| 摘要: |
| 几丁质脱乙酰酶（Chitin deacetylase, CDA）是一种重要的生物催化剂，能够在温和条件下将几丁质转化为具有更高生物活性的壳聚糖。然而，目前成功实现异源表达的CDA酶种类相对较少，这限制了对这类酶的深入研究和应用开发。本研究从实验室前期获得的几丁质降解菌Chitinilyticum aquatile CSC-1中挖掘了一个新颖的几丁质脱乙酰酶CaCDA1。利用PCR技术扩增上述几丁质脱乙酰酶及其截短体CaCDA1_CD的基因，构建了重组质粒，并转入E. coli BL21-CodonPlus(DE3)-RIPL中诱导表达。通过对硝基乙酰苯胺法确定了粗酶的最适温度为45 ℃，最适pH为5.0，其中CaCDA1的粗酶活为7.70±0.47 U/mL，CaCDA1_CD的粗酶活为6.12±0.57 U/mL，为进一步研究该几丁质脱乙酰酶提供了条件。 |
| 关键词: 几丁质脱乙酰酶 截短体 克隆 诱导表达 酶活 |
| DOI： |
| 投稿时间：2024-08-14修订日期：2025-02-19 |
| 基金项目: |
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| Table 1 Primers for PCR amplification |
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| Abstract: |
| Chitin deacetylase (CDA) is an important biocatalyst that can convert chitin into chitosan with higher biological activity under mild conditions. However, the number of CDA enzymes successfully expressed heterologously were relatively limited, which restricts the in-depth study and application development of these enzymes.. In this study, a novel chitin deacetylase, CaCDA1, was mined from the chitin-degrading bacterium Chitinilyticum aquatile CSC-1, which was obtained in our laboratory's previous work. The above chitin deacetylase gene and its truncated gene CaCDA1_CD were amplified using the PCR technology, and recombinant plasmids were constructed and transformed into E. coli BL21-CodonPlus(DE3)-RIPL for induced expression The optimal temperature of the crude enzyme was determined to be 45 ℃ and the optimal pH was 5.0 using the p-nitroacetanilide method. The crude enzyme activity of CaCDA1 was 7.70 ± 0.47 U/mL, while that of CaCDA1_CD was 6.12 ± 0.57 U/mL. These results provide the foundation for further studies on the chitin deacetylase. |
| Key words: Chitin deacetylase Truncated mutant Clone Induced expression Enzyme activity |
