Enhancement of the activity and pH-performance of chitosanase from Bacillus cereus strains by DNA shuffling.
DNA shuffling was carried out with two chitosanase genes belonging to glycoside hydrolase family eight from Bacillus cereus KNUC51 and B. cereus KNUC55. The shuffled products, YM18 and YM20, which showed higher activity than the parents at 40 degrees C, were selected for further studies. The 50 kDa chitosanases were purified using affinity chromatography with glutathione-Sepharose 4B. In general, the specific activity of YM18 is enhanced 250% and that of YM20 is 350% compared to the parents. YM20 exhibits a shift of the optimal pH level from 5.5 to 6.5. DNA sequence analysis revealed that YM18 and YM20 contained 2 amino acid substitutions (I13T and A87V for YM18; K66R and N352S for YM20). We presumed that these amino acid substitutions increase the specific activity and change the property of the two variants.
Park
Yu-Mi
YM
Department of Microbiology, College of Natural Sciences, Kyungpook National University, #1370 Sankyug-Dong, Buk-Gu, Daegu 702-701, Korea.
Ghim
Sa-Youl
SY
D019943:Amino Acid Substitution / Q000235:genetics; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D032621:DNA Shuffling; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D017422:Sequence Analysis, DNA; D013696:Temperature
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; C019868:chitosanase