[Expression of the genes CelA and XylA isolated from a fragment of metagenomic DNA in Escherichia coli].
The glycosyl hydrolase genes cel5A and xyl3A previously isolated by ourselves within a fragment of DNA from the methagenomic library of cow rumen microflora DNA were sub-cloned and expressed in E. coli. The recombinant proteins Cel5A and Xyl3A were purified and characterized. Cellulase Cel5A belongs to the Family 5 glycosyl hydrolases and is a one-module 38.2 kDa enzyme that hydrolyses the 1,4-glycoside bonds of soluble cellulose substrates and amorphous cellulose, showing its maximal activity (31200 u/mg) on lichenan, a soluble substrate with mixed (beta-1,3-1,4) bonds. The end product of the amorphous cellulose hydrolysis is cellobiose. Cel5A is inactive toward the crystal forms of cellulose. Cel5A is an endoglucanase capable of exohydrolysis. The molecular mass of beta-xylosidase Xyl3A belonging to the Family 3 glycosyl hydrolases is 83.7 kDa. The enzyme is active only on xylooligosaccharides, with the maximal activity shown on xylobiose, the end product of the reaction being xylose. No activity on xylane was hitherto observed. Recombinant Cel5A and Xyl3A are stable over a wide range of pH and temperatures, their maximal activity being observed at pH 6.5 and at 55 degrees C.
Shedova
E N
EN
Lunina
N A
NA
Berezina
O V
OV
Zverlov
V V
VV
Schwarz
V
V
Velikodvorskaia
G A
GA
D000818:Animals; D002417:Cattle; D002480:Cellulase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004187:Disaccharides / Q000737:chemistry / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012417:Rumen / Q000201:enzymology*; D013696:Temperature
D004187:Disaccharides; D011994:Recombinant Proteins; D002482:Cellulose; C074775:CelA endoglucanase; D002480:Cellulase; D043364:Endo-1,4-beta Xylanases; C004173:xylobiose