Mechanism Of Salt Stress In Aspergillus Oryzae And The Purification And Characterization Of A Salt-tolerant Protease

Soy sauce with a mellow palate and salty palatability is an essential condiment,and one of the most important species in soy sauce fermentation is Aspergillus oryzae 3.042.In china,the two widely used techiniques in fermentaing soy sauce were low-salt in a solid-state and high-salt with a diluted-state.Salt can inhibit the growth of miscellaneous bacteria and prevent sauce culture from putrefaction.However,high salt concentration could inhibit the growth of A.oryzae and also cause inhibition of protease activity,resulting into incomplete hydrolysis of raw materials and prolonged fermentation period.According to the previous studies,the salt stress mechanism of A.oryzae was still not fully understood.So,the approach of proteomics was used to analyze the salt stress potential response mechanism of A.oryzae.Separation,purification and characterization of a salt-tolerant protease from sauce koji were done by different chromatographic columns.The main outcomes of this article have been listed as follows:Through tandem mass tags?TMT?based comparative proteomics analysis,totally 4748proteins were identified,among which 4381 proteins were quantified.There were 458 and199 proteins differentially expressed under solid-state condition and submerged condition.These differentially expressed proteins were involved in carbohydrate metabolism,lipid metabolism,amino acid metabolism,fatty acid metabolism,vitamin metabolism,nucleic acid metabolism,methane metabolism,protein endoplasmic reticulum modification and other metabolic pathways,including MAPK signaling pathway and mitochondrial autophagy.The rest of the differential proteins are peptidases,oxidoreductases,hydrolases,cell wall proteins and transporters.Based on the function and involvements of proteins in metabolic pathways,the underlying salt stress mechanisms of A.oryzae 3.042 can be summarized to the following aspects:?1?Salt stress activated HOG-MAPK signaling pathway mediated by MAPK-Hog1,which regulated glycerol synthesis to resist salt stress;?2?In the high-salt environment,the amounts of osmotic protection agents,such as glycerol,proline,glutamic acid and glutathione increased significantly to relieve salt stress and prevent Na⁺from the cytotoxicity of bacterial cells;?3?K⁺channel protein,K⁺transporter and the kinases provided energy in high-salt environment would be up-regulated significantly,transport K⁺into the cell,increased intracellular osmotic pressure,and protected the cells from Na⁺damage;?4?Salt stress activated the calcineurin signaling pathway to resist the salt stress.The study provided a theoretical basis of A.oryzae salt stress molecular mechanism and the transformation of industrial microbial characters.In this study proteases were purified to homogeneity by ammonium sulfate precipitation,Q-HP anion-exchange chromatography and Superdex 75 gel chromatographies.MALDI-TOF/TOF mass spectrometry analysis suggested that the protease showed a high similarity to the calpain RIM13,which was a cysteine protease.The protease was separated and purified from sauce koji for the first time and maintained high enzyme activity in salt environments.The optimal temperature was 50?C and optimal pH was 6.5,and the stable temperature was40?C and stable pH was 7.0.The protease can be activated by Mn²⁺,and inhibited by Fe³⁺,Fe²⁺,Cu²⁺,Ca²⁺,K⁺and Na⁺.Moreover,all the metal ions mentioned above had different effects on the secondary structure of the protease.Km and Vm values of the protease were2.43 g/L and 103.09 mg/?L·min?.The protease activities were decreased to 77.22%,54.39%and 41.15%under 5%,10%and 15%salinities,respectively.The above enzymatic properties indicated that the protease was suitable for soy sauce fermentation and provided theoretical support for the application of salt-resistant protease in soy sauce fermentation.