RESUMO
High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48â mM (27.2â g/L) in a small-scale batch process (200â µL) within 24â h using 0.02 genzyme /gproduct . Through in-situ ATP regeneration, the amount of ATP (0.6â mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1â L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4â g/L UDP-galactose were produced within 23â h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein /gproduct . The costs for substrates per gram of UDP-galactose synthesized were around 0.26â /g.
Assuntos
Enzimas/metabolismo , Uridina Difosfato Galactose/biossíntese , Trifosfato de Adenosina/metabolismo , Reatores Biológicos , Sistema Livre de Células , Concentração de Íons de Hidrogênio , Oligossacarídeos/biossíntese , Estudo de Prova de Conceito , Uridina Difosfato Galactose/isolamento & purificaçãoRESUMO
Active pharmaceutical ingredients are either extracted from biological sources-where they are synthesized in complex, dynamic environments-or prepared in stepwise chemical syntheses by reacting pure reagents and catalysts under controlled conditions. A combination of these two approaches, where plant extracts containing reagents and catalysts are utilized in intensified chemical syntheses, creates expedient and sustainable processes. We illustrate this principle by reacting crude plant extract, oxygen, acid, and light to produce artemisinin, a key active pharmaceutical ingredient of the most powerful antimalarial drugs. The traditionally discarded extract of Artemisia annua plants contains dihydroartemisinic acid-the final biosynthetic precursor-as well as chlorophyll, which acts as a photosensitizer. Efficient irradiation with visible light in a continuous-flow setup produces artemisinin in high yield, and the artificial biosynthetic process outperforms syntheses with pure reagents.