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Site-directed Mutation of Gene Encoding Hypertherthermophiles Acid-stable α-Amy Lase from Thermococcus siculi HJ21

Author: YaoZuo
Tutor: LiHuaZhong；WangShuJun
School: Jiangnan University
Course: Microorganism
Keywords: Hyperthermophilic Archaeon Thermoactive and acid-stableα-amylase Site-directed mutagenesis Orientational manipulation
CLC: TQ925
Type: Master's thesis
Year: 2011
Downloads: 112
Quote: 0
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Abstract


α-amylase is one of the most important commercial enzymes widely used in textile, brewing, sugar and other industries. Thermostable and acid-stableα-amylases are very important for industrial uses.In order to study the molecular evolution in vitro and the relationship of structure and function of Thermococcus siculiHJ21α-amylase(TSA), the site-directed mutation library of TSA was constructed. The point mutations at the sites of 98, 109, 121, 125, 157 and 184 were individually generated by PCR amplification with the nucleotide primers, with plasmid pET28a(+)-TSA as the template. The mutation library was transformed and expressed in Escherichia coli BL21(DE3). The mutation was confirmed by DNA sequencing. From theα-amylase activity it was found that mutant K98R, A109V, Y121S, V125L and Y184H had higher activity but mutant A157V had lower activity than TSA. The multi-point mutants revealed higher enzyme activity than that of single point mutant except including mutation A157V. The mutant TSAM-23 which showed 4.75 timesα-amylase activity of TSA. Among the multi-point mutants, mutant TSAM-23 including mutation of K98R, A109V, Y121S, V125L and Y184H showed highest enzyme activity as much as 4.75 times of TSA.TSAM-23 and TSA had nearly the same characters . The optimal temperature , 90℃; the fitting reaction pH lowered to 5.0; the thermal stability had been raised by 12.8%(90℃,without Ca2+).Homologous modeling was made to the 457 amino acid sequence of TSAM-23. It’s three-dimensional structure and catalytic amino acid residues in active site were predicted subsequently.

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