Preliminary Study On Isolation,Purification And Salt Tolerance Mechanism Of Aspergillus Oryzae Salt-tolerant Protease

The salt tolerance of protease produced by Aspergillus oryzae is closely related to the soy sauce protein utilization rate and amino acids content.At present,the studies on the salt tolerance and salt tolerance mechanism of proteases from A.oryzae 3.042 are less and not deep enough.Therefore,it is urgent to breed A.oryzae with high-yield protease and study the salt tolerance mechanism of protease.A mutant strain with high-yield proteases was obtained by Atmospheric and Room Temperature Plasma?ARTP?using A.oryzae 3.042 as the starting strain.Two salt-tolerant proteases including aspartyl aminopeptidase?AAP?and alkaline protease?AP?were isolated,purified and identified.And their salt-tolerant mechanisms were preliminarily elucidated at the molecular level.?1?A mutant H8 with high yield of protease was screened by ARTP technology.A mutant strain H8 with high yield of protease,stable inheritance?Passage 15 times?and spore color change was obtained using a novel ARTP technique.The enzyme activities of the neutral protease,acid protease,alkaline protease and aspartyl aminopeptidase were increased by31.82%,19.36%,20.76%and 24.77%in comparison with those from the original strain A.oryzae 3.042.The biomass of the mutant strain H8 was 2.9%higher than that of the original strain.It was further confirmed by RT-qPCR that the relative transcription level of AAP and AP genes in mutant H8 was 27%and 30%higher than those of the initial strain,respectively,indicating that the increased transcription levels of AAP and AP genes were one of the key factors enhancing enzyme activities.ARTP might promote the transcriptional expression of AAP and AP genes in A.oryzae,thereafter enhance the production of AAP and AP.?2?The isolation,purification and molecular mechanism of salt tolerance of aspartyl aminopeptidase?AAP?in mutant H8 were preliminarily studied.AAP from A.oryzae H8 was purified by buffer solution and modern chromatography,and identified to have a molecular weight of about 57 kDa.Specific inhibitor experiments indicated that it was an aminopeptidase containing Zn²⁺.Its optimal and stable pH values and temperatures were 7 and50°C,respectively.A comprehensive analysis including protein homology modelling,molecular dynamics simulation,secondary structure,acidic residues and hydrophobicity of interior residues demonstrated that AAP had a greater stability than non-salt-tolerant protease in high salinity.Higher contents of ordered secondary structures,more salt bridges between hydrated surface acidic residues and specific basic residues and stronger hydrophobicity of interior residues were thought to be the salt-tolerance mechanisms of AAP.?3?The isolation,purification and salt tolerance mechanism of alkaline protease?AP?in mutant H8 were preliminarily studied.Salt-tolerant AP?approximately 29 kDa?from A.oryzae was purified and identified by buffer solution,modern chromatography and MALDI-TOF-MS.Specific inhibitor experiments indicated that it was a metal ion-independent serine protease.The optimal and stable pH values and temperatures of AP were 9 and 40°C,respectively.A comprehensive analysis including protein homology modelling,molecular dynamics simulation,secondary structure,acidic residues and hydrophobicity of interior residues demonstrated that AP had a greater stability than non-salt-tolerant protease in high salinity.Higher proportion of ordered secondary structures,more salt bridges and stronger hydrophobicity of interior residues might account for the salt-tolerance mechanisms of AP.It will provide reference for the optimization and regulation of soy sauce fermentation process and a novel ARTP technique for breeding of new strains in this study.