华根霉固态与液态培养产胞外蛋白的比较蛋白质组学分析

【目的】考察固态和液态两种培养方式对丝状真菌华根霉(Rhizopus chinensis)CCTCC M201021产胞外蛋白的影响,以加深对微生物固、液态培养特异性产酶的认识。【方法】利用成分相同的培养基对华根霉分别进行平板固态培养和液态培养,提取华根霉所产胞外蛋白,采用二维凝胶电泳(2-DE)结合质谱分析,分离鉴定差异蛋白。【结果】固态培养下华根霉所产胞外蛋白的蛋白酶活性是液态培养的9.2倍。胞外蛋白质组数据分析表明,华根霉固态和液态培养所产胞外蛋白存在显著差异,其中约70%是固态和液态培养下各自产生的特有蛋白。质谱鉴定进一步表明,固、液态培养对华根霉产胞外蛋白的种类和表达量都有显著影响,其中水解酶类所占比例较大,主要是与蛋白质降解相关的蛋白。【结论】固态和液态不同培养方式影响了华根霉产胞外蛋白的组成,有些基因只在特定培养方式下表达。固态培养下华根霉产胞外蛋白酶种类相对更多以及大部分蛋白酶的上调表达,可能是固态培养下胞外蛋白酶活性很高的原因。研究结果提示需要注意固液态不同培养方式下丝状真菌胞外酶的筛选和生产可能存在的不一致性。

Comparison of extracellular proteome from Rhizopus chinensis in solid-state and submerged cultures

[Objective] We studied the influence of solid-state and submerged cultures on the extracellular proteome of filamentous fungus Rhizopus chinensis CCTCC M201021, to understand the specificity of enzyme production by filamentous fungi in different cultures. [Methods] Using same media, we carried out solid-state agar-plate culture and submerged culture of R. chinensis. Two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF-MS were used to analyze the extracted extracellular proteins, and the differential proteins were identified. [Results] Protease activity of extracellular protein from the solid-state culture was much higher (9.2-fold) than that from submerged culture. The 2-DE gel maps indicate the differences in the extracellular proteomes between solid-state and submerged cultures, and most proteins (about 70%) were special from solid-state or submerged culture. After identification, the variety and expression level of extracellular proteins from the two different cultures were significantly different. Among these differential proteins, hydrolases were the majority, most of which were related with protein degradation. [Conclusion] Different cultures influenced the composition of extracellular proteome by R. chinensis, and some proteins were only expressed in a specific culture. Relatively more variety and higher expression level of some proteases under solid-state culture could be the reason, which resulted in the much higher protease activity of extracellular protein. These results suggested the possible inconsistencies of extracellular enzymes produced by filamentous fungi between the strain screening and submerged fermentation in industry production, when solid-state culture and submerged culture were used.