摘要
Abstract
Alkaline polygalacturonate lyase (PGL,E.C.4.2.2.2) is an important industrial enzyme, which can catalyze the acleavage of α-1,4 glycosidic bond in polygalacturonate.PGL has been widely used in food, textile, and papermaking industry.To improve the thermal stability, PGL Bacillus sp.WSHB04-02 was subjected to amino acid sequence alignment with three heat-resistant pectinase, and seven amino acid positions invariant in thermostable enzymes but not invariant in PGL (I58, N189, G191, S229, K314, S315 and I325).The seven amino acid residues were individually mutated to their counter parts in heat-resistant enzymes.Results showed that the thermal stabilities of all the PGL mutants were enhanced by the mutations.In contrast to the wild type enzyme, the half-life of N189E, S229K, and I325F at 55 ℃ were enhanced by 2.32, 2.11, and 1.89-fold, respectively.I58V and K314M respectively showed 19.4％ and 38.9％ enhanced half-life at 55 ℃, and their specific activity were improved by 65％ and 91％ respectively.Structure analysis indicated that all PGL mutants except S315G showed changes in quantities of several intra-molecular interactions.These results suggested that site-directed mutagenesis could effectively enhance thermal stability of PGL, and that the changes in molecular interactions might account for the improvement of enzyme properties.关键词
碱性果胶酶/氨基酸序列比对/定点突变/热稳定性/作用力Key words
alkaline polygalacturonate lyase/alignment of amino acid sequence/site-directed mutagenesis/thermal stability/interaction
