N-acetylglucosamine Units Research Articles

Cloning, purification and biochemical characterization of two β-N-acetylhexosaminidases from the mucin-degrading gut bacterium Akkermansia muciniphila

Two genes encoding the β-N-acetylhexosaminidases Am2301 and Am2446 were cloned successfully from the mucin-degrading bacterium Akkermansia muciniphila. The recombinant enzymes with molecular masses of 61 kDa and 78 kDa were isolated and biochemically characterised. The optimum temperature of both enzymes was 37 °C, and the optimum pH was determined to be pH 5.0 for Am2301 and pH 6.5 for Am2446. The addition of sodium dodecyl sulphate (SDS) reduced the enzymes' activity significantly. Cu2+-ions decreased the activity of Am2301 by 70%, while the activity of Am2446 was significantly reduced by Fe3+-ions. PugNAc strongly inhibited both enzymes already in the sub-micromolar concentration range. The enzymes catalysed the hydrolysis of β1,4-linked N-acetylgalactosamine and β1,6-linked N-acetylglucosamine from glycan standards, as well as β1,2-linked N-acetylglucosamine units from the non-reducing end of N-glycans. The present study describes the first functional characterisation of β-N-acetylhexosaminidases from this human gut symbiont.

Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life.

Hyaluronan (HA) is an acidic high molecular weight cell surface polysaccharide ubiquitously expressed by vertebrates, some pathogenic bacteria and even viruses. HA modulates many essential physiological processes and is implicated in numerous pathological conditions ranging from autoimmune diseases to cancer. In various pathogens, HA functions as a non-immunogenic surface polymer that reduces host immune responses. It is a linear polymer of strictly alternating glucuronic acid and N-acetylglucosamine units synthesized by HA synthase (HAS), a membrane-embedded family-2 glycosyltransferase. The enzyme synthesizes HA and secretes the polymer through a channel formed by its own membrane-integrated domain. To reveal how HAS achieves these tasks, we determined the biologically functional units of bacterial and viral HAS in a lipid bilayer environment by co-immunoprecipitation, single molecule fluorescence photobleaching, and site-specific cross-linking analyses. Our results demonstrate that bacterial HAS functions as an obligate homo-dimer with two functional HAS copies required for catalytic activity. In contrast, the viral enzyme, closely related to vertebrate HAS, functions as a monomer. Using site-specific cross-linking, we identify the dimer interface of bacterial HAS and show that the enzyme uses a reaction mechanism distinct from viral HAS that necessitates a dimeric assembly.

In vitro bioconversion of chitin to pyruvate with thermophilic enzymes

Chitin is the second most abundant organic compound on the planet and thus has been regarded as an alternative resource to petroleum feedstocks. One of the key challenges in the biological conversion of biomass-derived polysaccharides, such as cellulose and chitin, is to close the gap between optimum temperatures for enzymatic saccharification and microbial fermentation and to implement them in a single bioreactor. To address this issue, in the present study, we aimed to perform an invitro, one-pot bioconversion of chitin to pyruvate, which is a precursor of a wide range of useful metabolites. Twelve thermophilic enzymes, including that for NAD+ regeneration, were heterologously produced in Escherichia coli and semi-purified by heat treatment of the crude extract of recombinant cells. When the experimentally decided concentrations of enzymes were incubated with 0.5mgmL-1 colloidal chitin (equivalent to 2.5mM N-acetylglucosamine unit) and an adequate set of cofactors at 70°C, 0.62mM pyruvate was produced in 5h. Despite the use of a cofactor-balanced pathway, determination of the pool sizes of cofactors showed a rapid decrease in ATP concentration, most probably due to the thermally stable ATP-degrading enzyme(s) derived from the host cell. Integration of an additional enzyme set of thermophilic adenylate kinase and polyphosphate kinase led to the deceleration of ATP degradation, and the final product titer was improved to 2.1mM.

An Overview of the Protective Effects of Chitosan and Acetylated Chitosan Oligosaccharides against Neuronal Disorders.

Chitin is the second most abundant biopolymer on Earth and is mainly comprised of a marine invertebrate, consisting of repeating β-1,4 linked N-acetylated glucosamine units, whereas its N-deacetylated product, chitosan, has broad medical applications. Interestingly, chitosan oligosaccharides have therapeutic effects on different types of neuronal disorders, including, but not limited to, Alzheimer’s disease, Parkinson’s disease, and nerve crush injury. A common link among neuronal disorders is observed at a sub-cellular level, such as atypical protein assemblies and induced neuronal death. Chronic activation of innate immune responses that lead to neuronal injury is also common in these diseases. Thus, the common mechanisms of neuronal disorders might explain the general therapeutic effects of chitosan oligosaccharides and their derivatives in these diseases. This review provides an update on the pathogenesis and therapy for neuronal disorders and will be mainly focused on the recent progress made towards the neuroprotective properties of chitosan and acetylated chitosan oligosaccharides. Their structural features and the underlying molecular mechanisms will also be discussed.

Synthesis of novel muramic acid derivatives and their interaction with lysozyme: Action of lysozyme revisited.

The interaction of lysozyme with the N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) unit of peptidoglycan (PGN) polymer of the bacterial cell wall is of immense importance to understand the mechanism of lysozyme on PGN. The synthesis of three novel NAM derivatives containing fused oxazinone ring to the NAM moiety has been achieved. The synthesized compounds were evaluated for their potential as a glycomimetic acceptor of lysozyme using different biophysical and computational methods such as 1H NMR, STD NMR, DOSY and Molecular docking. Novel modified muramic acid derivatives have been synthesized in excellent yield containing fused cyclooxazine ring embedded on the muramic acid moiety using a newly developed hydrazinolysis reaction condition. From various biophysical studies, it has been established that the compound containing endo modified muramic acid moiety (compound 1) shows significant binding property for the lysozyme while the other isomer (compound 2) did not bind to the lysozyme. The catalytic residues Glu35 and Asp52 were found to be in the close proximity for the active molecule which justifies the selectivity of this molecule in conjunction to lysozyme enzymatic activity.

Comparison of Enzymatic Hydrolysis of Polysaccharides from Eggshells Membranes

Abstract AHyaluronic acid (HA) is part of the extracellular matrix of connective, epithelial and neural tissues, as well as the synovial fluid, skin, and cartilage. It is composed of repeating disaccharide units of D-glucuronic acid and N-acetyl glucosamine. Hyaluronic acid is used in abdominal surgery, ophthalmology, dermatology, rhinology; it is usable for the osteoarthritis treatment. The membranes of eggshell are a natural source of hyaluronic acid, collagen, glycosaminoglycan and collagenous proteins. In paper, we tested the possibility of extraction hyaluronic acid from the eggshell membranes by enzymatic hydrolysis. We identified optimal conditions of hydrolysis with trypsin at reaction temperature of 37 °C and pH 8; with pepsin at 40 °C and pH 3, as well as with papain at 60 °C and pH 7.5. The content of hyaluronic acid in samples was determined spectrophotometrically using the carbazole method. The experimental results showed a yield of ~ 4 -4.5 % hyaluronic acid per 1 g of dry eggshell membranes.

Homogeneous Functionalization of Biopolymers Utilizing Ionic Liquids

Recently there has been an increased interest in the utilization of natural polymers in order to move to a more sustainable economy. The natural polymers of interest are cellulose, a biopolymer consisting of repeating glucose units, and chitin, a biopolymer consisting of repeating N-acetylglucosamine units. Both polymers are currently used in a number of industrial areas, such as medical, textile, and food industries. Due to the strong inter- and intra- molecular interaction in the biopolymers, their dissolution has been limited, which has limited the degree to which they can be modified. Ionic liquids have opened the door to homogenous modification of biopolymers, which is leading to an increased degree of substitution and new modifications. Ionic liquids have been shown to dissolve various polymers, including cellulose and chitin.1-4 Their dissolution has opened up new avenues for the utilization of these biopolymers, including new composite materials and the homogenous modification mentioned previously. Heinze et al. have shown that using ionic liquids to dissolve cellulose and perform homogenous modification increases the degree of substitution (DS) for carboxymethylation and acylation reactions.5 , 6 An increased DS increases the effectiveness of the modification polymer. A modification of particular interest in our research is the addition of a chelating agent to the biopolymer, specifically the addition of the diethylaminoethyl functionality. Diethylaminoethyl-cellulose (DEAE-cellulose) is widely used as the stationary phase in ion exchange chromatography, where it functions as an anion exchanger. The addition of the DEAE branch to the cellulose backbone also increases its ability to chelate with metals in solution. The effectiveness of DEAE-cellulose to perform these functions is determined by its degree of substitution (DS). By increasing the DS, the effectiveness of the materials produced will be improved. Preliminary data, shown in figure 1, demonstrates in increase in the DS for the homogenous preparation of DEAE-cellulose in the ionic liquid, 1-butyl-3-methylimidazolium chloride ([C4mim][Cl]. There is an increase in the DS from 0.15 for the heterogeneous procedure to 0.35 for the homogenous procedure. The figure also shows the effect changes to the ionic liquid can have on the reaction. By changing the anion from chloride to acetate, there is a marked decrease in the DS of the cellulose. This research will explore the effects of various cations and anions on the homogenous modification of the biopolymers, cellulose and chitin. The DS for the modified biopolymers will be measured by titration and through FT-IR analysis. An investigation into the effectiveness of the modified polymers to remove various metals from aqueous solutions will also be discussed. (1) Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. J Am Chem Soc 2002, 124, 4974. (2) Xie, H.; Zhang, S.; Li, S. Green Chem. 2006, 8, 630. (3) Qin, Y.; Lu, X.; Sun, N.; Rogers, R. D. Green Chem. 2010, 12, 968. (4) Wang, W.-T.; Zhu, J.; Wang, X.-L.; Huang, Y.; Wang, Y.-Z. J. Macromol. Sci., Part B: Phys. 2010, 49, 528. (5) Heinze, T.; Schwikal, K.; Barthel, S. Macromolecular Bioscience 2005, 5, 520. (6) Barthel, S.; Heinze, T. Green Chem. 2006, 8, 301. Figure 1

Partial Characterization of Glycans from Daedalea quercina (L) Fr.

Heteroglycans were extracted and isolated from the fruiting bodies of wild mushroom Daedalea quercina (L) Fr. These were obtained from sequential extraction with hot water, 1% Ammonium oxalate then 5% Sodium hydroxide solution followed by ethanol or acidic precipitation affording crude extracts which were further purified by dialysis membrane (MWCO 12 400). Separation by charge and size were done using DEAE-cellulose column and Sephadex G-100 or Sepharose CL-4B. This resulted to water soluble neutral (W1P-1A) and acidic (W1P-2A) glycan isolates, ammonium oxalate soluble (A1P-1) and two alkali soluble (N1P-1 and N1P-2) glycan isolates. Characterization of the hydrolyzed isolates using HPAEC-PAD and MALDI TOF MS showed glucan-rich heteroglycans (W1P-1A and W1P-2A); glucan and mannan rich heteroglycan, (N1P-1) and galactan-rich heteroglycan (A1P-1). The latter being the first reported galactan-rich glycan found in ammonium oxalate mushroom extract. The hydrolysates had approximately 4-5 degree of polymerization with molecular weights ranging from 689.680 to 851.788 Da. The hydrolysate of isolate N1P-2 is a glucan-rich heteroglycan which also contain N-acetyl glucosamine units. It is composed of polymers with 4-5 hexose units as well as N-acetyl glucosamine containing polymers of 6-8 hexose units with molecular weight range of 689.799 to 851.915 Da and 1055.718 to 1379.584 Da, respectively.

Fungal chitinases: function, regulation, and potential roles in plant/pathogen interactions

In the past decades our knowledge about fungal cell wall architecture increased tremendously and led to the identification of many enzymes involved in polysaccharide synthesis and remodeling, which are also of biotechnological interest. Fungal cell walls play an important role in conferring mechanic stability during cell division and polar growth. Additionally, in phytopathogenic fungi the cell wall is the first structure that gets into intimate contact with the host plant. A major constituent of fungal cell walls is chitin, a homopolymer of N-acetylglucosamine units. To ensure plasticity, polymeric chitin needs continuous remodeling which is maintained by chitinolytic enzymes, including lytic polysaccharide monooxygenases N-acetylglucosaminidases, and chitinases. Depending on the species and lifestyle of fungi, there is great variation in the number of encoded chitinases and their function. Chitinases can have housekeeping function in plasticizing the cell wall or can act more specifically during cell separation, nutritional chitin acquisition, or competitive interaction with other fungi. Although chitinase research made huge progress in the last decades, our knowledge about their role in phytopathogenic fungi is still scarce. Recent findings in the dimorphic basidiomycete Ustilago maydis show that chitinases play different physiological functions throughout the life cycle and raise questions about their role during plant-fungus interactions. In this work we summarize these functions, mechanisms of chitinase regulation and their putative role during pathogen/host interactions.

Catalytic Depolymerization of Chitin with Retention of N-Acetyl Group.

Chitin, a polymer of N-acetylglucosamine units with β-1,4-glycosidic linkages, is the most abundant marine biomass. Chitin monomers containing N-acetyl groups are useful precursors to various fine chemicals and medicines. However, the selective conversion of robust chitin to N-acetylated monomers currently requires a large excess of acid or a long reaction time, which limits its application. We demonstrate a fast catalytic transformation of chitin to monomers with retention of N-acetyl groups by combining mechanochemistry and homogeneous catalysis. Mechanical-force-assisted depolymerization of chitin with a catalytic amount of H2SO4 gave soluble short-chain oligomers. Subsequent hydrolysis of the ball-milled sample provided N-acetylglucosamine in 53% yield, and methanolysis afforded 1-O-methyl-N-acetylglucosamine in yields of up to 70%. Our process can greatly reduce the use of acid compared to the conventional process.

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils.

Requirement of keratan sulfate proteoglycan phosphacan with a specific sulfation pattern for critical period plasticity in the visual cortex

Proteoglycans play important roles in regulating the development and functions of the brain. They consist of a core protein and glycosaminoglycans, which are long sugar chains of repeating disaccharide units with sulfation. A recent study demonstrated that the sulfation pattern of chondroitin sulfate on proteoglycans contributes to regulation of the critical period of experience-dependent plasticity in the mouse visual cortex. In the present study, we investigated the role of keratan sulfate (KS), another glycosaminoglycan, in critical period plasticity in the mouse visual cortex. Immunohistochemical analyses demonstrated the presence of KS containing disaccharide units of N-acetylglucosamine (GlcNAc)-6-sulfate and nonsulfated galactose during the critical period, although KS containing disaccharide units of GlcNAc-6-sulfate and galactose-6-sulfate was already known to disappear before that period. The KS chains were distributed diffusely in the extracellular space and densely around the soma of a large population of excitatory and inhibitory neurons. Electron microscopic analysis revealed that the KS was localized within the perisynaptic spaces and dendrites but not in presynaptic sites. KS was mainly located on phosphacan. In mice deficient in GlcNAc-6-O-sulfotransferase 1, which is one of the enzymes necessary for the synthesis of KS chains, the expression of KS was one half that in wild-type mice. In the knockout mice, monocular deprivation during the critical period resulted in a depression of deprived-eye responses but failed to produce potentiation of nondeprived-eye responses. In addition, T-type Ca2+ channel-dependent long-term potentiation (LTP), which occurs only during the critical period, was not observed. These results suggest that regulation by KS-phosphacan with a specific sulfation pattern is necessary for the generation of LTP and hence the potentiation of nondeprived-eye responses after monocular deprivation.

Chitin dipentanoate as the new technologically usable biomaterial.

In this article, the synthesis of novel biopolymer, chitin dipentanoate (Di-O-Valeryl Chitin, DVCH) has been described. DVCH is a chitin derivative esterified with two valeryl groups at positions 3 and 6 of the N-acetylglucosamine units and it is soluble in common organic solvents like ethanol, methanol, acetone, dichloromethane, 1,2-dichloroethane, N,N-dimethylmethanamide, N,N-dimethylacetamide and ethyl acetate. Highly efficient synthesis (degree of esterification close to 2) of DVCH was achieved by employing a huge excess of valeric anhydride used as both the acylation agent and the reaction medium in the presence of perchloric acid as catalyst. Studies on the DVCH synthesis were aimed at finding optimal conditions (temperature, reaction time) to obtain DVCH with high reaction yield and desirable physicochemical properties. Biological data demonstrate that DVCH is non-cytotoxic in vitro and doesn't exert irritating or allergic effects to animal skin. Thanks to its filmogenic properties, it can be used to manufacture threads, foils, foams and non-woven materials. Moreover, DVCH can be easily processed by salt-leaching method to prepare highly porous structures exhibiting open-cell architecture, that can be further employed in wound dressing therapies and scaffolds for tissue engineering.

Determination of the degree of acetylation and the distribution of acetyl groups in chitosan by HPLC analysis of nitrous acid degraded and PMP labeled products

Chitin is one of the most abundant polysaccharides on earth. It consists of repeating β-1,4 linked N-acetylated glucosamine (A) units. Chitosan is an N-deacetylated product of chitin. Chitosan and its derivatives have broad medical applications as drugs, nutraceuticals, or drug delivery agents. However, a reliable analytical method for quality control of medically used chitosans is still lacking. In current study, nitrous acid was used to cleave all glucosamine residues in chitosan into 2,5-anhydromannose (M) or M at the reducing end of di-, tri-, and oligosaccharides. PMP, i.e. 1-phenyl-3-methyl-5-pyrazolone, was used to label all the Ms. Online UV detection allowed quantification of all M-containing UV peaks whereas online MS analysis directly identified 11 different kinds of mono-, di-, tri-, and oligosaccharides that correlated each oligosaccharide with specific UV peak after HPLC separation. The DA (degree of acetylation) for chitosans was calculated based on the A/(A+M) value derived from the UV data. This newly developed method had several advantages for quality control of chitosan: 1. the experimental procedures were extensively optimized; 2. the reliability of the method was confirmed by online LC-MS analysis; 3. the DA value was obtainable based on the UV data after HPLC analysis, which was comparableto that of 1H NMR and conductometric titration analyses; 4. finally and most importantly, this method could be used to obtain the DA as well as chemical acetylation/deacetylation mechanisms for chitosan by any laboratory equipped with a HPLC and an online UV detector.

The Newly Bone Formation with Carbonate Apatite-Chitosan Bone Substitute in the Rat Tibia

Large bone defect still represent a major problem in orthopedics. A tissue engineering approach has been proposed where osteogenic cells, bioceramic scaffolds and growth factors can play in a role to the bone repair. Bone consist a mineral phase such as carbonate apatite and an organic phase such as collagen. Synthetic carbonate apatite ceramics are considered as promising alloplastic materials for bone substitute. Chitin is the organic matrix of the hard parts of exoskeleton of insect, crustacean and present in a small amounts in mushrooms. It is an insoluble, similar to cellulose and composed of N-acetylglucosamine unit. Partial deacetylation from chitin result in the formation of chitosan. Chitin’s properties as a flexible and strong material make it favourable as surgical thread. It has novel properties such as biocompatibility, biodegradability, anti bacterial, wound healing activity, tissue regeneration and hemostatic activitities. The composit from carbonate apatite and chitosan may have a great impact on human health care system as bioresorbable bone substitute. The aim of the study was to evaluate the newly bone formation on the bone healing of defect tibia treated with carbonate apatite-chitosan bone substitute. Eighteen Sprague Dawley rats, male, 3 months, weighing 250-300g used in this study. Bilateral defect were created in each tibia rat. The defects were filled with carbonate-apatite chitosan bone substitute. The rats were sacrificed after respectively 1, 2 and 3 weeks. The result of this study showed that carbonate apatite-chitosan significantly increased a number of osteoblast (p<0.05). Carbonate apatite-chitosan group showed that matrix deposition faster than the other groups and have a good interface with the old bone. These data indicate that carbonate apatite-chitosan are potential candidate for bone substitute

In vitro antitumor activity of heterochitooligosaccharides (a review)

This review presents an analysis of the literature data over the last decade in order to reveal the relationship between the structure and composition of chitin/chitosan oligomers and their antitumor activity. Oligosaccharides consisting of N-acetylglucosamine and/or glucosamine units connected to each other by β-1,4-O-glycoside bond are generally called chitooligosaccharides. Homochitooligosaccharides are the oligomers of N-acetylglucosamine (AGA) or glucosamine (GA). Heterooligosaccharides are a mixture of various oligomers differing in the degree of polymerization (DP), acetylation (DA), or deacetylation (DD), as well as in the location of N-acetyl residues in the oligomer chain. Heterochitooligomers with a polymerization degree of no more than 10 are usually water soluble. The solubility of chitooligosaccharides with a DP of more than 10 depends on the DA and pH of a solution. The pharmaceutical and food industries, as well as scientists engaged in basic research, are interested in the use of heterochitooligosaccharides. This is explained by their unique properties, such as good water solubility; minimal toxicity; biocompatibility; the ability to penetrate cell membranes, resulting in a high degree of absorption (unlike chitin and chitosan); and their biological activity. Therefore, in the last decade, researchers have focused their attention on studying the relationship of the structure of oligosaccharides and their specific activity, such as antitumor, antimicrobial, antioxidant, immunemodulatory, and other activities. This is shown by the number of publications, which has doubled compared to the number in 2001.

Effects of different nitrogen sources on the production of Hyaluronic acid by Streptococcus

Hyaluronic acid (HA) is a linear polysaccharide with high molecular weight composed of disaccharide units of D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). HA is naturally present in vertebrate organisms, as well as in bacteria. HA can be obtained commercially through three routes: human umbilical cords, rooster combs, and strains of group C Streptococcus .T his is an atural polysaccharide with extensive range of applications in the medical, pharmaceutical and cosmetics. Due to the viscoelastic and hydrophilic, the HA products and derivatives present high aggregated value ranging from US$ 2000 to 60000 Kg -1 , depending on their applications. The most frequently used bacteria in the industrial production of HA are Lancefield group A and C streptococci. These bacteria are nutritionally fastidious microorganisms which require complex nutrients due to their limited ability to synthesize specific aminoacids and B-vitamins. Additionally, there is the nutritional requirement with respect to organic nitrogen, which also supplies a large portion of carbon for their cellular biosynthesis. Recent studies seek alternatives to allow the cost of production of HA using agricultural derivatives and industrial waste. In this context, the objective of this study was to evaluate the effect of nitrogen sources yeast extract, soy protein, whey protein and corn steep liquor in the production of HA. Fermentations were carried out in 125 mL erlenmeyer flasks containing 25 mL culture medium. The culture medium comprised in ( gL -1 ): glucose, 30; nitrogen source (yeast extract, soy protein, whey protein or corn steep liquor), 30; K2HPO4 ,2 .5, NaCl, 2,0 and MgSO4 ,1 .5. The inoculum was 10% (v/v) and the fermentations occurred at 100 rpm, 37°C and pH 8.0 for 24 hours. The fermented medium was centrifuged and the cell free supernatant was treated with ethanol for the precipitation of HA which quantified using a colorimetric reagent Carbazole. The concentration of lactic acid, acetic acid and formic acid were also analyzed in a system of high performance liquid chromatography with IR detector, column Aminex HPX 87H at 60°C and a solution of H2SO4 0.005 mol L -1 as the mobile phase a flow rate of 0.7 mL min -1 . The results performed in triplicate were compared by the Tukey test at 5% probability level (p < 0.05). The highest production of HA, 0.534 g L -1 was obtained when using yeast extract as nitrogen source. Subsequently, experiments which resulted in a better yield of the polymer are those containing soybean protein (0.192 g.L -1 ) and whey protein ( 0.063 g.L -1 ). In medium containing corn steep liquor, no microbial growth or production of HA ocurred. The decreased production of HA was directly followed by a reduction of the production of lactic acid and acetic acid.

Enzymatic sequencing of partially acetylated chitosan oligomers

Chitosan oligosaccharides have diverse biological activities with potentially valuable applications, for example, in the fields of medicine and agriculture. These functionalities are thought to depend on their degree of polymerization and acetylation, and possibly on specific patterns of acetylation. Chitosan oligomers with fully defined architecture are difficult to produce, and their complete analysis is demanding. Analysis is typically done using MS or NMR, requiring access to expensive infrastructure, and yielding unequivocal results only in the case of rather small oligomers. We here describe a simple and cost-efficient method for the sequencing of μg amounts of chitosan oligosaccharides which is based on the sequential action of two recombinant glycosidases, namely an exo-β-N-acetylhexosaminidase (GlcNAcase) from Bacillus subtilis 168 and an exo-β-d-glucosaminidase (GlcNase) from Thermococcus kodakarensis KOD1. Starting from the non-reducing end, GlcNAcase and GlcNase specifically remove N-acetyl glucosamine (A) and glucosamine (D) units, respectively. By the sequential addition and removal of these enzymes in an alternating way followed by analysis of the products using high-performance thin-layer chromatography, the sequence of chitosan oligosaccharides can be revealed. Importantly, both enzymes work under identical conditions so that no buffer exchange is required between steps, and the enzyme can be removed conveniently using simple ultra-filtration devices. As proof-of-principle, the method was used to sequence the product of enzymatic deacetylation of chitin pentamer using a recombinant chitin deacetylase from Vibrio cholerae which specifically removes the acetyl group from the second unit next to the non-reducing end of the substrate, yielding mono-deacetylated pentamer with the sequence ADAAA.

Cost Effective Media Optimization for the Enhanced Production of Hyaluronic Acid Using a Mutant Strain Streptococcus zooepidemicus 3523–7: A Statistical Approach

The hydrated gel hyaluronic acid (HA) comprises repeating units of glucuronic acid and N-acetylglucosamine. HA production has a lot of significance in the present day scenario owing to its clinical applications. Fermentative optimization is carried out in two steps. Statistical optimization of media components for the production of HA using Taguchi and Response Surface Methodology (RSM) was studied using a mutant strain Streptococcus zooepidemicus 3523-7. Initial optimization was carried out using L27 orthogonal array. The significance of each factor with respect to HA production was identified using Taguchi design (Glucose, Yeast extract, K2HPO4, NaH2PO4, NaCl, NH4Cl, 1M MgSO4 and Trace metal mix (TMM)) and the productivity was increased 1.34 folds (738 to 988 mg/L) over native production medium. The outcome of Taguchi results indicates the glucose, yeast extract, K2HPO4 and NH4Cl are most influencing factors in the production of HA. Final optimization was carried out using RSM (Central Composite Rotatable Design) and the production was significantly enhanced to 1.4 folds (988 to 1386 mg/L). For the first time statistical optimization was carried out using a mutant strain Streptococcus zooepidemicus 3523-7 and high yield (1386 mg/L) of HA was achieved.

Metabolic control of hyaluronan synthases

Hyaluronan (HA) is a glycosaminoglycan composed by repeating units of D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) that is ubiquitously present in the extracellular matrix (ECM) where it has a critical role in the physiology and pathology of several mammalian tissues. HA represents a perfect environment in which cells can migrate and proliferate. Moreover, several receptors can interact with HA at cellular level triggering multiple signal transduction responses. The control of the HA synthesis is therefore critical in ECM assembly and cell biology; in this review we address the metabolic regulation of HA synthesis. In contrast with other glycosaminoglycans, which are synthesized in the Golgi apparatus, HA is produced at the plasma membrane by HA synthases (HAS1-3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. UDP-GlcUA and UDP-hexosamine availability is critical for the synthesis of GAGs, which is an energy consuming process. AMP activated protein kinase (AMPK), which is considered a sensor of the energy status of the cell and is activated by low ATP:AMP ratio, leads to the inhibition of HA secretion by HAS2 phosphorylation at threonine 110. However, the most general sensor of cellular nutritional status is the hexosamine biosynthetic pathway that brings to the formation of UDP-GlcNAc and intracellular protein glycosylation by O-linked attachment of the monosaccharide β-N-acetylglucosamine (O-GlcNAcylation) to specific aminoacid residues. Such highly dynamic and ubiquitous protein modification affects serine 221 residue of HAS2 that lead to a dramatic stabilization of the enzyme in the membranes.