Identification of key amino acid sites for 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。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 stability of GH46 family chitosanases, the chitosanase from Bacillus sp. DAU101 (optimum pH 7.5) was selected as template and the chitosanase Csn-BAC from Bacillus sp. MD-5 as 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 tolerance was significantly improved. These results indicate that the selected amino acid sites have an obvious effect on pH tolerance, and this method is a promising 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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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 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')
SequenceP68A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 
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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-BAC 7.25 556 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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