Casper Wilkens, Darrell Cockburn, Susan Andersen, Bent Ole Petersen, Christian Ruzanski, Robert A. Field, Ole Hindsgaul, Hiroyuki Nakai, Barry McCleary, Alison M. Smith, Maher Abou Hachem, Birte Svensson
Certain interactions between carbohydrate active enzymes and polysaccharides involve surface binding sites (SBS) situated on catalytic domains outside of the active site. We recently undertook to develop a toolbox for SBS identification and characterization. In affinity gel electrophoresis (AGE) SBS containing proteins are migrating slower in native polyacrylamide electrophoresis gels cast with polysaccharide versus without polysaccharide. Amylolytic enzymes from GH13 and GH77 and xylanases from GH10 and GH11 are the best studied GH families with respect to SBS, presenting about half of the reported SBSs. In GH13 SBSs have been seen in 17 subfamilies including SBSs with highly diverse functions in the same enzyme. Circumstantial evidence is provided for an SBS in the GH77 MalQ from Escherichia coli, the bacterial orthologue of Arabidopsis DPE2 involved in starch metabolism. Furthermore, Aspergillus nidulans α-L-arabinofuranosidase AnAbf62A-m2,3 of GH62 that has very high activity on wheat arabinoxylan (WAX) shows an unusually strongly retarded migration by WAX during AGE analysis. Using a recent GH62 crystal structure as template, Trp23 and Tyr44 in an AnAbf62A-m2,3 model are proposed to form an SBS situated about 30 Å from the catalytic site. Compared to wild-type, W23A/Y44A AnAbf62A-m2,3 retained 45% activity on WAX and was less retarded in AGE by WAX as well as by barley β-glucan and birchwood xylan, which are neither hydrolysed nor inhibiting activity towards WAX. The presence of a functional SBS agrees with W23A/Y44A AnAbf62A-m2,3 retaining only 3-25% activity for arabinoxylo-oligosaccharides (AXOS) of DP 3-5 possibly reflecting allosteric activation of wild-type through SBS occupation by AXOS.
Fructan was prepared from an extract of onion bulbs by preparative paper and gel permeation chromatographies. Fructan was a mixture of saccharides with degree of polymerization (DP) and MW range of approximately 7‒13 and 1,152‒2,124, respectively, as determined by matrix-assisted laser desorption ionization/time of flight mass spectrometry. It yielded fructose and glucose upon hydrolysis with acid or yeast β-fructofuranosidase. The ratio of D-fructose to D-glucose in the enzyme hydrolysate was 8.7:1, determined by high performance anion-exchange chromatography analysis. Structural determinations were made, based on 13C-nuclear magnetic resonance spectroscopy. Numerous signals corresponding to carbon-1 (C-1), C-2, C-3, C-4, C-5, and C-6 of the D-fructose residues of fructan were observed at δ 61.04‒61.81, 103.70‒104.41, 77.46‒78.10, 74.50‒75.15, 81.77, and 62.85‒62.96, respectively. These chemical shifts were similar to those of inulin. Moreover, weaker signals were detected at δ 92.90, 71.75, 73.10, 69.76 and 72.22 due to C-1, C-2, C-3, C-4, and C-5, respectively of the D-glucose residue. The chemical shifts are almost identical to those of the D-glucose carbons of neokestose, 6G(1-β-D-fructofuranosyl)2 sucrose, and 1F, 6G-di-β-D-fructofuranosyl sucrose. These findings were supported by analysis of the methanolysate from permethylated fructan using gas-liquid chromatography. The fructan from onion bulbs was composed of saccharides possessing approximately 6‒12 D-fructose residues, linked by β-2,1 bonds, and a non-terminal D-glucose residue bound with D-fructose residues at the C-1 and C-6 positions.
We reported previously that oral administration of high molecular weight proteoglycan (molecular weight ≥ 5,000,000) from salmon nasal cartilage showed excellent effects on the skin and joints. We have developed a new method for extraction of high molecular weight proteoglycan at high yield and low cost for utilization in food. Examination of the extraction method involving heating of shredded salmon nasal cartilage pieces dispersed in water indicated that high molecular weight proteoglycan (molecular weight ≥ 5,000,000) was extracted efficiently by controlling the heating temperature and time based on the molten state of cartilage pieces. Three proteoglycan fractions of different molecular weights from salmon nasal cartilage were purified from the hot water extract under optimal condition. Quantification of hyaluronic acid and collagen showed that the proteoglycan fraction with molecular weight ≥ 5,000,000 consisted of hyaluronic acid-collagen-proteoglycan complex. The hot water extraction method established in this study can be used to extract the hyaluronic acid-collagen-proteoglycan complex from salmon nasal cartilage, and we have designated the salmon nasal cartilage extract obtained by this process as “Hyaluco PG”. The only solvent necessary for the hot water extraction method is water and the process is simple, both of which can drastically reduce costs compared to processes using acid, alkali, enzymes, or organic solvents such as ethanol. The hot water extraction method will allow the development of novel food materials with beneficial effects on health and beauty.
Phosphoryl oligosaccharides of calcium (POs-Ca) is a calcium salt of phosphoryl maltooligosaccharides made from potato starch. POs-Ca has high solubility in water and it can supply both calcium ion and acidic oligosaccharides in an aqueous medium. In this study, we evaluated effects of POs-Ca on cultured normal human epidermal keratinocytes (NHEK) and human skin. Several in vitro studies using cultured NHEK demonstrated that POs-Ca promoted NHEK differentiation, tight junction formation, intercellular lipid production, and gene expression involved in stratum corneum condition, skin barrier function and hydration. Skin penetration study using a three-dimensional epidermal model demonstrated that POs-Ca was able to provide both calcium ion and phosphoryl oligosaccharides to the epidermis. Furthermore, an in vivo study demonstrated that POs-Ca improved human skin conditions including hydration, barrier function, stratum corneum condition, and skin texture. These results suggest that POs-Ca can be a superior active agent for healthy epidermis.
Salmon cartilage proteoglycan fractions have attracted attention as new ingredients of functional food and cosmetics. We recently developed methods for water-based delipidation and powderization for preservation of salmon head cartilage. In this study, global molecular images of proteoglycan in the water-based delipidated powder were analyzed by biochemical techniques and atomic force microscopy. Proteoglycans in this powder maintained their whole native structures including core proteins and glycosaminoglycans. Hyaluronan contained in this powder showed a distribution with high molecular weight like native hyaluronan in cartilage. Analytical data presented here provides assurance of the quality of the proteoglycan powder obtained using these methods.
Ten difructose anhydrides (DFAs) were the predominant products formed from the thermal treatment of equal amounts of D-glucose and D-fructose under melting conditions at 150°C for 60 min. The DFAs were isolated from the reaction mixture by carbon-Celite column chromatography and preparative high-performance liquid chromatography. The structures of the saccharides were confirmed by NMR measurements. We present the complete assignments of the 1H- and 13C-NMR signals of two of these DFAs for the first time.