Identification of key amino acid sites for pH stability of GH46 family chitosanase
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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。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.
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Key words:
- Chitosanase /
- Key amino acids /
- Sequence alignment /
- pH tolerance /
- Homology modeling
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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 P68AGCCCGTCACCCAATGCCTCGACATATCCATA P68A下游引物
Rv P68ATATGGATATGTCGAGGCATTGGGTGACGGGC A137G上游引物
Fw A137GCGAAATTCCTTATCATTTCCAAGCGACTTCCAGGCA A137G下游引物
Rv A137GTGCCTGGAAGTCGCTTGGAAATGATAAGGAATTTCG A203M上游引物
Fw A203MTTGGTGACCCGCCCATTTTTTTGTTCGTACGTTTAATCAAGGC A203M下游引物
Rv A203MGCCTTGATTAAACGTACGAACAAAAAAATGGGCGGGTCACCAA H234E上游引物
Fw H234EGTCACGGGTGTCCTCATTTGCCGGATTCATCAGATCG H234E下游引物
Rv H234ECGATCTGATGAATCCGGCAAATGAGGACACCCGTGAC 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]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 -
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