Marine fungal abilities to enzymatically degrade algal polysaccharides,proteins and lipids: a review

Journal of Applied Phycology - Over the last decades, metabolites with biotechnological application produced by marine resources and notably macroalgae have seen increasing interest. Among these...     

Size-exclusion liquid chromatography and capillary electrophoresis of pollen allergens

Allergens from the pollen of Phleum pratense, Dactylis glomerata. Arrhenatherum elatius, Secale cereale, Lolium perrene and Festuca sp. were analysed by size-exclusion chromatography (SEC) and capillary electrophoresis (CE). SEC was used for the determination of the molecular masses of main allergens. A CE method, using either 150 mmol/1 phosphoric acid (pH 1.8) or a micellar system consisting of 50 mmol/l sodium dodecyl sulphate-20 mmol/l borate (pH 9.35), was developed as a rapid and efficient alternative to SEC, especially for process control of allergenic preparations. The results obtained by the two methods confirmed similarities in the structures of the studied pollen allergens.     

Size-exclusion chromatography-a review of calibration methodologies

Recent developments are reviewed in size exclusion chromatographic calibration methodologies, including direct calibration by using narrow and broad polymer standards and various instrumental methods (nuclear magnetic resonance, mass spectrometry, light scattering) as well as universal calibration with and without viscometry detectors, for simple and complex polymers.     

Size-exclusion reaction chromatography (SERC): a new technique for protein PEGylation

A new, widely applicable process that combines reaction and separation in a single unit operation is described. The process, size-exclusion reaction chromatography (SERC), simultaneously allows control of the extent of reactions in which molecular size is altered and the separation of products and reactants. In SERC, a moving reaction zone is formed by injection of reactants onto a size-exclusion chromatography column. Reactants and products are partitioned differently within the mobile phase, resulting in different linear flow rates through the column. The products are therefore removed selectively from the reaction zone, minimizing their residence time in the reaction zone and allowing their separation in the downstream section of the column. For reactions such as protein PEGylation, in which successive addition of PEG groups to the protein results in significant molecular size increases, SERC potentially offers a method by which a dominant final PEGylated protein size can be produced at high yield. The SERC PEGylation of two model proteins, alpha-lactalbumin and beta-lactoglobulin, is demonstrated and results show that simultaneous reaction and separation was obtained.     

Size-exclusion chromatography of heparin oligosaccharides at high and low pressure

Recent findings on specific and non-specific interactions of glycosaminoglycans (GAGs) accentuate their pivotal role in biology and the call for improved sequencing tools. The present study evaluates size-exclusion chromatography (SEC) of heparin oligosaccharides at high and low pressure, requiring amounts as low as 0.2 microgram, using conventional UV detection after depolymerization with heparin lyases. Because of their high charge at physiological pH, SEC elution volumes of heparin oligosaccharides depend on both molecular size and charge repulsion from the matrix. As a consequence, SEC elution volumes of GAGs are smaller than those of globular proteins of similar molecular weight, and this might be exploited. Accordingly, larger heparin oligosaccharides are best separated according to their size at high ionic strength of the mobile phase (>30 mM); in contrast, disaccharides are best separated according to their charge at low ionic strength, compatible with on-line coupling to mass spectrometry. Optimized SEC affords separation of characteristic heparin trisaccharides that contain uronic acid at the reducing end and suggest cellular storage of heparin as a free glycan.     

Size-exclusion chromatography study of the molecular-weight distribution of gamma-irradiated pullulan

The polyelectrolyte behavior of gamma-irradiated pullulan in aqueous solutions leads to secondary effects of adsorption on a Shodex OHPak KB 806 column gel during size-exclusion chromatography. Suppression of the polyelectrolyte properties of gamma-irradiated pullulan is achieved by using a 0.05 M aqueous solution of NaH2PO4 (pH 4.95) as the mobile phase. Under these conditions, adequate molecular-weight distributions of gamma-irradiated pullulan samples are obtained.     

Size-exclusion chromatography as a linear transfer system: purification of human influenza virus as an example

Preparative size-exclusion chromatography suffers from low selectivity and productivity. Empirical optimization of operating conditions constitutes a laborious task due to many parameters. Here, a modeling framework based on linear systems theory is presented for predicting the influence of volume overloading. Impulse-responses characterizing system behavior are derived from experimental data by maximum entropy deconvolution. Theoretical derivations are validated experimentally by study of a model system and chromatography of human influenza virus. By application of the theory it is demonstrated how group separation operations can be optimized with respect to yield, purity, productivity and dilution of the product.     

Size-exclusion chromatography of biological samples which contain extremely alkaline proteins

An improved size-exclusion chromatography (SEC) was developed to isolate extremely basic (alkaline) proteins, such as trypsin (pI=10.5), lysozyme (pI=11), and histone (pI=10.8). Develosil 300 Diol-5 (300 x 8 mm I.D., 30 nm average pore diameter) column was used with an eluent of 0.1 M sodium phosphate, 1.5 M sodium chloride, glycerol (40%, v/v), 2-propanol (10%, v/v), and Brij-58 (1%, v/v). Under these conditions, the final apparent pH becomes to 4.0, and pH adjustment is not necessary. Column temperature and flow rate were 15 degrees C and 0.2 ml/min, respectively. This elution system is stable and reliable, and applications onto human pancreatic juice, human bile, and tissue homogenates were successfully achieved. Since this system is convenient for protein analysis, it is expected to be generally applicable to clinical and biochemical research for identifying protein components in combination with microsequencing.     

Regioselective esterification and etherification of cellulose: a review

Deep understanding of the structure-property relationships of polysaccharide derivatives depends on the ability to control the position of the substituents around the monosaccharide ring and along the chain. Equally important is the ability to analyze position of substitution. Historically, both synthetic control and analysis of regiochemistry have been very difficult for cellulose derivatives, as for most other polysaccharide derivatives. With the advent of cellulose solvents that are suitable for chemical transformations, it has become possible to carry out cellulose derivatization under conditions sufficiently mild to permit increasingly complete regiochemical control, particularly with regard to the position of the substituents around the anhydroglucose ring. In addition, new techniques for forming cellulose and its derivatives from monomers, either by enzyme-catalyzed processes or chemical polymerization, permit us to address new frontiers in regiochemical control. We review these exciting developments in regiocontrolled synthesis of cellulose derivatives and their implications for in-depth structure-property studies.     

Sorption of anionic polysaccharides by cellulose

The sorption of anionic polysaccharides pectin, alginate, and xanthan with cellulose were investigated in presence of calcium. Calcium sorption to cellulose was limited by the carboxyl group content in fibers. Atomic Absorption Spectroscopy (AAS) analysis was used to measure the calcium in cellulose fibers and chemical oxygen demand (COD) analysis reveals that the divalent ions calcium can bind the polysaccharide onto cellulose fibers. The amount of calcium and polysaccharide bound in Ca²⁺/polysaccharide modified cellulose fibers was 5.8-12.5 mM Ca²⁺/kg fibers and 1500-2400 mg polysaccharide/kg fibers, respectively. Fourier Transform Infrared Spectroscopy-Attenuated Total Reflectance (FTIR-ATR) analysis confirmed the presence of polysaccharide on calcium containing cellulose fibers. The results of alizarin dyeing experiments at the end of polysaccharide sorption further confirmed the presence of calcium in Ca²⁺/polysaccharide modified cellulose fibers. The basic phenomenon of interaction of soluble ionic polysaccharide and cellulosic fibers in presence of divalent cations such as calcium is a key to understand biological functions and technological applications.     

Liquid chromatography modelling: a review

Liquid chromatography modelling represents a real challenge to understand the phenomena encountered in the separation process of biomolecules, especially in non-linear adsorption cases, and to enhance production rates and recovery yields in preparative chromatography.

The three major ways to describe liquid chromatography are presented, i.e. the continuity equations, the theory of interferences and the plate theory. The former is described in detail, since it permits the inclusion of various physical and thermodynamical phenomena, and to handle non-linear adsorption problems. The numerical ways of solving the differential equations stated by the different models are also considered. The interference theory, despite its limitations to ideal situations, is still considered as a useful tool in non-linear multicomponents liquid chromatography modelling. The plate theory is not developed, for it is restricted to linear adsorption cases.     

The cellobiose-oxidizing enzymes CBQ and CbO as related to lignin and cellulose degradation — a review

Abstract: In this review properties of cellobiose:quinone oxidoreductase (CBQ) and cellobiose oxidase (CbO) are presented and their possible involvement in lignin and cellulose degradation is discussed. Although these enzymes are produced by many different fungi, their importance for wood-degrading fungi is the topic here. CBQ is a FAD enzyme, while CbO also contains a heine group of the cytochrome b type. Protease activity is reported to convert CbO to CBQ. During oxidation of cellobiose (emanating from cellulose) to cellobiono-l,5-lactone, both enzymes reduce quinones produced by laccase and peroxidase during lignin degradation to the corresponding phenols. Many phenoxy and cation radicals are also reduced. Quinone reduction is more rapid than oxygen reduction, although oxygen is slowly reduced to superoxide and/or hydrogen peroxide. Thus, a more appropriate name for CbO is cellobiose dehydrogenase. CbO also reduces Fe(III) and together with hydrogen peroxide produced by the enzyme Fenton's reagent may be formed, resulting in hydroxyl radical production. This radical can degrade both lignin and cellulose, possibly indicating that cellobiose oxidase has a central role in degradation of wood by wood-degrading fungi.     

重要食药用菌多糖降血糖分子机制研究进展

目前以糖尿病为代表的糖代谢紊乱疾病愈演愈烈,严重危害人体健康.食药用菌多糖因其具有良好的调节糖代谢作用而被关注,但其调节糖代谢的作用机制并未被很好地综述.本文从关键基因、蛋白、信号通路等方面综述了食药用菌活性多糖的降血糖机制,包括抑制蛋白酪氨酸磷酸酶1B(Protein Tyrosine Phosphatase-1B,...     

Role of water-soluble polysaccharides in bacterial cellulose production

Acetobacter xylinum BPR2001 produces water-insoluble bacterial cellulose (BC) and a water-soluble polysaccharide called acetan in corn steep liquor-fructose medium. Acetobacter xylinum EP1, which is incapable of acetan production was derived by disrupting the aceA gene of BPR2001. The BC production by EP1 (2.88 g/L) was lower than that by BPR2001 (4.6 g/L) in baffled-flask culture. When purified acetan or agar was added to the medium from the start of cultivation, the BC production by EP1 was enhanced and the final BC yield of EP1 was almost the same as that of BPR2001. A similar improvement of BC production by EP1 by the addition of agar was also confirmed by cultivation in a 50-L airlift reactor. From these results, the role of acetan in BC production is associated with the increase in the viscosity of the culture medium which may hinder coagulation of BC and cells in the culture, thereby accelerating the growth of BPR2001 and BC production by BPR2001.     

Identification of enzymatically produced peptide fragments by liquid chromatography and mass spectrometry

The qualitative and quantitative determination of peptide fragments of angiotensin I generated by rat lung dipeptidyl carboxypeptidase (angiotensin converting enzyme, EC 3.4.15.1) is described. Enzymatically formed peptide fragments, after derivatization with fluorescamine, were separated and isolated by reverse-phase high-performance liquid chromatography. The recovered fluorescamine derivative of histidyl-leucine was then further identified by mass spectrometry. It is anticipated that this approach would be widely applicable to other enzyme systems.     

Sulfated polysaccharides and their anticoagulant activity: A review

Published data on the sulfated polysaccharides of various origins that display an anticoagulant activity are summarized and analyzed. The methods used for producing semisynthetic derivatives are considered. A key role of the polysaccharide structure in the mechanisms of specific interaction with various blood plasma proteinases is discussed. The effects of the content and location of sulfate groups in polysaccharides and their molecular weight on the degree of the studied activity are assessed.     

Influence of anomeric configuration on mechanochemical degradation of polysaccharides: cellulose versus amylose

Cellulose and amylose are (1-->4)-linked polysaccharides that are used extensively in the textiles, paper, and food and feed industries and are finding increasing use as alternative fuels and so forth. At the molecular level, cellulose and amylose differ only in their anomeric configuration: beta in cellulose, alpha in amylose. During processing and end use, these polymers experience a variety of mechanochemical stresses, many through contact with transient elongational flow fields. Here, we subject solutions of both polysaccharides to extended periods of ultrasonic irradiation, as the cavitational bubble collapse characteristic of ultrasound experiments creates flow fields strictly analogous to those encountered in other transient elongational flow scenarios. With the use of multidetector size-exclusion chromatography, the effects of anomeric configuration on both the limiting molar mass, beyond which polymers do not degrade in transient elongation flow ( M lim), and the rate of degradation have been isolated in these (1-->4)-linked polysaccharides. This effect was found to be pronounced; for example, M lim (cellulose) = 5( M lim (amylose)). Also, while extensive change was observed in molar mass averages, distribution, polydispersity, and size of the analytes during degradation, their structure was found to remain invariant. A modified "path theory" of transient elongational flow degradation was proposed, with the persistence length identified as a parameter which embodies the minimum continuous path length and flexibility requirements of the theory.     

Size-exclusion effect of a substrate upon kinetics of trypsin immobilized on porous bead cellulose. 1. Influence of distribution coefficient of a substrate

A model of heterogeneous biocatalysis, in which kinetics and partition effects are connected via the size-exclusion principle, was worked up experimentally and theoretically. The present paper shows that the maximum relative activity of trypsin (EC 3.4.21.4) immobilized on porous bead (spherical) cellulose is directly proportional to the available distribution coefficient of the substrate. Providing that the excess of substrate is not sufficient (e.g.S/K_m ≈ 1) to safeguard saturated enzyme kinetics, the originally linear relationship of R_a versus K_av turns to an exponential one, without any dependence upon the manner of enzyme immobilization. It is suggested that the above may be a result of partition resistance and that the main factors determining the shape of the R_a versus K_av relation in conditions of substrate shortage are the size and geometry of the matrix. The physical characteristics of the porous carrier as well as the manner of covalent immobilization of the enzyme are all reflected in the constants applied in the derived equations.     

Engineering of routes to heparin and related polysaccharides

Anticoagulant heparin has been shown to possess important biological functions that vary according to its fine structure. Variability within heparin’s structure occurs owing to its biosynthesis and animal tissue-based recovery and adds another dimension to its complex polymeric structure. The structural variations in chain length and sulfation patterns mediate its interaction with many heparin-binding proteins, thereby eliciting complex biological responses. The advent of novel chemical and enzymatic approaches for polysaccharide synthesis coupled with high throughput combinatorial approaches for drug discovery have facilitated an increased effort to understand heparin’s structure–activity relationships. An improved understanding would offer potential for new therapeutic development through the engineering of polysaccharides. Such a bioengineering approach requires the amalgamation of several different disciplines, including carbohydrate synthesis, applied enzymology, metabolic engineering, and process biochemistry.     

Heavy metal adsorbents prepared from the modification of cellulose: a review

A number of industries currently produce varying concentrations of heavy metal laden waste streams with significant consequences for any receiving environmental compartment. In recent years, increasing emphasis has been placed on environmental impact minimisation and resulting from this the range and capability of natural and prepared materials capable of heavy metal removal has seen steady development. In particular considerable work has been carried out on the use of both natural materials and their modifications. These natural materials, in many instances are relatively cheap, abundant in supply and have significant potential for modification and ultimately enhancement of their adsorption capabilities. This review paper reviews the current state of research on the use of the naturally occurring material cellulose, its modified forms and their efficacy as adsorbents for the removal of heavy metals from waste streams. Adsorbents based on direct modification of cellulose are evaluated initially and subsequently modifications resulting from the grafting of selected monomers to the cellulose backbone with subsequent functionalisation are assessed. The heavy metal adsorption capacities for these modified cellulose materials were found to be significant and levels of uptake were comparable, in many instances, to both other naturally occurring adsorbent materials and commercial ion exchange type resins. Many of the modified cellulose adsorbents proved regenerable and re-usable over a number of adsorption/desorption cycles allowing recovery of the adsorbed heavy metal in a more concentrated form.