Project description:To analyze the molecular function of HDAC inhibitors in salt stress responses in Arabidopsis, we conducted microarray analysis using 4-day-old plants, which were treated with 1 μM Ky-9 or Ky-72 or water for 24 h, and then treated with or without 100 mM NaCl for 2 h
Project description:We investigated the whole genome sequence of a freshwater agar-degrading bacterium Cellvibrio sp. KY-GH-1 (KCTC13629BP) to explore genetic information encoding agarases which hydrolyze agar into its monomers. The complete genome of KY-GH-1 comprised 5,762,391 base pairs (bp) with 47.9% GC content, and contained 5080 protein-encoding sequences, including nine β-agarase genes and two α-neoagarobiose hydrolase (α-NABH) genes in an agarase gene cluster spanning approximately 77 kb. Based on these genetic information, the degradation of agar into monomers (D-galactose and 3,6-anhydro-L-galactose) by KY-GH-1 was predicted to be initiated by endolytic GH16 β-agarases and endolytic GH86 β-agarases, further processed by exolytic GH50 β-agarases, and then terminated by exolytic GH117 α-NABHs. This study reveals the diversity and abundance of agarase genes, and provides insight into their roles in the agar-degrading enzyme machinery of Cellvibrio sp. KY-GH-1.
Project description:Study of the secretome of Cellvibrio japonicus after growth on different chitins. Utilization of a novel plate method to enrich truly secreted proteins.
Project description:Neoagarobiose (NA2) is the repeating disaccharide unit of agarose and possesses various promising biological activities. To identify an efficient exolytic β-agarase required for NA2 production from agarose, the GH50A β-agarase gene from agar-degrading Cellvibrio sp. KY-GH-1 was overexpressed as a recombinant His-tagged protein using the Escherichia coli expression system. GH50A β-agarase that consists of 797 amino acids was able to produce predominantly NA2 from agarose at an optimal temperature and pH of 35 °C and 7.5, respectively. The enzyme was stable up to 35 °C and within a pH range of 7.0-9.0. The K m, V max, K cat, and K cat/K m values of the enzyme were 26.5 mg/mL, 16.9 U/mg, 25.2 s-1, and 1.2 × 105 s-1 M-1, respectively. The copresence of 5 mM MnSO4 and 10 mM tris(2-carboxyethyl)phosphine (TCEP) resulted in a 2.5-fold enhancement of the enzyme activity. For NA2 production, neoagaro-oligosaccharides (NAOSs) containing NA4-NA18 were preferred over agarose or agaro-oligosaccharides (AOSs) as substrates. NA2 was produced along with minor amounts of agarotriose (A3) after treatment of AOS with the enzyme, indicating that the exolytic digestion of AOS by the enzyme was initiated by releasing A3 from nonreducing ends. Enzymatic hydrolysis of 0.4% agarose (100 mL) using GH50A β-agarase (20 μg/mL) for 4 h under optimal reaction conditions (5 mM MnSO4, 10 mM TCEP, 35 °C, 20 mM Tris-HCl, and pH 7.5) and purification of NA2 from hydrolysis products by Bio-Gel P-2 column chromatography resulted in the recovery of 216 mg of NA2 (∼54% yield from agarose). Altogether, these results suggest that the recombinant GH50A β-agarase is useful to convert agarose to NA2.