莫海威芽孢杆菌XH1合成蛋白类生物破乳剂的代谢途径解析

For its non-secondary pollution, easily biodegradable and environmentally safe, the biodemulsifier is one of the most potential research and development orientations for demulsifiers. The application of high efficiency and environmental friendly biodemulsifier has important significance in improving the dewatering efficiency of reducing environment pollution. However, some key problems restrict the practical application of biodemulsifier such as unstable metabolic process of demulsifying bacteria and difficult analysis on complicated effective ingredients. In this project, effective ingredients secreted by protein biodemulsifier producing bacteria (Bacillus mojavens) XH1 will be extracted, purified then characterized. The extra extracellular proteome secreted by B. mojavens XH1 under the condations of with or without the inducing substrate will be studied by 2D-DIGE and MALDI-TOF-TOF MS. And the key factors that impact demulsification protein metabolic pathway, such as the key enzyme and functional protein, will be confirmed. Meanwhile, the anabolism pathy of demulsification protein will be establishment use the technic of HPLC-MS. The above research may providing fundamental basis for finding new demulsifying gene resources and in-depth knowledge of the mechanism for regulating demulsifying protein expression.

生物破乳剂是一种具有可生物降解性和安全性的绿色净水剂，它的应用对于提高含油污水的处理效率，减少不必要的环境污染具有重要意义。目前，生物破乳剂的实际应用受到破乳剂有效成分复杂、破乳菌代谢过程不稳定等问题的制约。本课题以蛋白类生物破乳剂产生菌(Bacillus mojavensis) XH1为研究对象，对其分泌的破乳蛋白进行纯化、鉴定并解析其微观结构；利用双向荧光差异凝胶电泳结合质谱技术，在不同底物诱导条件下开展XH1菌胞外蛋白质组的研究，分析不同诱导条件下XH1菌的胞外差异蛋白，从而确定XH1菌合成胞外破乳蛋白过程中的关键酶和功能蛋白；采用液质联用技术分析XH1菌合成胞外破乳蛋白过程中代谢中间产物和非目标产物的动态变化，通过质谱图库比较分析各关键产物的分子结构，从而构建XH1菌产蛋白类生物破乳剂的代谢路径。以上研究可为挖掘新的破乳基因资源、深入了解破乳蛋白的表达调控机制提供理论基础。

在高含水原油乳状液以及其它工业乳状液副产品的处理和处置中，应用生物破乳剂替代大量使用的化学破乳剂，对提高乳状液脱水率，降低环境污染风险具有深远意义。本项目以已筛选出的高效破乳菌Bacillus mojavensis XH1为研究对象，考察菌株B. mojavensis XH1 所产生的蛋白类生物破乳剂的主要有效成分，并对其代谢途径进行探究。结果如下：. 通过对菌株XH1进行全基因组及转录组的分析，对B. mojavensis XH1破乳基因进行探究。经全基因组分析发现B. mojavensis XH1基因组序列拼接后，其总长度为4,203,618 bp，GC百分含量为43.99%，并构建了B. mojavensis XH1菌株的基因组圈图。利用RNA-Seq技术，分析菌株XH1以有无液体石蜡诱导底物培养时其基因表达的差异情况。结果显示：与对照组相比，实验组上调表达基因2638个，下调基因326个；得到以液体石蜡为底物产生物破乳剂的代谢过程比以葡萄糖为底物的代谢途径在“碳水化合物”与“外源性物质降解和代谢”通路中大部分基因的表达量均有提高。在上调基因中发现草酸脱羧酶（Oxalate Decarboxylase）所对应基因有明显的上调，且log2 Ratio(EX/CX)=15. 41178，属于显著上调。. 利用比较蛋白质组学的相关理论及技术，分别在不同底物诱导条件下对菌株XH1进行扩大培养，对其胞外蛋白进行SDS-page电泳分析，利用“鸟枪”法对差异条带进行分析，初步确定XH1胞外分泌的草酸脱羧酶为一种破乳有效蛋白质。为进一步确定草酸脱羧酶的破乳功能，对草酸脱羧酶的碱基序列及氨基酸序列进行分析，构建重组质粒，热激法转入大肠杆菌BL21中进行基因表达，测得重组菌的破乳率及草酸脱羧酶酶活分别为60.2%和2.247μmol formate/min/mg，明确了草酸脱羧酶为一种具有破乳功能的蛋白。进一步通过气质联用技术，结合全基因组及转录组学的结果，初步分析得到了B.mojavens XH1产草酸脱羧酶类生物破乳剂的代谢途径。