Glycan Phosphorylases in Multi-Enzyme Synthetic Processes

Abstract

Glycoside phosphorylases catalyse the reversible synthesis of glycosidic bonds by glycosylation with concomitant release of inorganic phosphate. The equilibrium position of such reactions can render them of limited synthetic utility, unless coupled with a secondary enzymatic step where the reaction lies heavily in favour of product. This article surveys recent works on the combined use of glycan phosphorylases with other enzymes to achieve synthetically useful processes.

Keywords: Phosphorylase; biofuel; cellodextrin; disaccharide; high-value products; α-glucan.

Figure 1

Enzymes involved in glycosidic bond formation and cleavage. GT = glycosyltransferase; GP = glycoside phosphorylase; GH = glycoside hydrolase; GS = glycoside synthase; NDP = nucleotide diphosphate; OpNP = O-para-nitrophenyl.

Figure 2

Reaction mechanism of glycoside phosphorylases. R, retaining; I, inverting.

Figure 3

Structure illustration for CBP acceptors [25]. R₁ and R₂, which are linked to C-2 and C-6 respectively, are substituted by different functional groups for each of the aforementioned acceptors.

Figure 4

A) In situ generation of α-D-Glc-1-phosphate by SP coupling with galactosylation of N-acetylglucosamine [27]. β-(1→4)-GalT = β-(1→4)-galactosyltransferase; UDP = uridine diphosphate; UTP = uridine triphosphate; Pi = inorganic phosphate; SP = sucrose phosphorylase. B) Enzymatic production of nigerose from 4 different starting materials (cellobiose, starch, sucrose and maltose) [28]. Cellobiose, starch and sucrose were used for the production of α-D-Glc-1-phosphate, which was converted to β-anomer, and subsequently used by nigerose phosphorylase for nigerose production. Maltose was also phosphorolysed by maltose phosphorylase to generate β-D-Glc-1-phophate which can be used directly by nigerose phosphorylase. Pi = inorganic phosphate; CBP = cellobiose phosphorylase; SP = sucrose phosphorylase; MP = maltose phosphorylase; NP = nigerose phosphorylase; PGM = phosphoglucomutase.

Figure 5

Coupling SP with AGP for the enzymatic production of a variety of sugar-1-phosphate with axial selectivity at the anomeric position [29]. Sucrose was used by SP to generate α-D-Glc-1-phosphate, which was subsequently or simultaneously used in combination with 4 different acceptors, namely D-Man, D-GlcNAc, D-Gal or L-Fuc, by AGP for the production of the corresponding sugar-1-phosphates. SP = sucrose phosphorylase and AGP = α-glucose-1-phosphatase.

Figure 6

A) Two biosynthetic routes for GNB and LNB production proposed by Nishimoto and Kitaoka [35, 40]. For lacto-N-biose (LNB) production, LNBP and D-GlcNAc were used as the final catalyst and substrate respectively. LNBP and D-GlcNAc are substituted by GLNBP and D-GalNAc, respectively, for the production of GNB. B) Enzymatic production of GNB and sialylated-GNB proposed by Li and co-workers [41]. (1) BiGalK was used to convert D-Gal to α-D-Gal-1-P, which was utilised as a sugar donor by BiGLNBP with D-GalNAc as an acceptor to produce GNB. (2) Sialyl-GNB was formed by transferring Neu5Ac from CMP-Neu5Ac onto D-Gal of GNB via α-(2→3) linkage by the activity of PmST1. The sialyl-GNB was further sialylated at C-6 of D-GalNAc by Pd2,6ST via α-(2→6) linkage to form disialyl-GNB. BiGLNBP = GNB/LNB phosphorylase from Bifidobacterium infantis; BiGalK = galactokinase from Bifidobacterium infantis; NmCSS = CMP-Sia synthetase from Neisseria meningitides; PmST1 = α-(2→3)-sialyltransferase 1 from Pasteurella multocida; Pd2,6ST = α-(2→6)-sialyltransferase from Photobacerium damselae. C) Combination of immobilised LP from Euglena gracilis and SP can be used for the production of laminaribiose using sucrose as a starting material [42]. SP = sucrose phosphorylase; Pi = inorganic phosphate; LP = laminaribiose phosphorylase.

Figure 7

Metabolic engineering of E. coli for in vivo glycosylation platform of a flavonol molecule [49]. Underlined genes were introduced into E. coli for overexpression of the enzymes that are essential for production of desirable sugar metabolites, whereas erased genes were deleted from E. coli genome to prevent the breakdown of α-D-Glc-1-phosphate and UPD-Glc.

Figure 9

Reaction scheme depicting the conversion of starch to cellobiose using a combination of α-glucan phosphorylase and cellobiose phosphorylase [54]. Pi = inorganic phosphate.

Figure 8

Reaction scheme depicting the combination of cellobiose phosphorylase (CBP) and α-glucan phosphorylase (GP) for the synthesis of synthetic amylose [53]. Pi = inorganic phosphate.

Figure 10

Multi-enzyme one-pot reaction schemes for generation of α-D-Glc-1-phosphate. A) Enzymatic synthesis of α-D-Glc-1-phosphate from corn starch [55]. α-D-Glc-1-phosphate synthesis corn starch-mediated by thermostable α-glucan phosphorylase (αGP) from Thermotoga maritima. The hyper-thermostable isoamylase (IA) from Sulfolobus tokodaii was added for enhancing the α-D-Glc-1-phosphate yield. A third enzyme, 4-glucanotransferase (4GT) from Thermococcus litoralis was added to the reaction mixture for increasing the α-D-Glc-1-phosphate titre through maltose and maltotriose utilisation. B) One-pot scheme for enzymatic synthesis of α-D-Glc-1-phosphate from sucrose [56]. SP = sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum (EC 2.4.1.7); PGP = potato α-glucan phosphorylase (EC 2.4.1.1); GI = glucose isomerase (EC 5.3.1.5); GO = glucose oxidase (EC 1.1.3.4); CA = catalase (EC 1.11.1.6).

Figure 11

A) Combined use of potato α-glucan phosphorylase (αGP) and glycogen branching enzyme (GBE) from Deinococcus geothermalis for the generation of hyperbranched polysaccharides on silicon wafer [59]. (a) Oxidized Si wafer; (b) aminosilanized wafer; (c) maltoheptaose functionalised surface; (d) enzyme-catalysed growth of linear chain by αGP; (e) catalytic action of GBE; (f) resulting hyperbranched polysaccharide after the combined biocatalysis. B) Metabolic pathways associated with α-glucan and glycogen metabolism in Mycobacterium tuberculosis [61, 63]. The genes involved in the GlgE pathway are underlined. The pathway is negatively regulated by the serine/threonine kinase, PknB. The pathway has been exploited for generation of branched glycans using a combination of the GlgE and GlgB enzymes.

Figure 12

A) Production of H₂ from enzymatic degradation of cellulosic biomass [78]. Reaction products are in bold boxes. Pi = inorganic phosphate; CDP = cellodextrin phosphorylase; CBP = cellobiose phosphorylase; PGM = phosphoglucomutase; G6PDH = Glc-6-phosphate dehydrogenase; 6PGDH = 6-phosphogluconate dehydrogenase; NADP⁺ = nicotinamide adenine dinucleotide phosphate (oxidised form); NADPH = nicotinamide adenine dinucleotide phosphate (reduced form). B) Enzymatic degradation of cellulosic waste into valuable products [79]. Cellulose enzymatic hydrolysis leads to the production of ethanol by yeast fermentation, amylose by potato a-glucan phosphorylase (PGP), and single cell protein (SCP). All products are in bold boxes. EG = endo-glucanase; Pi = inorganic phosphate; CBP = cellobiose phosphorylase. C) Co-immobilisation of CBM3, PGP and CBP onto Avicel-nano magnetic particles (A-NMPs). CBM3 = carbohydrate binding module 3.