A library of Pseudoalteromonas carrageenovora arylsulfatase mutants was constructed by random mutagenesis using error-prone PCR.After screening,one mutant strain named 4-153 was obtained whose arylsulfatase had improved thermal stability.It was found that there were two amino acid substitutions in the mutant,including D84A and H260L.When p-nitrophenyl sulfate was used as a substrate,the optimal reaction temperature for the mutant enzyme was 55 ℃.Mutant arylsulfatase 4-153 (M4-153) retained 85％,83％,48％,and 13％ of its initial activity after incubation at 45,50,55 and 60 ℃ for 30 min,respectively.Meanwhile,wild-type arylsulfatase (WT) retained 79％,68％,21％,and 1％ of its initial activity after incubation at 45,50,55 and 60 ℃ for 30 min,respectively.These results showed that M4-153 had a better thermal stability than WT.M4-15 3 had an optimum pH of 8.0,and it was stable over the pH range of 5.0-9.0.Inhibition assay with EDTA indicated that metal ions played an important role in the catalytic process of the mutant enzyme.The recombinant arylsulfatase 4-153 showed a relatively strong tolerance to some detected detergents including Triton X-100,Tween 20,Tween 80,and Chaps.The desulfuration ratio of the crude polysaccharides from Gracilaria lemaneiformis by M4-153 was 79.5％.%利用易错聚合酶链式反应技术引入随机诱变,构建一个Pseudoalteromonas carrageenovora芳香基硫酸酯酶突变体库.经过筛选,获得一个芳香基硫酸酯酶热稳定性提高的突变株4-153.序列分析表明,该突变体有2个氨基酸替换,包括D84A和H260L.以对硝基苯硫酸钾为底物,突变酶4-153 (M4-153)的最适反应温度为55℃,在45、50、55、60℃处理30 min后,M4-153分别保留85％、83％、48％和13％的残留酶活力.野生型酶(WT)在45、50、55、60℃处理30 min后,分别保留79％、68％、21％和1％的残留酶活力.M4-153与WT相比具有更好的热稳定性.M4-153的最适反应pH值为8.0,在pH 5.0～9.0范围内保持稳定.EDTA对突变酶的抑制作用表明,金属离子在突变酶的催化过程中起重要作用.M4-153对一些洗涤剂,包括Triton X-100、Tween 20、Tween 80和Chaps,有好的耐受性.M4-153对龙须菜粗多糖硫酸基团的脱硫率为79.5％.
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