Cyclodextrin glycosyltransferase encoded by a gene of Paenibacillus azotofixans YUPP-5 exhibited a new function to hydrolyze polysaccharides with β-1,4 linkage

• Published in: Enzyme and microbial technology 2012-02, Vol.50 (2), p.151-157
• Main Authors: Zhou, Yi, Lee, Yong-Seok, Park, In-Hye, Sun, Zheng-xiang, Yang, Ting-xian, Yang, Pei, Choi, Yong-Lark, Sun, Ming
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acids; Amorphophallus konjac; bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Base Sequence; Biotechnology; carboxymethylcellulose; chitin; Cloning, Molecular; Culture Media; Cyclodextrin glycosyltransferase; cyclodextrins; Cyclodextrins - metabolism; cyclomaltodextrin glucanotransferase; Enzyme Stability; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fosmid library; galactomannans; gene expression; genes; glucomannans; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - isolation & purification; Glucosyltransferases - metabolism; Hydrolysis; Konjac glucomannan; Mannans - chemistry; Mannans - metabolism; Molecular Sequence Data; open reading frames; Paenibacillus - classification; Paenibacillus - enzymology; Paenibacillus - genetics; Paenibacillus - growth & development; Paenibacillus azotofixans; Polysaccharides - chemistry; Polysaccharides - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; rhizosphere; ribosomal DNA; sequence analysis; Sequence Analysis, DNA; signal peptide; Substrate Specificity; Thin layer chromatography; trisaccharides; xylan; Zymogram
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2011.12.001

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 β-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 β-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.