GH46家族壳聚糖酶影响产物聚合度的关键氨基酸挖掘及定向改造

Chitosanase, catalyzing the hydrolysis of chitosan, could cleave β-1,4 linkage to produce chitooligosaccharides (COSs), which have potential applications in food, pharmaceutical, and agriculture owing to their unique biological activities. COSs with different degrees of polymerization (DP) have been shown to have great effects on their qualities and applications, so preparing COS with specific DP by enzymatic methods and elucidating the catalytic mechanism are significant. Based on these aims, the chitosanases belonging to family GH46 were selected as the research object, and the method of phylogenetic analysis was used to investigate the diversity of their sequences in this study. Identification of the key amino acids of chitosanases belonging to family GH46 influenced their product specificity was conducted. Combined with the existing research of related chitosanases in family GH46, analysis of their sequence characteristics by biological informatics technology will be preformed to position the residues and their combinations in the structure of catalytic activity. And then these amino acid residues will be mutated to verify their influence on the DPs of products. With the assist of protein structures of chitosanases in family GH46, rational design, within the region of functional residues and their combinations of the recombinant chitosanases, will be performed to prepare the COSs with specific DPs. This research will promote the exploring and modification of chitosanase resources in family GH46, establish the enzymatic technologies for the preparation of COSs with specific DPs and high-value utilization of chitosan.

壳聚糖酶降解壳聚糖生成的壳寡糖具有独特的生物活性，是目前仅知唯一的碱性寡糖，在食品、医药、农业等领域有广泛的应用。不同聚合度壳寡糖对其功能和性质有很大影响，因此酶法获得特定聚合度壳寡糖并阐明其催化机制具有重要意义。基于此，本课题以GH46家族壳聚糖酶为研究对象，以系统进化分析的方法调研其序列多样性，开展GH46家族壳聚糖酶影响产物聚合度的关键氨基酸挖掘及定向改造研究。通过生物信息学手段分析其序列特征，结合GH46家族壳聚糖酶相关数据，分析定位催化活性架构中功能区的残基及其组合，对影响产物聚合度的关键氨基酸位点进行鉴定；再辅助以蛋白质结构对其进行酶分子改造，利用已克隆模板对功能区残基及其组合进行理性设计，制备特定聚合度壳寡糖。本研究将为GH46家族壳聚糖酶资源的开发与改造、特定聚合度壳寡糖的酶法制备及壳聚糖的高值化利用提供重要的理论依据和技术平台。

壳聚糖酶降解壳聚糖生成的壳寡糖具有独特的生物活性，是目前仅知唯一的碱性寡糖，在食品、医药、农业等领域有广泛的应用。不同聚合度壳寡糖对其功能和性质有很大影响，因此酶法获得特定聚合度壳寡糖并阐明其催化机制具有重要意义。但目前可用于高质量壳寡糖制备的壳聚糖酶还没有商品化，并且所得壳寡糖聚合度难以控制。基于此，本课题以GH46家族壳聚糖酶为研究对象，以系统进化分析的方法调研其序列多样性，开展了GH46家族壳聚糖酶影响产物聚合度的关键氨基酸挖掘及定向改造研究。（1）创建了基于系统进化分析预测产物特异性的方法，并成功用于构建多样性GH46家族壳聚糖酶资源库；（2）获得了针对不同聚合度壳寡糖制备的壳聚糖酶，为壳聚糖资源的高值化利用奠定了基础；（3）完成了壳聚糖酶Csn-PD的蛋白结晶和结构解析，确定了酶的结合口袋和催化中心；（4）筛选并鉴定了影响产物聚合度的关键氨基酸位点，获得了活性提高的人工设计酶。本课题为GH46家族壳聚糖酶资源的开发与改造、特定聚合度壳寡糖的酶法制备及壳聚糖的高值化利用提供了重要的理论依据和技术平台；同时为海洋多糖资源的高值化利用、研发特定聚合度海洋寡糖定制技术奠定了坚实的基础。