Centre d'Étude et de Valorisation de la Diversité Microbienne, Département de Biologie, Faculté des Sciences, Université de Sherbrooke, 2500 boulevard de l'Université, Sherbrooke J1K 2R1 Québec, Canada.
Appl Microbiol Biotechnol. 2013 Jul;97(13):5801-13. doi: 10.1007/s00253-012-4483-4. Epub 2012 Oct 18.
Chitosan raises a great interest among biotechnologists due to its potential for applications in biomedical or environmental fields. Enzymatic hydrolysis of chitosan is a recognized method allowing control of its molecular size, making possible its optimization for a given application. During the industrial hydrolysis process of chitosan, viscosity is a major problem; which can be circumvented by raising the temperature of the chitosan solution. A thermostable chitosanase is compatible with enzymatic hydrolysis at higher temperatures thus allowing chitosan to be dissolved at higher concentrations. Following an extensive micro-plate screening of microbial isolates from various batches of shrimp shells compost, the strain 1794 was characterized and shown to produce a thermostable chitosanase. The isolate was identified as a novel member of the genus Paenibacillus, based on partial 16S rDNA and rpoB gene sequences. Using the chitosanase (Csn1794) produced by this strain, a linear time course of chitosan hydrolysis has been observed for at least 6 h at 70 °C. Csn1794 was purified and its molecular weight was estimated at 40 kDa by SDS-PAGE. Optimum pH was about 4.8, the apparent Km and the catalytic constant kcat were 0.042 mg/ml and 7,588 min⁻¹, respectively. The half-life of Csn1794 at 70 °C in the presence of chitosan substrate was >20 h. The activity of chitosanase 1794 varied little with the degree of N-acetylation of chitosan. The enzyme also hydrolyzed carboxymethylcellulose but not chitin. Chitosan or cellulose-derived hexasaccharides were cleaved preferentially in a symmetrical way ("3+3") but hydrolysis rate was much faster for (GlcN)₆ than (Glc)₆. Gene cloning and sequencing revealed that Csn1794 belongs to family 8 of glycoside hydrolases. The enzyme should be useful in biotechnological applications of chitosan hydrolysis, dealing with concentrated chitosan solutions at high temperatures.
壳聚糖因其在生物医学或环境领域的应用潜力而引起了生物技术专家的极大兴趣。壳聚糖的酶解是一种公认的控制其分子量的方法，可以对其进行优化以适应特定的应用。在壳聚糖的工业水解过程中，粘度是一个主要问题；可以通过提高壳聚糖溶液的温度来解决。耐热壳聚糖酶与高温下的酶解相容，从而可以使壳聚糖在更高的浓度下溶解。在对来自不同批次虾壳堆肥的微生物分离物进行广泛的微孔板筛选后，对菌株 1794 进行了表征，并证明其产生耐热壳聚糖酶。根据部分 16S rDNA 和 rpoB 基因序列，该分离物被鉴定为新的短小芽孢杆菌属成员。使用该菌株产生的壳聚糖酶（Csn1794），在 70°C 下至少 6 小时观察到壳聚糖水解的线性时间过程。Csn1794 已被纯化，其分子量通过 SDS-PAGE 估计为 40 kDa。最适 pH 值约为 4.8，表观 Km 和催化常数 kcat 分别为 0.042 mg/ml 和 7,588 min⁻¹。在存在壳聚糖底物的情况下，Csn1794 在 70°C 下的半衰期>20 小时。壳聚糖酶 1794 的活性随壳聚糖的 N-乙酰化程度变化不大。该酶还水解羧甲基纤维素，但不水解几丁质。壳聚糖或纤维素衍生的六糖优先以对称方式（“3+3”）切割，但（GlcN）₆的水解速率比（Glc）₆快得多。基因克隆和测序表明，Csn1794 属于糖苷水解酶家族 8。该酶在高温下处理浓缩的壳聚糖溶液的壳聚糖水解生物技术应用中应该是有用的。
