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Purification and Characterization of Chitosanase from Bacillus sp . Strain KCTC 0377BP and Its Application for the Production of Chitosan Oligosaccharides. Yeon Jin Choi, 2004.For the enzymatic production of chitosan oligosaccharides from chitosan, a chitosanase-producing bacterium, Bacillus sp . strain KCTC 0377BP, was isolated from soil . The bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer . The production of chitosanase was increased from 1.2 U/ml in a minimal chitosan medium to 100 U/ml by optimizing the culture conditions . The chitosanase was purified from a culture supernatant by using CM-Toyopearl column chromatography and a Superose 12HR column for fast-performance liquid chromatography and was characterized according to its enzyme properties . The molecular mass of the enzyme was estimated to be 45 kDa by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme demonstrated bifunctional chitosanase-glucanase activities, although it showed very low glucanase activity, with less than 3% of the chitosanase activity . Activity of the enzyme increased with an increase of the degrees of deacetylation (DDA) of the chitosan substrate . However, the enzyme still retained 72% of its relative activity toward the 39% DDA of chitosan, compared with the activity of the 94% DDA of chitosan . The enzyme produced chitosan oligosaccharides from chitosan, ranging mainly from chitotriose to chitooctaose . By controlling the reaction time and by monitoring the reaction products with gel filtration high-performance liquid chromatography, chitosan oligosaccharides with a desired oligosaccharide content and composition were obtained . In addition, the enzyme was efficiently used for the production of low-molecular-weight chitosan and highly acetylated chitosan oligosaccharides . A gene (csn45) encoding chitosanase was cloned, sequenced, and compared with other functionally related genes . The deduced amino acid sequence of csn45 was dissimilar to those of the classical chitosanase belonging to glycoside hydrolase family 46 but was similar to glucanases classified with glycoside hydrolase family 8 . Rhizobium leguminosarum Has a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family. A. H. F. Hosie, 2002.Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (BraRl) . Characterization of the solute specificity of BraRl shows it to be the second general amino acid permease of R . leguminosarum . Although BraRl has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (L-glutamate, L-arginine, and L-histidine), in addition to neutral amino acids (L-alanine and L-leucine) . While amino and carboxyl groups are required for transport, solutes do not have to be  -amino acids . Consistent with this, BraRl is the first ABC transporter to be shown to transport
 -aminobutyric acid (GABA) . All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily . Also, transport by BraRl does not appear to be stereospecific as D amino acids cause significant inhibition of uptake of L-glutamate and L-leucine . Unlike all other solutes tested, L-alanine uptake is not dependent on solute binding protein BraCRl . Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRl membrane complex during L-alanine uptake . Overall, the data indicate that BraRl is a general amino acid permease of the HAAT family . Furthermore, BraRl has the broadest solute specificity of any characterized bacterial amino acid transporter .
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