橙黄小单孢菌 (<i>Micromonospora aurantiaca</i>) 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

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

王逗¹,
游锦若¹,
申铉日^{1, 2},
李永成^{1, 2},
夏光华^{1, 2},
何燕富^{1, 2},
张雪莹^{1, 2, ,}

1.
海南大学食品科学与工程学院，海南海口 570228
2.
南海水产资源高效利用工程研究中心，海南海口 570228

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
计量
- 文章访问数: 46
- 被引次数: 0
出版历程
- 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

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

WANG Dou¹,
YOU Jinruo¹,
SHEN Xuanri^{1, 2},
LI Yongcheng^{1, 2},
XIA Guanghua^{1, 2},
HE Yanfu^{1, 2},
ZHANG Xueying^{1, 2
, ,}

1.
School of Food Science and Engineering, Hainan University, Haikou 570228, China
2.
Hainan Engineering Research Center of Aquatic Resources Efficient Utilization in South China Sea, Haikou 570228, China

摘要

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K_m和V_max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。
- 橙黄小单孢菌 (Micromonospora aurantiaca) /
- 几丁质酶 /
- 克隆表达 /
- 酶学性质 /
- N /
- N-二乙酰基壳二糖
Abstract: In this study, a novel carbohydrate 18 family chitinase gene Machi3 was cloned from the genomic DNA of marine microorganism Micromonospra aurantiaca and successfully expressed in Escherichia coli. The optimal reaction temperature and pH for MaChi3 were 55 ℃ and 7.0, respectively. MaChi3 showed good stability below 55 ℃ and at pH of 6−9. The activity of MaChi3 was slightly promoted by Mg²⁺, Ca²⁺, Tween 40 and Tween 80. The recombinant chitinase showed hydrolytic activity toward α-chitin, colloidal chitin, shrimp shell powder, chitosan (50%~95% of deacetylation), starch and cellulose, among which the highest activity of 2.24 U mg⁻¹ was observed in colloidal chitin. The results of scanning electron microscopy suggests that the fiber structure of chitin became loose after pretreatment with HCl, so it was more favorable to the hydrolysis of MaChi3. The Km and Vmax values of MaChi3 toward colloidal chitin were 5.93 mg·mL⁻¹ and 8.58 μmol·(min·mg)⁻¹, respectively. In addition, the main product of colloidal chitin hydrolyzed by MaChi3 was N, N-diacetyl chitobiose with a yield of 285.54 mg·g⁻¹. MaChi3 shows good catalytic activity, which is beneficial for its development and application in the biotransformation of chitin.
- Micromonospora aurantiaca /
- Chitinase /
- Cloning and expression /
- Enzymatic properties /
- N /
- N-diacetyl chitobiose

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.

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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.

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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

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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

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图 5 重组酶MaChi3的底物特异性

Figure 5. Substrate specificity of recombinant chitinase MaChi3

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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)

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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)

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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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