Cyclodextrin glycosyltransferase encoded by a gene of Paenibacillus azotofixans YUPP-5 exhibited a new function to hydrolyze polysaccharides with β-1,4 linkage.
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.
Engineering Research Center of Wetland Agriculture in the Central Yangtze, Ministry of Education, College of Agriculture, Yangtze University, Jingzhou 434025, Hubei Province, PR China. yiyizhzh@yahoo.com.cn
Yi
Y
Zhou
Yong-Seok
YS
Lee
In-Hye
IH
Park
Zheng-xiang
ZX
Sun
Ting-xian
TX
Yang
Pei
P
Yang
Yong-Lark
YL
Choi
Ming
M
Sun
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D003470:Culture Media; D003505:Cyclodextrins / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D008351:Mannans / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003470:Culture Media; D003505:Cyclodextrins; D008351:Mannans; D011134:Polysaccharides; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase