ICCBM15, Hamburg, 2014
Sep. 17
th, Poster
1
Purification, Characterization and Crystallization of Scytalidium thermophilum Xylanase
Sutay Kocabas D1, Tur E1, Guder S2
1Department of Food Engineering, Karamanoglu Mehmetbey University, Karaman, Turkey; 2Department
of Chemistry, Karamanoglu Mehmetbey University, Karaman, Turkey
Xylanases are hydrolytic enzymes responsible for xylan depolymerization. Xylan is the complex polysaccharide of the plant cell wall mainly consisting of D-xylose as the monomeric unit and it is the most abundant non-cellulosic renewable polysaccharide on the earth (Beg et al. 2001, Dhiman et al. 2008). Fungal xylanases are favorable at industrial scale such as animal feed production, manufacture of bread, food and drinks, pharmaceutical and chemical applications, textiles, pulp and paper production (Polizeli et al. 2005). Microbial xylanases are preferred biocatalysts in industry due to their high substrate specificity, mild reaction conditions, conservation of substrate and insignificant side product formation (Kulkarni et al 1999).
S. thermophilum xylanases were produced as described previously with a slight modification (Sutay
Kocabas et al. 2008). Ground corn cob particles (<2 mm sizes) were used at 20 g/l concentration in the main culture as the carbon source. By using two-step chromatography technique including gel filtration and anion exchange, low molecular weight xylanase was purified ca. 21.8 fold with 9.6% recovery to apparent homogeneity as demonstrated by SDS-PAGE. The enzyme has a molecular weight of 21 kDa and an isoelectric point of pH 8.6. Optimum temperature and pH values of purified xylanase were found to be 65°C and 6.5, respectively. Xylanolytic activity was most stable at pH 7.0 and 40°C. Purified xylanase showed the maximum specificity towards beechwood xylan and wheat bran among investigated commercial and lignocellulosic substrates.
Xylanase crystal growth was screened using ready-to-use kits from Hampton Research (USA) by sitting drop vapor diffusion method. After dye test of the observed crystals, crystal forming conditions were optimized in 24-well plates by hanging drop vapor diffusion technique at 18°C by mixing an equal volume of the protein with reservoir solution. So far, best crystals, having potential for X-ray structure studies, were obtained using ammonium citrate dibasic (1.6-1.8 M) and sodium acetate trihydrate (0.10-0.12 M) at 18°C at 3 mg/ml protein concentration. An example of a xylanase crystal is shown in Figure 1.
(a) (b)
Figure 1. Crystal of S. thermophilum xylanase, (a) original crystal (b) crystal after dye treatment
References
Beg QK, et al., Appl. Microbiol. Biotechnol. 56, 326-338 (2001) Dhiman SS, et al., Bioresources 3, 1377-1402 (2008)
Kulkarni N, et al., FEMS Microbiol Rev 23, 411-456 (1999)
Polizeli MLTM, et al., Appl. Microbiol. Biotechnol. 67, 577-591 (2005) Sutay Kocabas D, et al., Appl. Microbiol. Biotechnol. 79, 407-415 (2008)
