内蒙古原始森林土壤宏基因组文库构建及低温高活性纤维素酶筛选和特征分析

Cellulose is the most potential solution of petrochemical energy shortage and environmental pollution problems. Existing cellulose enzyme activity is not high enough, which is the key limiting factor for cellulose cannot replace the fossil energy. For low temperature area, low temperature is another major limiting factor for cellulose using. Study of screening high enzyme activity of cold-adapted cellulase is the key to solve the problem. The current widely method used for screening of high efficiency cold-adapted cellulase based on the cultivation of microorganisms is not successful. While the metagenomics technology does not depend on the cultivation, and some highly active enzymes had been isolated by this technology. But the screening of cold-adapted cellulase based on the metagenomics technology has not been reported. There are many primeval forests with the climate characteristics of low temperature in Inner Mongolia. And there are rich varieties of cold-adapted cellulase resources in the primeval forests. This project is based on the metagenomics technology, and builds the metagenomic library through enrichment of lignocellulose-degrading microorganisms at low temperature. Screen high enzyme activity of cellulase by function driving assay method, analyze cellulase gene and predicted protein structure, and define the structure characteristics of specific genes and proteins to clarify the molecular mechanism of efficient degradation of cellulose by cellulase at low temperature. It will provide theoretical basis and practical basis for fully understanding and utilizing the cold-adapted cellulase resources in Inner Mongolia.

纤维素是解决石化能源短缺及其引起的环境污染问题的最具潜力的途径。现有的纤维素酶活性尚不够高，是现阶段纤维素无法替代石化能源的关键限制因素。而对于气温较低的地区，低温是纤维素利用的另一主要限制因素。研究筛选高酶活低温纤维素酶是解决问题的关键。当前普遍使用的、基于分离培养微生物而筛选高效低温纤维素酶并不成功。而宏基因组技术不依赖于培养，已获得了一些高活性的酶类。但基于宏基因组技术研究筛选低温纤维素酶尚未见研究报道。内蒙古原始森林较多，低温的气候特点突出，蕴藏有丰富多样的低温纤维素酶资源。本项目拟基于宏基因组技术，通过富集低温木质纤维素降解微生物及构建其宏基因组文库，然后以功能驱动方法筛选高酶活纤维素酶，并对其基因和预测蛋白结构进行分析，明确基因和蛋白的结构特点，阐明低温纤维素酶高效降解纤维素的分子机制，为充分认识和利用内蒙古地区低温纤维素酶资源提供理论依据和实践基础。

纤维素的开发利用是解决石化能源短缺及其引起的环境污染问题的最具潜力的途径，现有的纤维素酶活性尚不够高，是目前纤维素无法代替石化能源的关键限制因素。而对于气温较低地区，低温是纤维素利用的另一主要限制因素。所以筛选低温高酶活纤维素酶是解决问题的关键。本研究利用内蒙古九峰山、大兴安岭等原始森林土壤为菌源，以涂布划线手段筛选低温和常温纤维素降解微生物；利用DNS法测定菌株CMC酶活；采用宏基因组文库构建及测序技术手段，发掘纤维素酶基因并克隆表达。实验结果表明，利用传统微生物分离筛选手段获得893株纤维素降解菌，其中常温细菌有450株，常温真菌有106株，低温细菌有336株。常温纤维素降解细菌分属于变形菌门、放线菌门、厚壁菌门、拟杆菌门和酸杆菌门4个门，常温纤维素降解真菌分属子囊菌门、担子菌门和球囊菌门3个门，低温纤维素降解细菌分属放线菌门、变形菌门、厚壁菌门和拟杆菌门4个门。其中低温纤维素降解细菌最高纤维素酶活为31.11 U/mL，常温纤维素降解真菌的最高纤维素酶活能达到158.36 U/mL，常温纤维素降解细菌的纤维素酶活介于两者之间。利用宏基因组文库构建、宏基因组测序技术，通过碳水化合物活性酶注释，共得到215702条与纤维素降解有关的基因，共有5条纤维素酶基因克隆并表达成功，其中3条属于低温纤维素酶，重组酶23-1、93-5、23-5、103-2和E3653最适底物分别为CMC、pNPX、微晶纤维素、pNPX和微晶纤维素，最适温度分别为50 ℃、40 ℃、55 ℃、40 ℃和40℃；最适pH分别为5.0、6.0、9.0、5.0和9.0。分别以微晶纤维素、CMC和D-水杨苷分别筛选出具有外切葡聚糖酶、内切葡聚糖酶和β-葡聚糖苷酶的酶活性的菌株构建了27个高效低温降解复合系，滤纸酶活性最高的降解复合系为组合D13'1"和D13'2"，酶活性可达158.02U/mL，是其复合系中单菌株的5~10倍，为低温降解复合系最优菌群组合，具有较高的应用潜力。本研究为充分认识和利用内蒙古地区低温纤维素资源提供理论依据和实践基础。