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GH46家族壳聚糖酶酸碱耐受性的关键氨基酸位点的鉴定

程伊梦 孙慧慧 刘淇 赵玲 曹荣

程伊梦, 孙慧慧, 刘淇, 赵玲, 曹荣. GH46家族壳聚糖酶酸碱耐受性的关键氨基酸位点的鉴定[J]. 南方水产科学. doi: 10.12131/20210290
引用本文: 程伊梦, 孙慧慧, 刘淇, 赵玲, 曹荣. GH46家族壳聚糖酶酸碱耐受性的关键氨基酸位点的鉴定[J]. 南方水产科学. doi: 10.12131/20210290
CHENG Yimeng, SUN Huihui, LIU Qi, ZHAO Ling, CAO Rong. Identification of key amino acid sites for stability of GH46 family chitosanase[J]. South China Fisheries Science. doi: 10.12131/20210290
Citation: CHENG Yimeng, SUN Huihui, LIU Qi, ZHAO Ling, CAO Rong. Identification of key amino acid sites for stability of GH46 family chitosanase[J]. South China Fisheries Science. doi: 10.12131/20210290

GH46家族壳聚糖酶酸碱耐受性的关键氨基酸位点的鉴定

doi: 10.12131/20210290
基金项目: 国家重点研发计划“蓝色粮仓科技创新”重点专项 (2019YFD0901902)
详细信息
    作者简介:

    程伊梦 (1997—),女,硕士研究生,研究方向为壳聚糖酶的挖掘及改造。E-mail: 2804810307@qq.com

    通讯作者:

    孙慧慧 (1988—),女,副研究员,博士,从事水产动物精深加工与综合利用研究。E-mail: sunhh@ysfri.ac.cn

  • 中图分类号: TQ 936.2

Identification of key amino acid sites for stability of GH46 family chitosanase

  • 摘要: 壳寡糖具有多种生物活性,是目前仅知的唯一碱性寡糖,在食品、农业和生物医药都有广泛的应用。壳聚糖酶可以特异性切割壳聚糖中的β-1,4糖苷键,形成不同聚合度的壳寡糖,因此,获得具有良好稳定性的壳聚糖酶是大规模酶法制备壳寡糖的关键。为了鉴定影响糖苷水解酶 (Glycoside hydrolase, GH) 46家族壳聚糖酶酸碱耐受性的相关氨基酸位点,该研究选取来自芽孢杆菌 (Bacillus sp.) DAU101 (最适pH为7.5) 的壳聚糖酶为模板,以来自芽孢杆菌的壳聚糖酶Csn-BAC为研究对象,综合同源建模和序列比对的方法,选取了4个候选位点并构建了4个突变体 (V1: P68A; V2: A137G; V3: A203M; V4: H234E)。结果显示,与Csn-BAC相比,4个突变体的热稳定均出现了不同程度的降低,而酸碱耐受性有了明显的提升。该结果表明,选取的氨基酸位点对酸碱耐受性均产生了显著的影响,同时表明该方法在改造壳聚糖酶稳定性方面是一条有前途的途径。
  • 图  1  Csn-BAC生物信息学分析

    Csn-BAC同源模型 (a) 及其突变位点 (b);(c) Csn-BAC与GH46家族壳聚糖酶的多序列比对。

    Figure  1.  Bioinformatics analysis of Csn-BAC

    Homology model of Csn-BAC (a) and its mutation sites (b); Multiple sequence alignment of Csn-BAC and GH46 family chitosanases.

    图  2  Csn-BAC突变体的SDS-PAGE分析

    M. 标准蛋白Marker;1—3. Csn-BAC粗酶、Csn-BAC穿出液、Csn-BAC纯酶;4—6. V1粗酶、V1穿出液、V1纯酶;7—9. V2粗酶、V2穿出液、V2纯酶;10—12. V3粗酶、V3穿出液、V3纯酶;13—15. V4粗酶、V4穿出液、V4纯酶。

    Figure  2.  SDS-PAGE analysis of Csn-BAC variants

    M. Protein molecular weight markers (6.5~270 kDa); 1. Whole cell lysate of Csn-BAC; 2. Eluate component of whole cell lysate of Csn-BAC; 3. Purified Csn-BAC; 4. Whole cell lysate of V1; 5. Eluate component of whole cell lysate of V1; 6. Purified V1; 7. Whole cell lysate of V2; 8. Eluate component of whole cell lysate of V2; 9. Purified V2; 10. Whole cell lysate of V3; 11. Eluate component of whole cell lysate of V3; 12. Purified V3; 13. Whole cell lysate of V4; 14. Eluate component of whole cell lysate of V4; 15. Purified V4.

    图  3  Csn-BAC突变体酶活分析

    Figure  3.  Enzyme specific activity analysis of Csn-BAC variants

    图  4  Csn-BAC突变体酶学性质分析

    (a) Csn-BAC突变体最适反应温度;(b) Csn-BAC突变体温度稳定性;(c~f) 突变体V1~V4最适反应pH;(g~j) 突变体V1~V4 pH稳定性。

    Figure  4.  Enzymatic property Csn-BAC variants

    Effect of temperature on variants activity (a) and stability (b); (c-f) The optimum pH of V1, V2, V3, V4, respectively; (g-j) pH stability of V1, V2, V3, V4, respectively.

    表  1  定点突变引物

    Table  1.   Primers for directed evolution

    引物
    Primer name
    序列 (5'—3')
    Sequence
    P68A上游引物
    Fw P68A
    gcccgtcacccaatgcctcgacatatccata
    P68A下游引物
    Rv P68A
    tatggatatgtcgaggcattgggtgacgggc
    A137G上游引物
    Fw A137G
    cgaaattccttatcatttccaagcgacttccaggca
    A137G下游引物
    Rv A137G
    tgcctggaagtcgcttggaaatgataaggaatttcg
    A203M上游引物
    Fw A203M
    ttggtgacccgcccatttttttgttcgtacgtttaatcaaggc
    A203M下游引物
    Rv A203M
    gccttgattaaacgtacgaacaaaaaaatgggcgggtcaccaa
    H234E上游引物
    Fw H234E
    gtcacgggtgtcctcatttgccggattcatcagatcg
    H234E下游引物
    Rv H234E
    cgatctgatgaatccggcaaatgaggacacccgtgac
    下载: 导出CSV

    表  2  Csn-BAC及其突变体反应动力学参数

    Table  2.   Reaction kinetic parameters of chitosanase Csn-BAC and its mutants

    酶 
    Enzyme 
    米氏常数
    Km/ (mg·mL−1)
    周转率
    Kcat/s−1
    催化效率
    Kcat/Km/[mL·(mg·s−1) −1]
    野生型 Csn-BAC7.2555676.65
    突变体 V13.68307.2283.37
    突变体 V23.13409.49130.66
    突变体V37.27540.8874.39
    突变体 V43.16322.68102.19
    下载: 导出CSV
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  • 收稿日期:  2021-10-09
  • 修回日期:  2021-12-01
  • 录用日期:  2021-12-15
  • 网络出版日期:  2021-12-09

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