Discovery Of Novel Glycoside Phosphorylases And Their Use For The Synthesis Of Distinctive Indoxy-based Structures

Glycoside phosphorylases(GPs)are a major group of enzymes which catalyze the reversible formation and cleavage of glycosidic linkages,thus combining a dual functionality of both glycoside hydrolases(GHs)and glycosyltransferases(GTs).This unique advantage of reversibility,alongside with the strict regioselectivity,and the use of simple sugar phosphate donors,makes phosphorylases valuable catalysts in the synthesis of various carbohydrate structures,most notably oligosaccharides,by utilizing an appropriate donor sugar 1-phosphate together with suitable carbohydrate acceptors.Using these types of enzymes,it was possible to synthesize oligosaccharides from naturally abundant sugars using sequential conversion of one cheap sugar substrate to another more expensive sugar oligomer product.Although these types of enzymes are still under-explored compared to the glycoside hydrolases and the glycosyltransferases,recently more discovered phosphorylases had been reported leading to more possibilities of synthesizing difficult glycosides.The current study covers the eloning,expression and purification of novel phosphorylases from diverse microbial sources,leading to the finding of two previously uncharacterized enzymes,a mannoside phosphorylase from GH family 130 and a maltose phosphorylase from GH family 65.Special attention was brought for the discovery of the first enzyme,as it possesses a remarkable function by generating and hydrolyzing ?-1,3-mannosidic linkages,with relaxed acceptor substrate specificity and the capability of oligomerization.The study also explores the synthetic diversity of both enzymes particularly with anomerically modified glycosides(X-sugars),which was achieved by utilizing the well-known reversibility function of phosphorylases.1.Screening for novel phosphorylases:cloning,expression,and purification of recombinant proteinsThis chapter presents the basic molecular cloning of recombinant proteins,which is the most common approach in the search for new active enzymes.We showed a screening of various putative genes,which have been selected based on protein homology search.A common cloning protocol was followed on an initial number 36 genes remotely similar to previously characterized phosphorylases,using a generic cloning vector for gene propagation,followed by ligation into pET30a expression vector and transformation into E.coli BL21 cells.A total of 16 genes were successfully cloned,recombinantly expressed and subsequently purified by Ni2+-nitrilotriacetate agarose affinity chromatography.The resulting purified protein was further investigated for activity tests in the following chapters.2.Discovery and biochemical characterization of a mannose phosphorylase catalyzing the synthesis of novel ?-1,3-mannosidesGlycoside hydrolase family GH130 is the most recent established family of carbohydrate active enzymes.Members of this family share the common substrate product mannose-1-phosphate.Herein we report for the first time,a new member of GH130 cloned from the marine flavobacterium Zobellia galactanivorans.This novel enzyme exhibits an exclusive ?-1,3-linkage specificity towards oligomannose structures,accompanied with a wide acceptor substrate promiscuity.The reversible phosphorolysis of this enzyme revealed the generation of various free di-and oligosaccharides as well as the synthesis of 10 indoxy-based sugars.Structural analysis using NMR spectroscopy of two purified indoxy-disaccharid.es(X-Man-man and X-GlcNAc-man)proved the nature of the ?-1,3-mannoside linkage.The exquisite functionality of this protein added a new catalytic tool that could be utilized for the synthesis of new unfamiliar mannosides.3.Enzymatic synthesis of indoxyglycosides catalyzed by a novel maltose phosphorylase from Emticicia oligotrophicaMaltose phosphorylases(EC 2.4.1.8)catalyze the reversible conversion of maltose to glucose and glucose-1-phosphate in the presence of inorganic phosphate.Herein,we describe for the first time the use of a maltose phosphorylase for the synthesis of various anomerically modified diglycosides.The maltose phosphorylase used was isolated from the bacterium Emticicia oligotrophica and showed a high selectivity towards the phosphorolysis of maltose,whereas no phosphorolysis was observed using other glucose-containing disaccharides such as cellobiose,melibiose,sucrose and trehalose.The addition of glucose to various 5-bromo-4-chloro-3-indolyl-glycosides(X-sugars)was used to evaluate the promiscuity of the maltose phosphorylase,and product formation was verified by LC-ESI-MS and MALDITOF-MS.The simple expression and purification protocol and the use of maltose as an inexpensive starting material make this maltose phosphorylase from E.oligotrophica a valuable novel biocatalyst for the synthesis of glucose-containing glycosides.