| In order to adapt to the changing environment, microorganisms can change their physiological activities,it result in the proteins associated with metabolism will also be changed accordingly. Streptomyces roseolus DH was a wild strain isolated from soil near the East China Sea, it can produce the related enzymes and grow by different metabolic and regulatory mechanisms when glucose or chitin(or chitosan) is used as the sole carbon source. Chitosanase(EC3.2.1.132), obtained from S. roseolus,is a endo-type acting enzyme and can catalyze specific the hydrolysis of chitosan. Chitosan oligosaccharide, the degradation products of chitosan, has no toxicity, no heat source, no variation and the physiological activity of anticancer, antibacterial and so on. So it is widely used in the fields of agriculture, food, medicine and other fields. Currently, researches about S. roseolus induced chitosan are focused on identification and isolation of chitosan decomposition enzyme, and optimization of fermentation process of decomposition enzyme. However, it is lack of understanding of the metabolic mechanism of the strain using chitosan, and the production of decomposition enzyme is still insufficient. In this paper, a preliminary study on proteomics of S. roseolus by induction of chitosan was made for the first time.(1)The conditions of two-dimensional electrophoresis for total proteins from S. roseolus were optimized from several aspects, such as the purification method, the staining method, the loading quantities and the separation range of strips. The method for analysis of total proteins was established as following: The total cellular proteins were extracted by ultrasonic with the lysate[7mol/L urea, 2 mol / L thiourea, 4%(w / V)CHAPS, 2%(V/V)IPG buffer, 40mmol/L DTT and 0.5%(V/V)protease inhibitor mixture], and purified by 2D clean-up kit to remove the effects of salt ions, polysaccharides, lipids and other impurities. IPG strips with pH 4-7 and 80μg protein sample volume was selected to the isoelectric focusing electrophoresis. After isoelectric focusing electrophoresis, the strips were transferred to 12% SDS-PAGE for vertical electrophoresis. The end of two-dimensional electrophoresis, the gel was stained with silver staining method. Finally, 2D-gels with clear background, high resolution were obtained. The results of three biological replicates experiment of total cellular protein from S. roseolus were analyzed by Image Master2 D Platinum7.0. The repetition rate was 89.4%, which indicated that the optimized 2-DE technique had good reproducibility, and could be used for the analysis of total cellular protein from S. roseolus.(2)The isolation of the total protein from S. roseolus under the condition of normal and induction with chitosan by using the established two dimensional electrophoresis technique. The electrophoretic patterns of cellular proteins were analyzed by the Platinum7.0 Image Master2 D software. The analysis results showed that: 697±25 clear protein spots were detected on the total protein map of S. roseolus under the no induced condition, and 723±14 protein spots were detected on the total protein map of S. roseolus induced by chitosan. 10 proteins whose expression levels differed more than 5 times in the induced strain were selected and identified by mass spectrometry. Through the analysis of SwissProt2015, Uni Prot and DAVID, it turned out: the expressions of Elongation factor G, Serine hydroxymethyltransferase, N,N’-diacetylchitobiose phosphorylase and Enolase and other proteins were increased. These proteins played an important role in protein synthesis, carbohydrate metabolism and stress response. This showed that S. roseolus was in response to induction by chitosan and produced proteins that can decompose chitosan. It promoted the expression of proteins related to protein synthesis increased. In addition, the absorption and utilization of the degradation products need to be through a series of metabolic activities, such as glycolysis and tricarboxylic acid cycle. These resulted in a significant increase in the expression of proteins associated with these metabolic activities. These proteins with differential expression in the culture of chitosan maintained the normal physiological activities of S. roseolus.(3)S. roseolus was cultured in seed medium and chitosan fermentation medium and the culture medium supernatant was collected for two-dimensional electrophoresis. By comparing the results of three methods of extracellular protein purification, TCA- acetone method was used for two dimensional gel electrophoresis analysis of extracellular proteins. The isolation of the extracellular protein from S. roseolus under the condition of normal and induction with chitosan by using the established two dimensional electrophoresis technique. The electrophoretic patterns of cellular proteins were analyzed by software. The analysis results showed that: 391±13 clear protein spots were detected under the no induced condition, and 427±10 protein spots were detected under the condition induced by chitosan. After matching and difference analysis, 15 proteins detected in induced strain were in differential expression. Among these, 13 proteins were up-regulated and 2 proteins were down-regulated. 10 proteins whose expression levels differed more than 5 times were selected and identified by mass spectrometry and 7 effective proteins were identified successfully. Through the analysis of SwissProt2015, UniProt and DAVID, it turned out: the abundances of 30 S ribosomal protein S13, Elongation factor Ts, Fructose-bisphosphate aldolase and Malate dehydrogenase were increased. This was in agreement with the results of the differential expression of the total protein of S. roseolus.In this study, the protein information about the metabolism of chitosan was obtained. This discovery provided biological information for revealing the pathway for chitosan metabolism of S. roseolus, and also provided the theoretical basis for production related decomposition enzymes, so as to lay the foundation for the study of the gene transformation and the improvement of enzyme production capacity. |
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