反向遗传学策略解析S. maltophilia DHHJ 菌株SMBP蛋白及其膜受体功能

禽类羽毛主要成份为角蛋白（86%-97%），是地球上最大的氮源废弃物之一，炼制产物用途广泛，生物炼制由于多种优点而成为目前潮流。但羽毛生物炼制的机理一直是该领域的难解点，因此炼制效率难以提高，限制了该工艺应用。申请人前期研究得到了羽毛角蛋白降解酶特征及基因序列，并发现了S. maltophilia DHHJ 菌株中与羽毛降解相关蛋白—肽链结合蛋白（SMBP），本项目研究在此基础上，利用RNAi技术分析SMBP蛋白对角蛋白降解作用，包括了对酶表达及角蛋白降解效率的影响；同时利用荧光定量PCR技术初步确定角蛋白进入细胞的通道蛋白。从而进一步阐明角蛋白降解的分子过程，为后续菌种改造，角蛋白生物炼制生化机制的全面解析奠定基础，同时为角蛋白作为新型氮源利用具有重要的实用价值。

As encrusted protein, feather keratin has the characteristics of no-dissolve, anti-decomposition and anti-pain, and it also cannot be dissolved in normal conditions. So feather keratin is very difficult to be deal with. And at the same time a lot of keratin materials, as wastes, are released in China, such as feathers from poultry farms, leather heels, and so on. To protect our environment and make full use of the resources，most of researchers will turn the wastes to treasures with biotechnology method because of its advantages of no pollution and easy to control. In our initial research，a novel kerationlytic bacterium, Stenotrophomonas maltophilia DHHJ had been isolated and identified (Cao et al, 2009). The bacterium strain is very well to degrade feather. Traditionally, it thinks enzymes are the key factor to degrade macromolecular material. Therefore, more than 50 keratin hydrolysis enzymes (keratinase) had been identified by different laboratories all over the world. But feather keratin can`t bedegraded by these enzymes in vitro. Another side, E.coli that transfer keratinase gene hasn`t the capability to degrade feather through different research in several laboratory. So, researchers think some other molecular works to degrade feather. We find a peptide binding protein, named SMBP, which is a key factor to degrade feather in S. maltophilia DHHJ. We would like to do some research based this: Firstly, the function of SMBP would be sure through RNA interference method. Secondly, SMBP would be sited through fusion expressed with GFP. In other side, keratin monomer was labeled with FITC. It would be observed about the molecular movement in the cultural liquid with bacteria. Thirdly, the membrane receptor of SMBP would be researched by molecular blotting method. The research would help people to understand the mechanism of feather keratin biodegraded. Further, it would provide a base to researcher improve the ability to degrade feather in bacterium.