Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.
Purification and Characterization of a Thermostable Laminarinase from Penicillium rolfsii c3-2 (1) IBRL.
Lee, K. C., Arai, T., Ibrahim, D., Kosugi, A., Prawitwong, P., Lan, D., Murata, Y. & Mori, Y. (2014). BioResources, 9(1), 1072-1084
A laminarinase (endo-β-1,3-glucanase) was purified to homogeneity from Penicillium rolfsii c3-2(1) IBRL, which was originally produced in liquid culture containing 1% xylan from birchwood, via anion-exchange chromatography, gel filtration on Sephacryl S-100, and hydrophobic interaction chromatography. A single protein band with a molecular weight of 75 kDa was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which had an optimum catalytic activity at pH 4.0 to 5.0 and 70°C. This purified enzyme was most stable in the pH range 4 to 7, while it was thermostable up to 55°C and retained up to 90% of its activity after 4 h pre-incubation. A substrate laminarin kinetic study yielded estimated Km and Vmax values of 0.0817 mg/mL and 372.2 µmol/min/mg, respectively. Laminari-oligosaccharide degradation, which was analyzed by thin layer chromatography, yielded the major hydrolysis products laminaribiose and glucose.
Flavobacterium johnsoniae as a model organism for characterizing biopolymer utilization in oligotrophic freshwater environments.
Sack, E. L. W., van der Wielen, P. W. J. J. & van der Kooij, D. (2011). Applied and Environmental Microbiology, 77(19), 6931-6938.
Biopolymers are important substrates for heterotrophic bacteria in oligotrophic freshwater environments, but information on bacterial growth kinetics with biopolymers is scarce. The objective of this study was to characterize bacterial biopolymer utilization in these environments by assessing the growth kinetics of Flavobacterium johnsoniae strain A3, which is specialized in utilizing biopolymers at µg liter-1 levels. Growth of strain A3 with amylopectin, xyloglucan, gelatin, maltose, or fructose at 0 to 200 µg C liter-1 in tap water followed Monod or Teissier kinetics, whereas growth with laminarin followed Teissier kinetics. Classification of the specific affinity of strain A3 for the tested substrates resulted in the following affinity order: laminarin (7.9 × 10-2 liter·µg-1 of C·h-1) >> maltose > amylopectin ≈ gelatin ≈ xyloglucan > fructose (0.69 × 10-2 liter·µg-1 of C·h-1). No specific affinity could be determined for proline, but it appeared to be high. Extracellular degradation controlled growth with amylopectin, xyloglucan, or gelatin but not with laminarin, which could explain the higher affinity for laminarin. The main degradation products were oligosaccharides or oligopeptides, because only some individual monosaccharides and amino acids promoted growth. A higher yield and a lower ATP cell-1 level was achieved at ≤10 µg C liter-1 than at >10 µg C liter-1 with every substrate except gelatin. The high specific affinities of strain A3 for different biopolymers confirm that some representatives of the classes Cytophagia-Flavobacteria are highly adapted to growth with these compounds at µg liter-1 levels and support the hypothesis that Cytophagia-Flavobacteria play an important role in biopolymer degradation in (ultra)oligotrophic freshwater environments.