Das Poonam, Das Manisha, Sahoo Sheela Kumari, Dandapat Jagneshwar, Pradhan Jyotsnarani
Department of Biotechnology, Utkal University, Vani Vihar, Bhubaneswar 751004, Odisha, India.
Centre of Excellence Integrated Omics and Computational Biology, Utkal University, Bhubaneswar, Odisha, India.
Curr Res Microb Sci. 2024 Nov 22;8:100325. doi: 10.1016/j.crmicr.2024.100325. eCollection 2025.
Chitosan is a promising biopolymer with wide range of applications. It is the deacetylated product of chitin. Commercially, it is produced from chitin via a harsh thermochemical process that has several shortcomings and heterogenous deacetylation product. Chitin can be transformed into chitosan through enzymatic deacetylation using chitin deacetylase (CDA), enabling the production of chitosan with a specific degree of deacetylation. CDA is primarily extracted from fungi followed by bacteria and insects. The extraction of CDA from fungus is more complex, possess several health risks for human including skin lesions. Therefore, screening of potent bacterial CDA is the need of the hour. In this study, for the first time we have isolated a bacterial strain Aneurinibacillus aneurinilyticus from the rinsed water of marine crab shell, and it was found to be a potent CDA producer. The extracellular CDA from has been partially purified and the specific activity of the enzyme was found to be 569.73 U/ mg protein. SDS-PAGE profiling of the purified sample depicts two isomers of CDA with molecular weights of 27 kD and 45 kD. The pH and temperature optima of the purified CDA were found to be 7.4 and 37 °C, respectively. The partially purified enzyme has Km and Vmax values of 98.455 µM and 909.09 µmole/min, for non-chitinous substrate such as p-nitroacetanilide. For chitinous substrates like glycol chitin, N-acetyl glucosamine hexamer and colloidal chitin, the enzyme exhibited K of 96.96, 111.75 and 127.86 µM, respectively, V for these substrates were 23.31, 10.12 and 10.772 µmole/min, respectively. Metal ions like Mn and Mg considerably boost the production and activity of CDA, whereas Cd and Co strongly inhibit its activity. Insights from this study further substantiate that this enzyme follows Michaelis-Menten equation and has potential for industrial applications.
壳聚糖是一种具有广泛应用前景的生物聚合物。它是几丁质的脱乙酰产物。在商业上，它是通过一种苛刻的热化学过程从几丁质中生产出来的，该过程存在几个缺点且脱乙酰产物不均一。几丁质可以通过使用几丁质脱乙酰酶（CDA）进行酶促脱乙酰转化为壳聚糖，从而能够生产具有特定脱乙酰度的壳聚糖。CDA主要从真菌中提取，其次是细菌和昆虫。从真菌中提取CDA更为复杂，对人类有多种健康风险，包括皮肤损伤。因此，筛选高效的细菌CDA是当务之急。在本研究中，我们首次从海蟹壳冲洗水中分离出一株解硫胺素芽孢杆菌菌株，发现它是一种高效的CDA生产者。已对该菌株的胞外CDA进行了部分纯化，发现该酶的比活性为569.73 U/mg蛋白质。纯化样品的SDS-PAGE分析显示有两种分子量分别为27 kD和45 kD的CDA异构体。纯化的CDA的最适pH和温度分别为7.4和37℃。对于非几丁质底物如对硝基乙酰苯胺，部分纯化的酶的Km和Vmax值分别为98.455 µM和909.09 µmole/min。对于几丁质底物如乙二醇几丁质、N-乙酰葡糖胺六聚体和胶体几丁质，该酶的K值分别为96.96、111.75和127.86 µM，这些底物的V值分别为23.31、10.12和10.772 µmole/min。锰和镁等金属离子显著提高CDA的产量和活性，而镉和钴则强烈抑制其活性。本研究的见解进一步证实该酶遵循米氏方程，具有工业应用潜力。
