Amylase solution (1 mL) was incubated at 70 °C with 0.5% soluble starch in Tris–HCl buffer (pH 10.0) containing 10% NaCl. Aliquots were drawn at different time intervals, and hydrolysis was stopped by boiling at 100 °C. After centrifugation at 12 000 g for 15 min, each sample was selleck kinase inhibitor analyzed by HPLC analysis on a micro

Bond pack Amino Carbohydrate column (4.1 × 300 mm). Samples (15 μL) were injected and eluted with acetonitrile/water (70 : 30 ratio) at a flow rate of 1 mL min−1. The hydrolyzed products were detected using a refractive index detector. Glucose, maltose, maltotriose, and maltopentaose (Sigma) were used as standards. Based on morphological, physiological, and biochemical characteristics, the isolate LY20 is a Gram-positive, motile, rod-shaped and aerobic bacterium.

Colonies are http://www.selleckchem.com/hydroxysteroid-dehydrogenase-hsd.html light yellow, uniformly round, circular, and convex on CM agar plate. It was able to grow in medium containing 0.5–25% (w/v) NaCl and grew optimally at 10% (w/v) NaCl. No growth was observed in the absence of NaCl. Thus, this bacterium can be considered as a moderately halophilic microorganism (Ventosa et al., 1998). Optimal temperature and pH for bacterial growth were 37 °C and 10.0. H2S production, methyl red, and Tween-80 hydrolysis were negative, while Voges–Proskauer test, nitrate reduction, oxidase, catalase, and gelatin hydrolysis were positive. Acid is produced from maltose, fructose, sucrose, and glucose. learn more Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that the isolate LY20 belonged to Salimicrobium species and was most closely related to Salimicrobium halophilum DSM 4771T (98.9% 16S rRNA gene sequence similarity; Fig. 1). As shown in Fig. 2, both enzymes started

to produce from the early-exponential phase of bacterial growth (4 h for amylase and 10 h for protease) and reached a maximum level during the early-stationary phase (42 h). Both enzymes were purified by ammonium sulfate precipitation, Q-Sepharose ion exchange, and Sephacryl S-200 gel filtration chromatography. The amylase was purified 21.5-fold with recovery of 31.9% and specific activity of 573.5 units mg−1 protein, while protease was purified 27.5-fold with recovery of 32.4% and specific activity of 832.7 units mg−1 protein. Molecular weights of the β-amylase and protease were determined to be 81 and 30 kDa, respectively (Fig. 3, lanes 2 and 3), corresponding with those determined by gel filtration. These results indicated that both enzymes from LY20 were monomeric ones. Also, zymographic activity staining revealed the activity bands for purified samples at corresponding positions on SDS-PAGE (Fig. 3, lanes 4 and 5). The amylase hydrolyzed soluble starch to form maltose as the main product. This product was readily apparent during the early stages of the reaction and increased in concentration along with the time course of the reaction.