title: Enhancing the thermostability of a novel beta-agarase AgaB through directed evolution.
abstract: To increase the thermostability of beta-agarase AgaB by directed evolution, the mutant gene libraries were generated by error-prone polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) shuffling. Mutants with high thermostability were screened by a simple method based on agarase-degrading agar to generate a clear zone on the agar plate. A mutant S2 was obtained through two rounds of error-prone PCR and a single round of DNA shuffling and selection. It has higher thermostability and slightly increased catalytic activity than wild-type AgaB. Melting temperature (T(m)) of S2, as determined by circular dichroism, is 4.6 degrees C higher than that of wild-type AgaB, and the half-life of S2 is 350 min at 40 degrees C, which is 18.4-fold longer than that of the wild-type enzyme. Saturation mutagenesis and hydrophobic cluster analysis indicated that hydrophobic interaction might be the key factor that enhances the enzyme stability.
authors.affiliation: The Department of Molecular Biology, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Shi
authors.forename: Xinzhi
authors.initials: X
authors.lastname: Lu
authors.forename: Cuiping
authors.initials: C
authors.lastname: Ma
authors.forename: Yiming
authors.initials: Y
authors.lastname: Ma
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Fu
authors.forename: Wengong
authors.initials: W
authors.lastname: Yu
mesh_terms: D019020:Directed Molecular Evolution / Q000379:methods*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D013696:Temperature
chemical_list: D006026:Glycoside Hydrolases; C019624:agarase