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橙黄小单孢菌 (Micromonospora aurantiaca) 几丁质酶基因的克隆、表达、鉴定及应用

王逗 游锦若 申铉日 李永成 夏光华 何燕富 张雪莹

王逗, 游锦若, 申铉日, 李永成, 夏光华, 何燕富, 张雪莹. 橙黄小单孢菌 (Micromonospora aurantiaca) 几丁质酶基因的克隆、表达、鉴定及应用[J]. 南方水产科学. doi: 10.12131/20230029
引用本文: 王逗, 游锦若, 申铉日, 李永成, 夏光华, 何燕富, 张雪莹. 橙黄小单孢菌 (Micromonospora aurantiaca) 几丁质酶基因的克隆、表达、鉴定及应用[J]. 南方水产科学. doi: 10.12131/20230029
WANG Dou, YOU Jinruo, SHEN Xuanri, LI Yongcheng, XIA Guanghua, HE Yanfu, ZHANG Xueying. Cloning, expression and application of a chitinase gene from Micromonospora aurantiaca[J]. South China Fisheries Science. doi: 10.12131/20230029
Citation: WANG Dou, YOU Jinruo, SHEN Xuanri, LI Yongcheng, XIA Guanghua, HE Yanfu, ZHANG Xueying. Cloning, expression and application of a chitinase gene from Micromonospora aurantiaca[J]. South China Fisheries Science. doi: 10.12131/20230029

橙黄小单孢菌 (Micromonospora aurantiaca) 几丁质酶基因的克隆、表达、鉴定及应用

doi: 10.12131/20230029
基金项目: 海南省自然科学基金项目 (320QN203,322RC587);海南大学科研启动基金项目 [KYQD (ZR) 20046]
详细信息
    通讯作者:

    张雪莹 (1989—),女,讲师,博士,研究方向为水产品加工与贮藏。E-mail: 994257@hainanu.edu.cn

  • 中图分类号: Q556.2

Cloning, expression and application of a chitinase gene from Micromonospora aurantiaca

  • 摘要: 从海洋微生物橙黄小单孢菌 (Micromonosprra aurantiaca) 的基因组DNA中克隆到一条新型的碳水化合物18家族几丁质酶基因Machi3,并成功在大肠杆菌 (Escherichia coli) 中表达。该酶的最适反应温度和pH分别为55  ℃和7.0,在低于55  ℃和pH 6.0~9.0范围内稳定性较好。镁离子 (Mg2+)、钙离子 (Ca2+)、吐温40 (Tween 40) 和吐温80 (Tween 80) 对MaChi3的酶活力有轻微地促进作用。该重组酶对α-几丁质、胶体几丁质、虾壳粉、不同脱乙酰度 (50%~95%) 的壳聚糖、淀粉及纤维素均具有水解活性,其中以胶体几丁质为底物时酶活力最高 (2.24 U·mg−1)。由扫描电镜结果可知,几丁质经盐酸 (HCl) 预处理后得到的胶体几丁质纤维结构变的松散,更有利于MaChi3的水解作用。以胶体几丁质为底物时动力学参数KmVmax值分别为5.93 mg·mL−1和8.58 μmol·(min·mg)−1。此外,胶体几丁质经MaChi3酶解后生成的主产物为N, N-二乙酰基壳二糖,产率 (几丁质) 为285.54 mg·g−1。该酶展现出良好的酶学特性,为其在几丁质资源的开发和应用中奠定了基础。
  • 图  1  MaChi3基因PCR扩增产物 (a)、保守域分析 (b) 和部分催化域氨基酸多序列比对 (c)

    注:(a)图中 M为DNA分子量标准;(c)图中列出的几丁质酶氨基酸序列来源于Bacillus circulans WL-12 (PDB ID:1ITX),C. shinanonensis (PDB ID:6KXL),B. thuringiensis (PDB ID:6BT9),Paenibacillus sp. FPU-7 (PDB ID:5GZU) 和L. lactis ssp. Lactis (NCBI登录号AAK06048)。保守基序SxGG和DxDxE在底部用带下划线的红色字母标识,保守的酪氨酸残基在上部用红色※标识。

    Figure  1.  PCR products (a), conserved domain analysis (b) and sequence alignment of partial predicted catalytic domain  (c) of MaChi3

    Note: M is DNA molecular mass standard in Fig. 1 (a). The listed sequences in Fig. 1 (c) include the chitinases from Bacillus circulans WL-12 (PDB ID: 1ITX), C. shinanonensis (PDB ID: 6KXL), B. thuringiensis (PDB ID: 6BT9), Paenibacillus sp. FPU-7 (PDB ID: 5GZU) and L. lactis ssp. Lactis (NCBI accession NO.: AAK06048). The conserved sequence motifs SxGG and DxDxE are showed at the bottom with red color underlined, and the conserved tyrosine residue is indicated by a red asterisk.

    图  2  重组酶MaChi3的SDS-PAGE分析

    M. 蛋白分子量标准。

    Figure  2.  SDS-PAGE analysis of purified recombinant chitinase MaChi3

    M. Protein molecular mass standard.

    图  3  重组几丁质酶MaChi3的最适反应温度 (a) 及热稳定性 (b)

    Figure  3.  Optimal temperature (a) and thermal stability (b) of recombinant chitinase MaChi3

    图  4  重组酶MaChi3的最适反应pH (a) 及pH稳定性 (b)

    Figure  4.  Optimal pH (a) and pH stability (b) of recombinant chitinase MaChi3

    图  5  重组酶MaChi3的底物特异性

    Figure  5.  Substrate specificity of recombinant chitinase MaChi3

    图  6  α-几丁质 (a) 和胶体几丁质 (b) 以及MaChi3酶解后的α-几丁质 (c) 和胶体几丁质 (d) SEM图

    Figure  6.  SEM images of α-chitin (a), colloidal chitin (c), MaChi3-hydrolyzed α-chitin (b) and MaChi3-hydrolyzed colloidal chitin (d)

    图  7  胶体几丁质水解产物分析 (a) 和 (GlcNAc)1-6标准品及水解产物的HPLC图 (b)

    Figure  7.  Hydrolysis products of colloidal chitin (a) and HPLC spectra of (GlcNAc)1-6 stands and hydrolysis products (b)

    表  1  化学试剂和金属离子对重组酶MaChi3酶活力的影响

    Table  1.   Effects of metal ions and chemical reagents on activity of recombinant chitinase MaChi3

    化学试剂 Chemical reagent终浓度/体积分数Final concentration相对酶活力 Relative activity/%
    对照 Control 100
    尿素 Urea 200 mmol·L−1 70.9±0.8
    乙二胺四乙酸 EDTA 10 mmol·L−1 94.2±1.6
    十二烷基硫酸钠 SDS 10 mmol·L−1 58.1±0. 5
    吐温20 Tween 20 1% 90.4±0.8
    吐温40 Tween 40 1% 104.1±1.6
    吐温60 Tween 60 1% 79.6±1.0
    吐温80 Tween 80 1% 108.4±0.8
    曲拉通 X-100 Triton X-100 1% 101.0±0.9
    钾离子 K+ 1 mmol·L−1 86.4±0.7
    银离子 Ag+ 1 mmol·L−1 43.3±0.4
    镁离子 Mg2+ 1 mmol·L−1 117.7±0.7
    亚铁离子 Fe2+ 1 mmol·L−1 65.4±0.7
    钙离子 Ca2+ 1 mmol·L−1 107.3±0.4
    钴离子 Co2+ 1 mmol·L−1 87.2±0.7
    钡离子 Ba2+ 1 mmol·L−1 99.5±1.3
    锌离子 Zn2+ 1 mmol·L−1 97.6±1.1
    铜离子 Cu2+ 1 mmol·L−1 45.0±0.7
    铁离子 Fe3+ 1 mmol·L−1 59.3±1.1
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  • 收稿日期:  2023-03-01
  • 修回日期:  2023-04-07
  • 录用日期:  2023-05-10
  • 网络出版日期:  2023-05-15

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