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

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

程伊梦^{1, 2,},
孙慧慧^1, ,,
刘淇¹,
赵玲¹,
曹荣¹

1.
中国水产科学研究院黄海水产研究所，山东青岛 266071
2.
中国海洋大学食品科学与工程学院，山东青岛 266003

基金项目: 国家重点研发计划项目 (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
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- 文章访问数: 185
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- 被引次数: 0
出版历程
- 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
- 刊出日期: 2022-04-01

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

CHENG Yimeng^{1, 2
,},
SUN Huihui^{1
, ,},
LIU Qi¹,
ZHAO Ling¹,
CAO Rong¹

1.
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
2.
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

摘要

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、农业和生物医药领域应用广泛。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，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个突变体的热稳定性均出现了不同程度的下降，而酸碱耐受性有了明显的提升。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，同时表明该策略在改造壳聚糖酶稳定性方面是一种有效的方法。
- 壳聚糖酶 /
- 关键氨基酸位点 /
- 序列比对 /
- 酸碱耐受性 /
- 同源建模
Abstract: Chitooligosaccharides, which have a variety of biological activities, are the only known basic oligosaccharide widely used in food, agriculture and biomedicine. Chitosanases can cleave the β-1,4 glycosidic bonds in chitosan specifically to form chitooligosaccharides with different degrees of polymerization. Therefore, obtaining chitosanases with good stability is the key for the large-scale enzymatic preparation of chitooligosaccharides. In order to identify the amino acid sites affecting the pH stability of GH46 family chitosanases, the chitosanase from Bacillus sp. DAU101 (optimal pH 7.5) was selected as template and the chitosanase Csn-BAC from Bacillus sp. MD-5 as the research object. By combining homology modeling and sequence alignments, four candidate sites were selected, and the corresponding mutants were obtained (V1: P68A; V2: A137G; V3: A203M; V4: H234E). Compared with Csn-BAC, the thermal stabilities of four mutants showed varying degrees of reduction, while the pH stability was significantly improved. These results indicate that the selected amino acid sites have an obvious effect on pH tolerance, and this strategy is an effective way to modify the stability of chitosanase.
- Chitosanase /
- Key amino acids /
- Sequence alignment /
- pH tolerance /
- Homology modeling

HTML全文

图 1 Csn-BAC生物信息学分析

Figure 1. Bioinformatics analysis of Csn-BAC

下载: 全尺寸图片幻灯片

图 2 Csn-BAC突变体的SDS-PAGE分析

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

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图 3 Csn-BAC突变体酶活分析

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

下载: 全尺寸图片幻灯片

图 4 Csn-BAC突变体酶学性质分析

Figure 4. Enzymatic property of Csn-BAC variants

下载: 全尺寸图片幻灯片

表 1 定点突变引物

Table 1. Primers for directed evolution

引物 Primer name	序列 (5'—3') Sequence (5'—3')
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 K_m/(mg·mL⁻¹)	周转率 K_cat/s⁻¹	催化效率 K_cat/K_m/[mL·(mg·s)⁻¹]
野生型 Csn-BAC	7.25	556.00	76.65
突变体 V1	3.68	307.22	83.37
突变体 V2	3.13	409.49	130.66
突变体 V3	7.27	540.88	74.39
突变体 V4	3.16	322.68	102.19

下载: 导出CSV

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