ABSTRACT
The bacteria with hydrolysis activity to glucomannan were isolated from the rhizosphere of Amorphophallus konjac through enrichment cultivation. One strain with strong activity in degrading glucomannan was identified preliminarily as Paenibacillus azotofixans YUPP-5 according to the sequence analysis of 16S rDNA. This strain is able to hydrolyze many polysaccharide with ß-1,4 linkage, including glucomannan, galactomannan, xylan, carboxymethyl cellulose, and chitin. One hydrolytic enzyme band of approximately 70 kDa was examined from the supernatants of YUPP-5 by using zymogram with mixture polysaccharides as substrate. The encoding gene had an open reading frame of 2157 bp, which deduced cyclodextrin glycosyltransferase (CGTase), including 718 amino acids with a signal peptide in the N-terminal region. When the gene was expressed in Escherichia coli BL21, the recombinant CGTase exhibited strong activity in degrading polysaccharides with ß-1,4 linkage, and in forming cyclodextrin by using carboxymethyl cellulose as substrate. This CGTase exhibited some new functions. Finally, the hydrolytic oligosaccharides from galactomannan or glucomannan were detected by thin layer chromatography. Pentasaccharide, tetrasaccharide, trisaccharide, and disaccharide could be examined as reaction time went on.
Subject(s)
Glucosyltransferases/metabolism , Paenibacillus/enzymology , Polysaccharides/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Biotechnology , Cloning, Molecular , Culture Media , Cyclodextrins/metabolism , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Glucosyltransferases/chemistry , Glucosyltransferases/genetics , Glucosyltransferases/isolation & purification , Hydrolysis , Mannans/chemistry , Mannans/metabolism , Molecular Sequence Data , Paenibacillus/classification , Paenibacillus/genetics , Paenibacillus/growth & development , Polysaccharides/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Substrate SpecificityABSTRACT
A new near-infrared (NIR) dye, 1,1'-disulfobutyl-3,3,3',3'-tetramethylindotricarbocyanine (DTCY) has been developed for the quantitation of proteins in solution. The method is based on the binding of DTCY to proteins under acidic conditions. The binding of DTCY to proteins causes a new band at 814 nm. The maximum binding number of bovine serum albumin (BSA) with DTCY was measured as 100. The linear range is 0.3-40 microg mL(-1) for BSA and human serum albumin (HSA), respectively. Except for Fe(2+), Cu(2+), and cetyltrimethylammonium bromide, all of the examined coexisting substances show no interference in the assay. The method has been applied to the quantitation of proteins in serum and urine with recoveries between 96 and 105%.