食品科学 ›› 2017, Vol. 38 ›› Issue (10): 18-23.doi: 10.7506/spkx1002-6630-201710004

• 生物工程 • 上一篇    下一篇

Pseudoalteromonas carrageenovora芳香基硫酸 酯酶突变文库热稳定性提高突变体的筛选及鉴定

乔超超,王新侠,李鹤宾,倪 辉,肖安风,朱艳冰   

  1. 1.集美大学食品与生物工程学院,福建 厦门 361021;2.厦门医学院药学系,福建 厦门 361023
  • 出版日期:2017-05-25 发布日期:2017-05-23
  • 基金资助:
    国家自然科学基金青年科学基金项目(31401632);福建省高校新世纪优秀人才支持计划项目(B15139)

Screening and Characterization of Mutant with Improved Thermostability from a Random Mutant Library of Pseudoalteromonas carrageenovora Arylsulfatase

QIAO Chaochao , WANG Xinxia , LI Hebin , NI Hui , XIAO Anfeng , ZHU Yanbing   

  1. 1. College of Food and Biological Engineering, Jimei University, Xiamen 361021, China; 2. Department of Pharmacy, Xiamen Medical College, Xiamen 361023, China
  • Online:2017-05-25 Published:2017-05-23

摘要: 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%。

关键词: 芳香基硫酸酯酶, 易错聚合酶链式反应, 热稳定性提高, 突变体性质

Abstract: 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. M4-153 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%.

Key words: arylesterase, error-prone PCR, improved thermostability, mutant property

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