Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

<p>Abstract</p> <p>Background</p> <p>Fungal β-<it>N</it>-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-<it>N</it>-acetylhexosaminidase. The fungal β-<it>N</it>-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from <it>Aspergillus oryzae </it>was purified and its sequence was determined.</p> <p>Results</p> <p>The complete primary structure of the fungal β-<it>N</it>-acetylhexosaminidase from <it>Aspergillus oryzae </it>CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the <it>N</it>-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.</p> <p>Conclusion</p> <p>Whereas the intracellular bacterial β-<it>N</it>-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-<it>N</it>-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and <it>N</it>-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys⁴⁴⁸with Cys⁴⁸³stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.</p

Running the debts in the company Enviro-Ekoanalytika, s. r.o.

Accounting terms are very important for every company in the sphere of company finance, accounting terms are claims and accounts payable. The aim of this work was detailed description of claims, then show the control system of the claims of ENVIRO-EKOANALYTIKA and propose improvements to the existing claims management system. The main reasons why claims come into existence are business relations, but they can be caused by provided deposits, loans and bank credits. Claims from business relations are necessary parts of belongings, because every businessman needs goods and services from others. A company can analyse high earnings and profit in accounting but his financial resources can be essentially different. For a company is very important to have a sufficient number of financial resources and to this a company ensures of claims and then restricts entrepreneurial risk. The ensuring of claims means that contractual partner is obliged to realise his account payable. General forms to ensure claims are for example: penal clause, liability, acknowledge a debt, bank quarantee or altitude of debt. We can divide claims in risk claims and abandoned claims. During trading a lot of profits are formed, these profits can be evoked by for example: political or economical events, announcing a financial insolvency or natural catastrophe. Between the most frequent cause of risk claims are mainly the absence of morality in business relations. If the payment of claim is not done until fixed date, the claim gets the bad debt. The only way how to anticipate abandoned claims is, that busines men do not let them come into existence. The proceedings also include claims recovery. The main objective of recovery is to get customers to pay, and as early as possible. Most companies use a standard procedure for recovery, which includes telephone reminders, terminating the supply of credit, written reminders, the transmission of judicial or extrajudicial recovery

Zdokonalenie systému riadenia výroby a technologických procesov pri výrobe medi v n. p. Kovohuty Krompachy

PrezenčníNeuvedenoNeuveden

Work with claims of selected entrepreneurial subject

Accounting terms are very important for every company in the sphere of company finance, accounting terms are claims and accounts payable. The aim of this work was detailed description of claims which are parts of belongings of any company. The work has two parts, theoretical and practical part. Practical part predominate in this work, the main themes are claims, their operating,ensuring and debt recovery. In the practical part I describe claims of MICo company and I made my own project how to deal with claims. The main reasons why claims come into existence are business relations, but they can be caused by provided deposits, loans and bank credits. Claims from business relations are necessary parts of belongings, because every businessman needs goods and services from others. A company can analyse high earnings and profit in accounting but his financial resources can be essentially different. For a company is very important to have a sufficient number of financial resources and to this a company ensures of claims and then restricts entrepreneurial risk. The ensuring of claims means that contractual partner is obliged to realise his account payable. General forms to ensure claims are for example: penal clause, liability, acknowledge a debt, bank quarantee or altitude of debt. We can divide claims in risk claims and abandoned claims. During trading a lot of profits are formed, these profits can be evoked by for example: political or economical events, announcing a financial insolvency or natural catastrophe. Between the most frequent cause of risk claims are mainly the absence of morality in business relations. If the payment of claim is not done until fixed date, the claim gets the bad debt. The only way how to anticipate abandoned claims is, that busines men do not let them come into existence. MICo, spol. s.r.o. divides debts into short-term and long-term claims. The term of expiration of claims is established at intervals 14 {--} 45 days, it depends on which customer it is. The company of MICo tryes to aviod claims after maturity, therefore MICo makes use of ensuring claims as a penal clause, debt approval, bank quarantee or deposit. In conclusion, I would like to recommend to any company to have an evidence of claims and pay attention mainly to a prevention of claims because the ambit of czech legislation for a protection of rights of an accounting payee and the recovery of claims is generally regarded as deficient. Bankruptcy proceedings is slow and ineffective. During the competition accounting payees have small or even no rights and basically they are degraded inactive role and therefore the whole bankruptcy proceedings is regarded as a paradise for debtors

Závislost koncentrací polutantů ve vzorcích mlžné vody na vodním obsahu a celkovém povrchu kapiček vody v mlze

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Facile synthesis of nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranosides from ManNAc-oxazoline

The synthetic procedures for a large-scale preparation of o- and p-nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranoside are described. The synthetic pathway employs the glycosylation of phenol with ManNAc oxazoline, followed by nitration of the aromatic moiety yielding a separable mixture of the o- and p-nitrophenyl derivative in a 2:3 ratio

Ionic liquids as cosolvents for glycosylation by sucrose phosphorylase: balancing acceptor solubility and enzyme stability

Over the past decade, disaccharide phosphorylases have received increasing attention as promising biocatalysts for glycoside synthesis. Unfortunately, these enzymes typically have a very low affinity for non-carbohydrate acceptors, which urges the addition of cosolvents to increase the dissolved concentration of these acceptors. However, commonly applied solvents such as methanol and dimethyl sulfoxide (DMSO) are not compatible with many intended applications of carbohydrate-derived products. In this work, the solubility of a wide range of relevant acceptors was assessed in the presence of ionic liquids (ILs) as alternative and 'green' solvents. The IL AMMOENG 101 was found to be the most effective co-solvent for compounds as diverse as medium-and long-chain alcohols, flavonoids, alkaloids, phenolics and terpenes. Moreover, this IL was shown to be less deleterious to the stability and activity of sucrose phosphorylase than the commonly used dimethyl sulfoxide. To demonstrate the usefulness of this solvent system, a process for the resveratrol glycosylation was established in a buffer containing 20% AMMOENG 101, 1 M sucrose and saturated amounts of the acceptor. A single regioisomer 3-O-alpha-D-glucopyranosyl-(E)-resveratrol was obtained as proven by NMR spectroscopy

Rapid enzymatic hydrolysis of masked deoxynivalenol and zearalenone prior to liquid chromatography mass spectrometry or immuniassay analysis

Recently it has been shown that conjugates (‘masked mycotoxins’) may contribute to the total daily intake of hazardous mycotoxins. Therefore, there is an urgent need for rapid analysis methods that assess the level of both free and masked mycotoxins in food and feed. However, the analysis of masked mycotoxins by either immunoassays or instrumental methods, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), is severely hindered by the lack of standards and the unpredictable cross-reactivity profiles of the available antibodies. In this work, 26 enzymes were explored for rapid hydrolysis of masked mycotoxins using deoxynivalenol-3-glucoside (DON-3G) as model compound. Following initial screening, the most promising enzyme, a fungal 1,3-ß-glucanase (laminarinase), was investigated in detail and found to be fit-for-purpose, providing complete conversions in minutes rather than hours according to LC-MS/MS analyses. As a proof of concept, the enzymatic pretreatment was applied to an extract of beer containing DON-3G. In addition, the feasibility of a fully automated enzymatic pretreatment of masked mycotoxin standards in an autosampler was demonstrated in an imaging surface plasmon resonance immunoassay set-up. Such an automated pretreatment was found to be equally applicable to other mycotoxin conjugates, as shown by the conversion of zearalenone-14-ß-D-glucopyranoside and zearalenone-14-sulphate, in the latter case using a sulphatase enzyme. It is envisaged that laminarinase could be useful for other masked mycotoxins as well