZy GH20, EC 3.2.1.52) are exo-glycosidases specific for cleaving N-acetylglucosamine and N-acetylgalactosamine moieties of various substrates. The β-N-acetylhexosaminidase from the filamentous fungus Talaromyces flavus (TfHex), a model enzyme in this study, has a broad substrate flexibility and outstanding synthetic ability. We have designed and characterized seven glycosynthase-type variants of TfHex mutated at the catalytic aspartate residue that stabilizes the oxazoline reaction intermediate. Most of the obtained enzyme variants lost the majority of their original hydrolytic activity towards the standard substrate p-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside (pNP-β-GlcNAc); moreover, the mutants were not active with the proposed glycosynthase donor 2-acetamido-2-deoxy-d-glucopyranosyl-α-fluoride (GlcNAc-α-F) either as would be expected in a glycosynthase. Importantly, the mutant enzymes instead retained a strong transglycosylation activity towards the standard substrate pNP-β-GlcNAc. In summary, five out of seven prepared TfHex variants bearing mutation at the catalytic Asp370 residue acted as efficient transglycosidases, which makes them excellent tools for the synthesis of chitooligosaccharides, with the advantage of processing an inexpensive, stable and commercially available pNP-β-GlcNAc.
- MeSH
- aktivace enzymů MeSH
- beta-N-acetylhexosaminidasy genetika metabolismus MeSH
- houby enzymologie genetika MeSH
- hydrolýza MeSH
- katalýza MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- mutace * MeSH
- proteinové inženýrství MeSH
- substrátová specifita MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This "solid-state" enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of "bio-quality." Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the "Solid-State-Catalysis" concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.
- MeSH
- Aspergillus niger enzymologie genetika MeSH
- biokatalýza * MeSH
- disacharidy chemie metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- Pichia genetika metabolismus MeSH
- průmyslová mikrobiologie metody MeSH
- quercetin chemie metabolismus MeSH
- rutin chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
