The Study Of Actinomucor Elegans AS3.2778 Aminopeptidases Based On RNA-Seq Technology

Actinomucor elegans, synonyms creeping Actinomucor, is one of the best fungi that is commonly used in traditional Chinese food bean curd brewing. This filamentous fungus can secrete many kinds of proteases, which could hydrolyze soy protein and other plant proteins better, and play an important rol in the formation of bean curd flavor compounds in the post-fermented process. Since there is no clear information about the genetic background of Actinomucor elegans, and the composition of proteases and associated genes are not reported before, we have limited studies of its proteases in molecular level.In this paper, we had Actinomucor elegans AS3.2778 which grown on bran solid medium for 48 h transcriptome sequenced for the first time based on RNA-Seq technology, and we got its protease enzyme system with different transcriptional expression. By analyzing the data for transcriptome, we found 106 protease genes in this mucor transcriptome, of which were 21 aminopeptidase genes. The study further analyzed the aminopeptidases on their sequence features, evolution and classification. In addition, we analyzed and compared aminopeptidases of four culturing periods on the differences of their expression level and enzyme activity by real-time PCR and enzyme activity assay experiments respectively. Through this, we can better explain Mucor requires to produce the appropriate aminopeptidases to meet their growth and physiological needs in different growth stages. Transcriptome data showed that Mucor produced a total of 21 aminopeptidases, which belonged to seven protease families, specifically were families as M1, M17, M18, M24, M28, S9 and C1, and a great number of them were metalloproteinases. The phylogenetic tree was constructed by neighbor-joining method, indicating that proteases in the same family often gather on an evolutionary branch. Morphological observation revealed that Mucor has white and thick mycelia on the bran solid medium on the second day, reaching its exuberant period. We observed Mucor with electron microscope and found it started to produce spores on the second day and spores number increased with the prolongation of culture time. Besides, quantitative real-time PCR was used to validate aminopeptidase gene expression differences during 1d, 2d, 3d and 4d four periods. Results showed that the number 5 gene reached the highest gene expression level of the 21 aminopeptidases on the third day, and most genes had their maximum expression levels on the third to the fourth day. Determination the enzyme activity of the four periods proved that: Mucor crude enzyme in the p H6.5 and p H8.0 conditions had significantly higher degree of hydrolysis to the ten selected substrates than in the p H5.0 and p H9.0 buffer solutions. When the p H was 6.5, Mucor crude enzyme had the highest hydrolysis degree to Phe-p NA, so Phe-p NA was described as its optimum substrate and p H 6.5 was considered as its best p H. Besides, aminopeptidases of different time point had different enzyme activity to the same substrate under different buffer conditions. The above results suggest that Actinomucor elegans has a rich protease enzyme system, and it has vigorous growth in the first 2-3 days, both aminopeptidase expression level and enzyme activity are relatively high, and relevant researches lay a foundation to further understanding the protease genes of this strain and its molecular cloning research.