Dickeya dadantii DCE-01菌株苎麻脱胶关键果胶酶鉴定及其理化机制

Degumming is basic and key in the process of ramie. Biological degumming represents the development direction. There are problem that the ramie gum was biodegraded halfway, and it is resulted by the mismatch between key enzymes and the ramie gum complex. Because enzyme system for ramie degumming refers to various enzymes different in property, structure and function, it is hard to fit the ramie gum. Pectinase as the initiating factor is one of most critical enzyme. Therefore, it is very necessary to identify the key pectinase for ramie degumming and clarify their physical and chemical mechanism. An efficient strain Dickeya dadantii DCE-01 for ramie bio-degumming had been selected in our laboratory, and nine extracellular pectinase were obtained by prokaryotic expression. Furthermore, two dominant pectate lyases of Pel419 and PelG403 were selected using ramie pectin as the substrate. Based on the above results, this project is to knockout the pactate lyase genes pel419 and pelG403 of D. dadantii DCE-01 by homologous recombination, construct engineering mutations of Δpel419 and ΔpelG403; Comparing the difference between the wild type and mutants of ramie degumming, the key pecate lyases were verified; Chemical modification and amino acid substitution analyze the key amino acid sites or peptides of pectate lyases, reveal the matching law of the key amino acids and ramie gum degradation.

脱胶是苎麻加工过程中基础而关键的环节，生物脱胶是其发展方向。现有苎麻生物脱胶技术存在胶质去除不彻底的问题，究其原因是脱胶酶种类多，结构与功能差异大，难以找到与苎麻胶质复合体匹配的关键酶系。果胶酶作为前期引发因子是脱胶关键酶。因此，筛选苎麻脱胶关键果胶酶及明确其理化机制十分必要。实验室前期选育到脱胶高效菌株Dickeya dadantii DCE-01，通过原核表达获得了9种有生物活性的胞外果胶酶，用苎麻果胶做底物，从中筛选到优势果胶裂解酶Pel419和PelG403。本项目拟在上述基础上，采用同源重组的方法敲除果胶裂解酶基因pel419和pelG403，构建突变工程菌株Δpel419和ΔpelG403。通过比较野生型与突变菌株的苎麻脱胶效果差异，验证脱胶关键果胶裂解酶，再结合化学修饰法和氨基酸替换法分析关键果胶裂解酶的氨基酸位点或肽段，揭示关键氨基酸与苎麻胶质底物降解的匹配规律。

果胶酶作为前期引发因子是苎麻脱胶的关键酶，筛选苎麻脱胶关键果胶酶及明确其理化机制十分必要。本项目以苎麻脱胶高效菌种Dickeya dadantii DCE-01为出发材料，用同源重组法敲除的果胶裂解酶基因pel419和pelG403，获得正确的突变工程菌株MutΔpel419和MutΔpelG403。比较野生型D. dadantii DCE-01和2个突变工程菌株的苎麻生物脱胶效果，通过生理生化指标监测、脱胶纤维性能分析、转录组测序及数据分析，证明了2个果胶裂解酶DdPel419和DdPelG403均是苎麻脱胶过程的关键果胶酶。采用原核表达果胶裂解酶基因pel419和pelG403，“两步法”纯化，成功获得电泳纯DdPel419和DdPelG403。DdPel419的最适反应温度为50℃；保温1h，酶活稳定温度为45℃；最适pH为9；稳定pH为8.5-10；Ca2+、Zn2+和NH4+促进酶活力，Ca2+最适浓度为2mmol/L，Fe3+、Pb2+和EDTA严重抑制酶活力；PGA•Na为最适底物。DdPelG403最适反应温度为55℃；保温1h，稳定温度为60℃；最适反应pH为9.5；稳定pH为9-10；Ca2+能最大幅度提高酶活力，其最适浓度为1.5mmol/L，Pb2+和EDTA能严重抑制酶活力；苹果果胶为最适底物。结合多种生物信息学方法预测DdPel419和DdPelG403关键氨基酸位点(肽段)；通过点突变和化学修饰法，明确了该两种果胶裂解酶分别属于PelB和PelC，其8个保守的氨基酸构成了DdPelG403和DdPel419的活性口袋，其中DdPelG403对应的3个Asp162/164/203是Ca结合位点，5个氨基酸为催化位点，分别是Lys223、Arg252、Pro254、Arg257和Phe291；DdPel419对应的3个Asp151/153/192是Ca结合位点，5个氨基酸为催化位点，分别是Lys212、Arg240、Pro242、Arg245和Phe279。总之，通过实施该项目，我们获得了苎麻脱胶关键果胶酶DdPelG403和DdPel419，并全面解析了其理化特征，为阐明苎麻生物脱胶机理提供了理论依据。