竹类资源脱胶用高效重组基因工程菌构建及其重组酶脱胶机理研究

Resource depletion and environmental pollution are so far the biggest issue that we as human beings have to confront. Development and utilization of natural resources based on environment-friendly biotechnology has become a research hotspot. China is well known for the abundant bamboo resource.'Replacing cotton with bamboo' could not only alleviate the shortage of cotton fibers which rely on the cultivated land, but also open up a new high-value application area for bamboo resource. .The essential to successfully extract bamboo fiber through bio-degumming greatly depends on the possibility to make a breakthrough to effectively strengthen the bond between the applied enzymes or bacteria and bamboo structural constituents. The theories and methods of microbiology, molecular biology, enzymology and materialogy are employed in this project. Strains with high degumming potential are screened and isolated from retted and rotted bamboo and related soil system.The gene sequence for enzyme production is decoded and the recomibnant genetic engineering strains for high-yield enzyme production are constructed, thus a large quantity of recombinant enzymes are obtained. Mechanism of enzymatic degumming is revealed by the chracterization of enzymatic adsorption towards bamboo and the investigation of the kinetics of enzymatic degradation on the noncellulosic substances in bamboo under different degumming systems including individual-emzyme treatment, complex enzyme one-bath treatment and enzymatic multi-stage treatment, as well as the analysis of effect of pretreatment on the microsturcture and micromorphology of bamboo and the construction of pretreament model in bamboo system. Additionally, a suitable and comprehensive evaluation system is established for enzymatic degumming. This project lays the theoretical foundation for the development of eco-friendly bamboo fiber preparation technology and meanwhile provide a valid reference for the reasonable development of other vegetable fibers.

资源枯竭和环境污染是人类迄今面临的最大问题。采用环境友好型生物方法开发和利用天然资源成为目前研究的热点。我国竹类资源丰富，"以竹代棉"不仅可以有效缓解以耕地为基础的棉纤维短缺问题，而且为竹材高值利用开辟新途径。.竹纤维生物脱胶成功与否取决于所采用的酶或菌与竹在结构组分上的有机结合能否取得突破。本项目基于微生物学、分子生物学、酶工程及材料学等方法，从沤竹、腐竹及其土壤体系中筛选脱胶优势菌种，克隆产酶基因序列，构建高效产酶重组基因工程菌并大量制备脱胶用酶；研究酶对竹的吸附特性及在单一酶/复合酶共浴/多酶分阶段脱胶体系下对竹中非纤维素类伴生物的降解动力学机制；分析预处理方法对竹微观结构和形态的影响，建立竹体系预处理模型，探讨预处理增强酶脱胶效用的机制,揭示酶脱胶机理并构建适合的酶脱胶效果评定体系，为发展竹纤维绿色环保制取技术提供基础理论依据，也为其他植物纤维的合理开发提供有效的借鉴方法。

发展竹纤维生物制取技术，关键问题是获取适合竹脱胶体系的酶制剂以及增强酶的作用力度。本项目从筛选获得的高效脱胶菌株Streptomyces sp. FA1中分离纯化出一种木聚糖酶（XynA），作为竹脱胶主要用酶之一。研究其酶学性质表明XynA的最适pH为5.5，最适温度为60℃；适用pH范围广且50℃稳定性较好。对纯化所得的目的蛋白XynA进行肽指纹图谱鉴定，构建基因工程菌E.coli BL21(DE3)/pET-20b-xynA，其胞外酶活达到213.5 U/mL，是野生菌Streptomyces sp. FA1发酵上清液酶活的11倍。为了实现XynA的高效胞外分泌表达，对E.coli BL21(DE3)/pETDuet-1/cut/xynA的发酵产酶诱导条件及发酵培养基进一步优化，最终酶活达到780.2U/mL，是目前大肠杆菌摇瓶发酵产木聚糖酶的最高表达水平。由于竹子结构致密，且竹中各成分物质相互交缠，单一的木聚糖酶作用竹的效果有限，需要与其他酶进行复配并辅助以预处理的方式才能增加酶对竹子的水解。因此，筛选出酸性纤维素酶Suhong Cellish L和中性纤维素酶纤柔酶8000L与木聚糖酶进行复配，结合高温蒸汽、微波、超声波等作为预处理方式共同作用于竹上。结果表明，不论是对单一酶处理还是复合酶的共浴处理以及多酶的分阶段处理，预处理均能达到增强酶作用的效果，以高温蒸汽预处理的增强效应最为显著。经过预处理的竹子结构膨润松散，使得酶能够吸附到竹子表面，并渗透到竹子的内部，进而能够作用于内部结构中的特定物质，增强酶处理的力度。复合酶共浴处理中，只有中性纤柔酶8000L与木聚糖酶之间相互协同作用，有望通过纤柔酶的作用打开竹中的结构，增加木聚糖酶的作用位点，进而提升作用效果。在多酶分阶段处理过程中，纤维素酶的添加提高了木聚糖酶的利用率，增强的木聚糖酶解效果。本项目以糖分析为主，酶蛋白吸附评定为辅，结合竹子结构分析，建立酶处理的效果综合评价体系，研究酶脱胶的机制，诠释了预处理对酶脱胶增效作用。最后，在本项目的支持下，为拓展竹质基材在纳米材料及医用抗菌功能材料的领域应用进行了探索研究。本项目的开展将为竹类资源的综合利用及其纤维的生物制取奠定坚实的基础。基于本项目研究成果，共发表和录用论文15篇（其中SCI论文12篇），申请专利2件。本项目实施中，培养研究生6人，3人已答辩。