Synthesis and characterization of lactose and lactulose derived oligosaccharides by glucansucrase and trans-sialidase enzymes

Human milk oligosaccharides (hMOS) have an essential role in infants’ health by stimulating growth of health-beneficial gut bacteria for the human host. These hMOS also directly reduce pathogenic microbial infections by serving as antiadhesives against pathogenic microbes, and stimulating immune responses. Many babies have limited access to human milk, and alternative sources for hMOS are not available in nature. Infant formula with the commercial prebiotics lack pathogen exclusion and immune and barrier modulating effects exerted by hMOS. Development of efficient routes for synthesis of hMOS or structurally/functionally effective mimics is currently focus of attention. At present, whole cell biosynthetic routes and single/multiple enzyme biocatalytic systems for synthesis of hMOS (mimics) are studied. In this project, the glucansucrases Gtf180-ΔN and GtfA-ΔN from Lactobacillus reuteri were used to glucosylate lactose or GOS. The mixture of glucosylated lactose derivatives obtained showed selective effects on growth of various gut bacteria, including lactobacilli, bifidobacteria and commensal bacteria. Mutational analysis of Gtf180-ΔN revealed that three amino acid residues (N1029, W1065 and Q1140) play important roles in determining linkage specificity in lactose trans-glycosylation. Finally, trans-sialidase from Trypanosoma cruzi was used to transfer sialic acid to mixtures of glucosylated-lactose, galactosylated-lactulose and galacto-oligosaccharide (Vivinal GOS) molecules. The newly synthesized galactose-containing oligosaccharides and sialylated oligosaccharides hold strong potential for further applications as hMOS mimics. Glucansucrase and trans-sialidase enzymes are promising tools as biocatalysts for efficient synthesis of new functional oligosaccharides.


Chapter 1 Introduction Human gastrointestinal tract
The human digestive system is a complex series of organs and glands that processes food ( Figure 1). After entering the mouth with physical breakdown by chewing, food continues its way through stomach and intestine where it is partly digested by human digestive enzymes, i.e. salivary enzymes, pancreatic enzymes and enzymes excreted in the small intestine. The undigested food ends up in the large intestine or colon where it is fermented by various microorganisms. The gut microbiome is the largest microbial community of the human body with approximately 1,000 bacterial species; most of the gut microbiome resides in the large intestine. 1 A healthy gut microbiome provides a barrier against colonization by pathogens through competition, assists the GI tract by degradation of complex nutrients providing energy and essential vitamins, contributes to lipid metabolism and lipid absorption by lowered pH as a result of short-chain fatty acids secretion, and stimulates the immune system. 2,3 Figure 1: Scheme of the human digestive system (source: www.Storyblocks.com).

Human gut microbiota
The composition of the intestinal microbiota of infants is largely regulated by the diet. 4 At birth the digestive tract of human is sterile and soon after becomes colonized by microbes originating from the mother's vagina and feces, as well as from the environment. The infant may be fed by breast milk or an alternative source like formula milk, resulting in different microbiota compositions. With breast-fed infants, gut microbiota composition is more dominated by bifidobacteria; in contrast, formula-feeding without added health beneficial oligosaccharides leads to the development of a gut microbiota with a more adult type of distribution. 5 formula-fed infants. 8,9,10 Bifidobacteria and lactobacilli are considered the most important health-beneficial bacteria for the human host, whereas staphylococci and clostridia are potentially pathogenic. 11 Human breast milk thus is an important source of oligosaccharides for the neonate's developing microbiota. 12 The intestinal microbiota is known to be very important for the development of the gut physiology and the immune system. Attempts have been made to mimic the intestinal microbiota of breast-fed infants by formula-feeding.
The composition of the intestinal microbiota can be influenced either by administration of health-promoting bacteria, so-called probiotics, or the dietary ingredients, so called prebiotics. 13 They have shown beneficial effects in infants' health, providing protection against infections, 14,15,16 that can cause diarrhea, 17,18 and necrotizing enterocolitis, 19 as well as reducing atopic dermatitis. 20,21,22 Probiotic bacteria, which are able to survive the gastrointestinal tract exert their biological activity by interaction with the surface of the small intestine, and colonize the colon.
The most common probiotic bacteria that have been studied and used are bifidobacteria or lactobacilli. However, to maintain colonization it is essential to keep them alive. 23 Upon ingestion they are confronted with physical and chemical barriers such as gastric acid, bile acids. Reaching the colon, they still have to compete for nutrients and colonization sites with the host's resident species. As a result, a small proportion of ingested probiotic bacteria successfully colonizes the colon. 24,25,26 An alternative approach which partly overcomes the limitations of probiotics is the use of prebiotics. 27 Prebiotics are generally defined as "a substrate that is selectively utilized by host microorganisms conferring a health benefit". 28 Those substrates that are non-digestible during the passage through the small intestine without being absorbed or utilized, reach the colon, and stimulate selectively health promoting colonic bacteria. 29

Human milk oligosaccharides
The best known natural prebiotic compounds are oligosaccharides from human breast milk. Human milk oligosaccharides (hMOS) have been well studied and documented for their prebiotic, and particularly bifidogenic effects. 30 There are currently several GOS products on the market such as Vivinal ® GOS, Oligomate 55 and Bimuno. 57,58,59 This group of oligosaccharides has been widely studied and shown to have stimulatory effects on the growth of probiotic bacteria to various extents. 60,61,22 Another group of oligosaccharides that has attracted much commercial interest as prebiotics are FOS. These oligosaccharides can be obtained Chapter 1 13 from natural sources like chicory root derived inulin or synthesized enzymatically from sucrose by bacterial fructansucrase enzymes. 62,77 Inulin normally consists of a sucrose core with one or more (β2→1) linked fructosyl unit elongations, but there is another type of FOS lacking the terminal glucose part of the sucrose. The degree of polymerization of FOS varies between 2 and 60 units (Figure 3). 63 The stimulatory effect toward bifidobacteria (Bifidogenic effects) of FOS have been widely studied. 64,65 The most common disaccharide used as prebiotic is lactulose. This is a synthetic disaccharide in the form Gal(β1→4)-Fru ( Figure 3). Lactulose was shown to be resistant to digestion in the small intestine, and showed selective stimulation towards growth of lactobacilli and bifidobacteria. 66,67,68,69 These are prebiotics with well-known status supported by a significant number of human, double-blind and placebo controlled trials. 70,71 However, there has been a growing search for new carbohydrates which could be considered as emerging prebiotics such as lactosucrose; isomalto-oligosaccharides; resistant starch; xylo-oligosaccharides, arabinoxylo-oligosaccharides and pectic-oligosaccharides. 71,72,73,74,75,76 Where studied, most of these prebiotics however lack the pathogen exclusion and immuneand barrier modulating effects that hMOS possess.

Synthesis of Prebiotics and hMOS mimics
Prebiotics and hMOS mimics can be either chemically or enzymatically synthesized.
However, chemical synthesis is cumbersome because it requires many synthetic steps and a lot of effort to get rid of side products. 77 The high selectivity and regiospecificity of enzymatic routes has advantages over the chemical approach. 77,78 The microbial whole cell engineered biosynthetic routes, with outstanding features to scale-up for economic production, appeared to be the preferred choices to produce hMOS compounds like 2'-fucosyllactose (2'-FL). 79,80 However, prebiotic synthesis using whole cell biosynthetic approaches requires a rigorous removal of the production strain before their application in infant food, and a clear proof that no genetically modified organisms remain is challenging. Application of isolated and highly specific enzymes for synthesis of oligosaccharides may simply overcome this obstacle. In addition, it is easier to control various incubation conditions, such as reaction conditions (enzyme/substrate concentrations) and environmental conditions (pH, temperature, metal ion), when using enzymes for synthesis of hMOS mimics compared to the whole-cell biocatalysts. 81 From a practical viewpoint, glycosidase enzymes are the preferred choice, they are generally more available, and less expensive than glycosyl-transferases, and do not require expensive nucleotide-sugar donors. 82 The choice of suitable substrates and highly active glycosidases clearly plays a key role in allowing the synthesis of 'tailor-made' hMOS mimics of high interest for application in the food industry. Lactose is always at the reducing end of human milk oligosaccharides, this compound is considered as the initial substrate for hMOS synthesis. 83 Moreover, galactose is present in a high content in hMOS. Thus, lactose and lactose derivatives like GOS are potential candidates for transglycosylation to mimic hMOS.

Glucansucrase and trans-glycosylation
Glucansucrases belong to glycoside hydrolase family 70 (GH70) (http://www.CAZy.org) and are extracellular trans-glycosidases found in lactic acid bacteria. 84,85 GH70 glucansucrases belong to the α-amylase superfamily based on amino acid sequence similarity and structure analogy. 86 They are structurally and mechanistically related to GH13 and GH77 enzymes. 87 To date, three-dimensional structures of four microbial glucansucansucrases were obtained by crystallization of the recombinantly produced and truncated forms of these proteins, including those from Lactobacillus reuteri 180, 88 L. reuteri 121, 89 Streptococcus mutans, 90 and Leuconostoc mesenteroides NRRL B-1299. 91 The three-dimensional structures of truncated glucansucrases revealed that they linkage. 88 The anomeric configuration of the donor is conserved in the product. 92,93 Glucansucrase enzymes in family GH70 transfer glucose from sucrose to the nonreducing end of oligosaccharides in a processive manner, retaining the αregiospecificity. 94 Depending on the nature of the acceptor substrate, glucansucrase enzymes catalyze three types of reactions: hydrolysis of sucrose with water as acceptor, polymerization with growing α-glucan chains as acceptor, or transglycosylation with sucrose as donor substrate and other compounds as acceptor substrates (including oligosaccharides). 87 The glucosidic linkage type formed in the product is dependent on the acceptor substrate and the enzyme specificity. These enzymes synthesize not only α-glucan polymers but also efficiently catalyze transfer of glucose moieties from sucrose as donor substrate to numerous hydroxylgroup containing molecules. 100,101,102,103,104,105 In case of these small sugar acceptor substrates, low molecular mass oligosaccharides are synthesized with different types of linkage, size, branching, and physicochemical properties. 106 Maltose is considered to be the most effective acceptor substrate of glucansucrase enzymes, synthesizing various products (DP 3-6) such as panose or other isomaltooligosaccharides. 107,108,109 Other acceptor substrates that were studied include isomaltose, nigerose, methyl α-D-glucoside, 1,5-anhydro-D-glucitol, D-glucose, turanose, methyl β-D-glucoside, cellobiose, and L-sorbose. 110 Lactose, raffinose, melibiose, D-galactose, and D-xylose are also used as acceptor substrate by these Gtf enzymes but only give a single glucosylated product each. 110 More recently it was reported that dextransucrases from Leuconostoc mesenteroides and Weissella confusa also use lactose as their acceptor substrate synthesizing 2-α-D-glucopyranosyl-lactose. 111 The most common application of α-glucans is the use as sweetening, stabilizing, viscosifying, emulsifying or water-binding agents in food as well as non-food industries. 115,116,117,118 Moreover, α-glucans and oligosaccharides synthesized by glucansucrases have shown evidence of prebiotic properties, stimulating growth of beneficial intestinal bacteria such as Bifidobacterium and Lactobacillus. 119 Isomaltooligosaccharides (IMOs) are composed of glucose monomers linked by (α1→6) glucosidic linkages, and have been widely studied as potentially prebiotic. 119,73,120 Another group of gluco-oligosaccharides, which are synthesized by glucansucrases from Leuconostoc mesenteroides using sucrose as donor substrate and maltose as acceptor substrate, also has potential stimulatory effects on gut bacteria. 121,122 In another study, the addition of an α-glucan product to animal feed improved the weight gain of piglets and broilers. 123 A lactose-derived trisaccharide compound, i.e.
2-glucosyl-lactose, synthesized by L. mesenteroides dextransucrase using lactose as acceptor substrate showed selective stimulatory effects on growth of Bifidobacterium breve. 111 Prebiotic effects of gluco-olicosaccharides were shown to be inversely dependent on the size of the oligosaccharides synthesized by alternansucrase and dextransucrase, with DP3 possessing the highest prebiotic potential towards bifidobacteria i.e. B.
bifidum, B. longum, B. angulatum. 124,125 Therefore, α-glucans and oligosaccharides synthesized by glucansucrases with a large variety of structures hold great potential for food applications, more particularly for prebiotic applications.

Trans-sialidase
In human milk, lactose and hMOS backbones can be decorated with sialic acid to become acidic oligosaccharides. 38 There is increasing evidence for the functional effects of this group of oligosaccharides on human health. 126,127,128,129 Sialylated oligosaccharides are able to prevent intestinal attachment of pathogens by acting as receptor analogs competing with epithelial ligands for bacterial binding. 130,131,132,133 Binding of Cholera toxin was inhibited by 3′-sialyllactose. 134  The trans-sialidases (EC 3.2.1.18) are glycosidases that naturally catalyze the transfer of sialyl residues from one sialo-glycan to the terminal Gal residue of another asialo-glycan. 137 In micro-organisms, these enzymes are virulence factors that enable spreading and infection of host cells. 138 Trans-sialidase was first identified in and isolated from Trypanosoma species. Trans-sialidase from Trypanosoma cruzi preferentially catalyzes the reversible transfer of (α2→3)-linked sialic acids from donor glycans directly to terminal β-Gal-containing acceptor molecules, thereby giving rise to new (α2→3) glycosidic linkages ( Figure   4). 139,140 When the acceptor substrate is absent, the enzyme acts as a hydrolase transferring sialic acid to water. 137  In chapter 2, we investigated the ability of the Gtf180-ΔN and GtfA-ΔN enzymes to use lactose as acceptor substrate for trans-glucosylation, using sucrose as donor substrate. The results showed that both enzymes synthesized similar transfer products with a degree of polymerization (DP) of 3 to 4, therefore called GL34 mixture. New linkage types were observed when using lactose as acceptor than observed in the α-glucan products from sucrose of these enzymes, i.e.
Further reaction and process engineering is required to optimize conversion and product yields.
The newly synthesized GL34 mixture maybe of interest for the food industry, more particularly they may find application in infant foods, or in animal feed. We therefore studied its prebiotic potential (chapter 3) by analyzing the stimulatory effects of the GL34 mixture synthesized by Gtf180-ΔN on growth of selected gut bacteria, including lactobacilli, bifidobacteria and commensal bacteria. The mixture was also challenged with common carbohydrate degrading enzymes and showed resistance to most of the tested enzymes, including α-amylase from porcine pancreas. Bifidobacteria strains clearly grew better on the GL34 mixture than lactobacilli and commensal bacteria. Particularly B. adolescentis grew effectively on GL34.
When using lactose as acceptor substrate, the linkage specificity of these glucansucrases changed to also produce (α1→2)-linkages, which is totally new for these enzymes. Previous studies have shown that mutagenesis of residues in the glucansucrase active site pocket may change its linkage specificity . 153