Enhancement of the thermostability of a recombinant beta-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis.
Random mutagenesis was performed on beta-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K-T307I were approx. 140, 190 and 200%, respectively, of wild type beta-agarase (661 U/mg) at 40 degrees C. All three mutant enzymes were stable up to 50 degrees C and E99K-T307I had the highest thermostability. The melting temperature (Tm) of E99K-T307I, determined by CD spectra, was increased by 5.2 degrees C over that of the wild-type enzyme (54.6 degrees C). Activities of both the wild-type and E99K-T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl2. The E99K-T307I enzyme was stable at 55 degrees C with 1 mM CaCl2, reaching 260% of the activity the wild-type enzyme held at 40 degrees C without CaCl2.
Jang
Min-Kyung
MK
Department of Pharmaceutical Engineering, College of Medical Life Sciences, Silla University, Busan, 617-736, Korea.
Lee
Seung Woo
SW
Lee
Dong-Geun
DG
Kim
Nam-Young
NY
Yu
Ki Hwan
KH
Jang
Hye Ji
HJ
Kim
Suhkman
S
Kim
Andre
A
Lee
Sang-Hyeon
SH
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002122:Calcium Chloride / Q000378:metabolism; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D041965:Flavobacteriaceae / Q000201:enzymology* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D016296:Mutagenesis; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics; D020125:Mutation, Missense; D055550:Protein Stability; D044366:Transition Temperature
D001426:Bacterial Proteins; D050505:Mutant Proteins; D006026:Glycoside Hydrolases; C019624:agarase; D002122:Calcium Chloride