TAILIEUCHUNG - Báo cáo khoa học: Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase

We have obtained two types of thermostable mutant lactate oxidase – one that exhibited an E-to-G point mutation at position 160 (E160G)through error-prone PCR-based random mutagenesis, and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 (E160G/V198I)through DNA shuffling-based random mutagenesis – both of which we have previously reported. | Eur. J. Biochem. 270 3628-3633 2003 FEBS 2003 doi Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase Hirotaka Minagawa1 Jiro Shimada1 and Hiroki Kaneko2 1 Fundamental Research Laboratories NEC Corp. Tsukuba Japan department of Applied Physics College of Humanities and Sciences Nihon University Tokyo Japan We have obtained two types of thermostable mutant lactate oxidase - one that exhibited an E-to-G point mutation at position 160 E160G through rrror-prone PCR-bamd random mutagenesis and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 E160G V198I through DNA shui lling-baeed random mutagenesis - both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the b a 8 barrel structure but a thermal-inactivation experiment on the five kinds of single-mutant lactate oxidase at position 160 E160A E160Q E160H E160R and E160K showed that the side-chain volume ofthe amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis and analysed the thermostability of wild-type V198I E160G and E160G V198I lactate oxidase enzymes. The half-life of E160G V198I lactate oxidase at 70 C was about three times longer than that of E160G lactate oxidase and was about 20 times longer than that of wild-type lactate oxidase. In contrast the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability but does affect it when combined with the E160G mutation. Keywords lactate oxidase thermostability site-directed mutagenesis random mutagenesis. Lactate oxidase is widely .

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