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.
Chitinase from Bacillus licheniformis DSM13: Expression in Lactobacillus plantarum WCFS1 and biochemical characterisation.
Nguyen, H. A., Nguyen, T. H., Nguyen, T. T., Peterbauer, C. K., Mathiesen, G. & Haltrich, D. (2012). Protein Expression and Purification, 81(2), 166-174.
The gene chi, coding for a GH18 chitinase from the Gram-positive bacterium Bacillus licheniformis DSM13 (ATCC 14580), was cloned into the inducible lactobacillal expression vectors pSIP403 and pSIP409, derived from the sakacin-P operon of Lactobacillus sakei, and expressed in the host strain Lactobacillus plantarum WCFS1. Both the complete chi gene including the original bacillal signal sequence as well as the mature chi gene were compared, however, no extracellular chitinase activity was detected with any of the constructs. The chitinase gene was expressed intracellularly as an active enzyme with these different systems, at levels of approximately 5 mg of recombinant protein per litre of cultivation medium. Results obtained for the two different expression vectors that only differ in the promoter sequence were well comparable. To further verify the suitability of this expression system, recombinant, His-tagged chitinase Chi was purified from cell extracts of L. plantarum and characterised. The monomeric 65-kDa enzyme can degrade both chitin and chitosan, and shows properties that are very similar to those reported for the native chitinase purified from other B. licheniformis isolates. It shows good thermostability (half lives of stability of 20 and 8.4 days at 37 and 50°C, respectively), and good stability in the pH range of 5–10. The results presented lead the way to overproduction of chitinase in a food-grade system, which is of interest for the food and feed industry.
Heterologous expression and characterization of an N-acetyl-β-D-hexosaminidase from Lactococcus lactis ssp. lactis IL1403.
Nguyen, H. A., Nguyen, T. H., Křen, V., Eijsink, V. G. H., Haltrich, D. & Peterbauer, C. K. (2012). Journal of Agricultural and Food Chemistry, 60(12), 3275-3281.
The lnbA gene of Lactococcus lactis ssp. lactis IL1403 encodes a polypeptide with similarity to lacto-N-biosidases and N-acetyl-β-D-hexosaminidases. The gene was cloned into the expression vector pET-21d and overexpressed in Escherichia coli BL21* (DE3). The recombinant purified enzyme (LnbA) was a monomer with a molecular weight of approximately 37 kDa. Studies with chromogenic substrates including p-nitrophenyl N-acetyl-β-D-glucosamine (pNP-GlcNAc) and p-nitrophenyl N-acetyl-β-D-galactosamine (pNP-GalNAc) showed that the enzyme had both N-acetyl-β-D-glucosaminidase and N-acetyl-β-D-galactosaminidase activity, thus indicating that the enzyme is an N-acetyl-β-D-hexosaminidase. Km and Kcat for pNP-GlcNAc were 2.56 mM and 26.7 s-1, respectively, whereas kinetic parameters for pNP-GalNAc could not be determined due to the Km being very high (>10 mM). The optimal temperature and pH of the enzyme were 37°C and 5.5, respectively, for both substrates. The half-life of activity at 37°C and pH 6.0 was 53 h, but activity was completely abolished after 30 min at 50°C, meaning that the enzyme has relatively low temperature stability. The enzyme was stable in the pH 5.5–8 range and was unstable at pH below 5.5. Studies with natural substrates showed hydrolytic activity on chito-oligosaccharides but not on colloidal chitin or chitosan. Transglycosylation products were not detected. In all, the data suggest that LnbA’s role may be to degrade chito-oligosaccharides that are produced by the previously described chitinolytic system of L. lactis.
An extremely alkaline novel chitinase from Streptomyces sp. CS495.
Pradeep, G. C., Choi, Y. H., Choi, Y. S., Suh, S. E., Seong, J. H., Cho, S. S., Bae M. & Yoo, J. C. (2014). Process Biochemistry, 49(2), 223-229.
An extremely alkaline chitinase from Streptomyces sp. CS495 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular chitinase was purified 7.0 fold with a 33.9% yield by Sepharose Cl-6B column. The molecular mass of the enzyme (Ch495) was approximately 41 kDa. Ch495 was found to be stable over a broad pH range (5–12.5) and to 50°C and have an optimum temperature of 60°C. Ch495 had Km and Vmax values of 1.34 ± 2.9 mg/mL and 889 ± 3.6 mmol/min, respectively using different concentrations of colloidal chitin. N-terminal sequence of Ch495 was APREKINLLYFLGYF. HPLC and TLC analysis of Ch495 shows the production of produced N-acetyl D-glucosamine (GlcNAc) as minor and diacetylchitobiose (GlcNAc)2 as major products. Ch495 shows antifungal activity against Fusarium solani and Aspergillus brasiliensis which can be used for the biological control of fungus. As being simple in purification, extreme alkalophilic, stable in broad range of pH, ability to produce oligosaccharides, and antifungal activity shows that Ch495 has potential applications in industries as for chitooligosaccharides production used as medical prebiotics or/and for the biological control of plant pathogens in agriculture.