构建引入纤维素酶分离因子的海洋黑曲霉新表达体系及性能研究

Marine cellulase is a kind of cellulase with better tolerances,which is produced by Aspergillus Niger and has a good application field. At present, there are few studies on the properties, structure and function of conventional Aspergillus Niger cellulase, but almost blank for marine Aspergillus Niger, which affects the performance analysis and application of marine cellulase. One of the main reasons is that cellulase is a combined enzyme and it is difficult to isolate and purify the cellulase component.The aim of this project is to establish a homologous expression system of marine Aspergillus Niger, and to introduce the cellulase separation factor into the marine Aspergillus Niger, and to express the corresponding cellulase components. By this method, the process of separation and purification can be greatly simplified, and the purity of the protein more than 95% can be obtained. The purified cellulose components can be used to analyze the structure and enzymatic properties of cellulase, especially to find the basic reason of good tolerance. At the same time, the salt tolerance mechanism of marine cellulase will be explained by molecular simulation..The establishment of homologous expression of cellulase system is an effective way to introduce separation factors from upstream process to solve the difficulty of separation and purification in downstream process. The method can also be used for reference in the construction of other enzyme expression systems and is an example of the integration of bioreaction and bioseparation.

海洋纤维素酶是一类具有更好耐受性的纤维素酶，由海洋黑曲霉产生，有较好的应用领域。目前即使对常规的黑曲霉纤维素酶性质、结构和功能的研究也很少，而对于海洋黑曲霉几乎空白，影响了对这类纤维素酶的性能分析和应用，一个主要原因在于纤维素酶是一个组合酶，很难分离纯化得到其中的纤维素酶组分。本项目旨在通过建立海洋黑曲霉的同源组成型表达系统，把纤维素酶的分离因子植入海洋黑曲霉中，可以定向表达相应的纤维素酶组分，在大幅度简化下游分离纯化过程的同时还能得到蛋白纯度大于95%以上纤维素酶组分，可用于纤维素酶的结构解析和酶学性质，尤其找到耐受性较好的原因。同时还将通过分子模拟的手段尝试阐释海洋纤维素酶的耐盐机制。同源表达纤维素酶系统的建立是从上游过程引入分离因子来解决下游过程分离纯化难点的一种有效手段，从源头上解决了后续过程的困难。该方法对其它酶表达系统的构建也有借鉴作用，是生物反应与分离集成化的一个实例。

通过四年的研究，完成了所有预定的研究计划，包括目标对象海洋黑曲霉的选定，海洋黑曲霉的全基因测定，把基因同源表达方法作为定向表达纤维素酶酶组分的分离因子，建立了海洋黑曲霉酶组分表达的新体系，成功实现了酶组分的分泌表达，定向表达了6个酶组分，简化了相应酶组分的分离纯化程序，经1-2步分离纯化得到了纯度较高的纤维素酶组分，开展了酶组分的简单结晶方法，得到了酶组分的结晶体，结合分子模拟方法确定了相关酶组分耐盐和耐温的分子机理，同时将海洋黑曲霉菌丝球用于六价铬的生物还原，研究了生物还原的机理，实现了在pH中性条件下生物还原六价铬的过程。开展了转录组和代谢组学研究，分析了黑曲霉菌丝球对Cr(Ⅵ)的消除机理以及磷酸盐影响黑曲霉菌丝球消除Cr(Ⅵ)的机理。