Dynamic Temporal Variations in Bovine Lactoferrin Glycan Structures.

It has been reported previously that glycosylation of bovine lactoferrin changes over time. A detailed structural overview of these changes over the whole course of lactation, including predry period milk, is lacking. In this study, a high-throughput analysis method was applied to the glycoprofile of lactoferrin isolated from colostrum, mature, and predry period mature milk, which was analyzed over two subsequent lactation cycles for 8 cows from diverse genetic backgrounds. In addition, comparisons are made with commercial bovine lactoferrin samples. During the first 72 h, dynamic changes in lactoferrin glycosylation occurred. Shifts in the oligomannose distribution and the number of sialylated and fucosylated glycans were observed. In some cows, we observed (α2,3)-linked sialic acid in the earliest colostrum samples. The glycoprofiles appeared stable from 1 month after delivery, as well as between cows. In addition, the glycosylation profiles of commercial lactoferrins isolated from pooled mature milk were stable over the year. Lactoferrin glycosylation in the predry period resembles colostrum lactoferrin. The variations in lactoferrin glycosylation profiles, lactoferrin concentrations, and other milk parameters provide detailed information that potentially assists in unraveling the functions and biosynthesis regulation of lactoferrin glycosylation.

Prebiotic galactooligosaccharides activate mucin and pectic galactan utilization pathways in the human gut symbiont Bacteroides thetaiotaomicron.

Galactooligosaccharides (GOS) are prebiotic carbohydrates that impart changes in the gut bacterial composition of formula-fed infants to more closely resemble that of breast-fed infants. Consuming human milk oligosaccharides (HMOs) provides specific bacterial strains with an advantage for colonizing the infant intestine. These same effects are seen in infants after GOS consumption, however GOS are very complex mixtures and the underlying molecular mechanisms of how GOS mimic HMOs are relatively unknown. Here we studied the effects of GOS utilization on a prominent gut symbiont, Bacteroides thetaiotaomicron, which has been previously shown to consume HMOs via mucin O-glycan degradation pathways. We show that several pathways for targeting O-mucin glycans are activated in B. thetaiotaomicron by GOS, as well as the galactan utilization sytem. Characterization of the endo-galactanase from this system identified activity on various longer GOS substrates while a subset of GOS compounds were identified as potential activators of mucin glycan metabolism in B. thetaiotaomicron. Our results show that GOS functions as an inducer of mucin-glycan pathways while providing a nutrient source in the form of ß-(1 → 4)-galactan. These metabolic features of GOS mixtures may serve to explain the beneficial effects that are seen for GOS supplemented infant formula.

Carbohydrate-binding module 74 is a novel starch-binding domain associated with large and multidomain α-amylase enzymes.

Microbacterium aurum B8.A is a bacterium that originates from a potato starch-processing plant and employs a GH13 α-amylase (MaAmyA) enzyme that forms pores in potato starch granules. MaAmyA is a large and multi-modular protein that contains a novel domain at its C terminus (Domain 2). Deletion of Domain 2 from MaAmyA did not affect its ability to degrade starch granules but resulted in a strong reduction in granular pore size. Here, we separately expressed and purified this Domain 2 in Escherichia coli and determined its likely function in starch pore formation. Domain 2 independently binds amylose, amylopectin, and granular starch but does not have any detectable catalytic (hydrolytic or oxidizing) activity on α-glucan substrates. Therefore, we propose that this novel starch-binding domain is a new carbohydrate-binding module (CBM), the first representative of family CBM74 that assists MaAmyA in efficient pore formation in starch granules. Protein sequence-based BLAST searches revealed that CBM74 occurs widespread, but in bacteria only, and is often associated with large and multi-domain α-amylases containing family CBM25 or CBM26 domains. CBM74 may specifically function in binding to granular starches to enhance the capability of α-amylase enzymes to degrade resistant starches (RSs). Interestingly, the majority of family CBM74 representatives are found in α-amylases originating from human gut-associated Bifidobacteria, where they may assist in resistant starch degradation. The CBM74 domain thus may have a strong impact on the efficiency of RS digestion in the mammalian gastrointestinal tract.

Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains.

The bacterium Microbacterium aurum strain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomain M. aurum B8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded that M. aurum B8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation.

Enzymatic degradation of granular potato starch by Microbacterium aurum strain B8.A.

Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules.