苎麻韧皮微生物高效脱胶过程分子机理及优化控制

Ramie fiber is a kind of natural high quality fiber which is unique to China and its advantages are beyond comparison. It needs to be obtained through the ramie bast fiber degumming process. However, the traditional chemical degumming process has several severe problems such as high energy consumption and environmental pollution. Microbial degumming can greatly promote energy conservation and emission reduction, nevertheless, the mechanism of microbial degumming process is not yet clear, the efficiency of microbial degumming needs to be improved and the quality of the ramie fiber is not very stable. The high effectively degumming strain Bacillus sp. HG-28 which was obtained by autonomous selection and has been industrially applicated with proven success was chosen as the research object in this project. Firstly, the key genes, series of degumming enzymes and their expression timing and intensity of the strain in high effectively degumming process were determined by the transcriptome analysis under different degumming stages, the measurement of degumming enzymes’ activities and the comparative analysis of their degradation products’ contents. Then, visualization technology will be used to analyze the dynamic changes of ramie bast fiber surface structure, key degumming enzymes and their degradation products, then confirming the microscopic degumming rules of key degumming enzymes. Further more, through analyzing the interaction between these key degumming enzymes and the impacts of pectin degradation products on enzymes activities to determine the main factors influencing the activities of key degumming enzymes and degumming effects as well as the optimal control strategy. Finally revealing the molecular mechanism of microbial high effectively degumming process and achieving optimal control. It has important theoretical significance and practical guidance to promote high efficient and stable microbial degumming technology application.

苎麻纤维是我国特有天然纤维，优于其他纤维，需通过韧皮脱胶获得，但传统化学脱胶存在污染大、能耗高等突出问题，微生物脱胶可促进节能减排，但由于其脱胶过程机理尚不清楚，导致脱胶效率有待提高且纤维品质尚不是很稳定。本项目以自主选育的已实现工业应用的高效脱胶菌株芽孢杆菌HG-28为对象，首先通过对该菌株在高效脱胶过程的不同脱胶阶段进行转录组分析、脱胶酶活性及胶质降解物含量动态变化的比较分析，确定该菌株脱胶过程系列关键脱胶酶及其表达的时序和强度；进而采用可视化观测分析脱胶过程中苎麻韧皮表面结构和关键脱胶酶及胶质降解物的动态变化，揭示关键脱胶酶的微观脱胶规律；最后通过分析关键脱胶酶及其与胶质降解物之间相互作用，确定影响关键脱胶酶活性和脱胶效果的主要因素及其优化控制方法，最终揭示苎麻韧皮微生物高效脱胶过程分子机理，实施优化控制，对于促进微生物高效稳定脱胶技术应用具有重大理论和实践意义。

苎麻纤维具有强力大、轻盈透气等优点，被誉为“天然纤维之王”，是纺织工业不可替代的原料。但苎麻产业长期受传统脱胶制约，存在“污染重、能耗高、品质低”的瓶颈问题，随着国家节能减排要求日益严格，苎麻产业面临严重危机。微生物脱胶具备条件温和、绿色环保等优点，是当前的研究热点。但是目前报道的脱胶微生物存在效率低、不稳定、脱胶机理不明等问题，很难实现大规模工业应用。针对这些问题，本研究通过系统研究苎麻韧皮微生物脱胶过程机理及高效稳定脱胶的优化控制方法，以期实现微生物脱胶的工业生产，主要研究结果如下：.① 本研究选育获得一株优势脱胶菌株胡萝卜软腐果胶杆菌HG-49，在最优脱胶条件下，胶质去除率高达82.16%。.② 通过分析HG-49脱胶过程生物量、酶活性、胶质含量、细菌群落结构的动态变化，发现菌株HG-49的丰度始终>80%，抗杂菌能力强；酶表达具有时序性，果胶酶先表达且活性较高，甘露聚糖酶与木聚糖酶表达滞后且活性都偏低；果胶去除率高达97.05%，但半纤维素去除率为73.54%，还有待提高；.③ 发现HG-49对各类胶质组成中包含的单糖和多糖均具有较好利用能力，并且脱胶过程中各种单糖浓度都极低，表明胶质降解物能被HG-49很好的利用，不会影响脱胶酶活。.④ 结合基因组和转录组测序分析，发现23个果胶酶，2个β-木糖苷酶及3个β-1,4-内切葡聚糖酶基因，且根据过程表达趋势及表达量，初步确定9个果胶酶及2个内切葡聚糖酶是脱胶关键酶，从基因水平及转录水平进一步说明HG-49具有丰富的果胶酶系，但半纤维素酶系不足；.⑤ 通过扫描电镜、原子力显微镜和激光共聚焦显微镜对酶分布和韧皮结构的可视化动态分析，明确了脱胶的关键酶主要为果胶酶，揭示了果胶酶在降解果胶的同时会去除部分其他胶质，阐明了苎麻韧皮结构特征形貌之间的覆盖性和各类多糖之间的交错连接性会影响酶与胶质的接触，进而影响脱胶效率。.基于上述脱胶机理，建立了苎麻韧皮湿热-拷打的预处理方法，松散了结构；同时，验证了编号为4349的内切葡聚糖酶为多功能酶，具有较高甘露聚糖酶活，另外，筛选到外源高活性木聚糖酶与乙酰木聚糖酯酶，并且构建了具有组成分泌型表达系统的工程菌，丰富了半纤维素酶系，优化了工程菌复合协同脱胶，与原始菌HG-49相比，胶质去除率提高6.76%，半纤维素去除率提高10.88%，为苎麻纤维的绿色生产提供理论及实践指导。