Intracellular Dynamic Response Of Glycosylase High Yield Aspergillus Niger Under The Concentration Gradient Of Substrates And Dissolved Oxygen

Glycosylase is one of amylase and it has the ability to hydrolyze all d-glucose bonds.The glycosylase produced in industry have the characteristics of thermal stability,pH stability,substrate specificity and so on.Amylase Market has developed into the second largest market of industrial enzymes,with great economic and research value.However,the yield of laboratory scale products cannot be achieved in the large-scale amplification of glycosylase produced by Aspergillus Niger.Inadequate mixing is a key factor affecting amplification production,and the dynamic response of microorganisms in fluctuating environments may be a key cause of amplification difficulties.Therefore,the concentration gradient(substrate and dissolved oxygen gradient)in large-scale fermentation process is simulated by Scale-down experiment,and the dynamic response of microorganisms and the effect on the yield of the product are studied.In this work,the dynamic response of intracellular and extracellular metabolites of A.niger was investigated after sudden exposure to high and low excess glucose concentrations in chemostats(?=0.08 h-1).It was found that the steady state pathway turnover time of the carbon flux through the central carbon metabolism(CCM)was PP pathway 50 s,EMP pathway 20 s,and TCA cycle 189 s,and an upper limit for individual metabolite concentrations in the CCM was estimated.The ATP paradox,known from other organisms,was also observed in the studied A.niger strain.Then,a periodic substrate pulse is carried out by a self-designed pulse device,which simulates the substrate gradient existing in mass production.Microorganisms undergo a"Feast/Famine"cycle and find that most intracellular metabolites respond in the same way as single low-concentration glucose pulses.After a long period of fluctuating environment,biomass was decreased by around 6%,while the concentration of glycosylase increased by 12%.In the determination of intracellular amino acids,the decrease of precursor amino acid(Glu and Lys)in fungal synthesis and the increase of precursor amino acid(Ala and Leu)in glycosylase synthesis were discovered,which may be the reason for the decrease of biomass and the increase of yield.Dissolved oxygen is another important factor affecting the growth and metabolism of fungi.A dissolved oxygen fluctuation device was designed to simulate the dissolved oxygen gradient produced by mass production.The results of dissolved oxygen periodic pulse-limiting experiment showed that the restriction of dissolved oxygen had a promoting effect on the formation of products and by-products,and the rate of glycosylated enzyme production increased by 37%,which also had a great influence on fungal growth,which reduced the bacterial concentration by 72%.The restriction of dissolved oxygen led to a negative response in most intracellular metabolites in the cell,in which the metabolites such as ASP and ATP were significantly correlated with dissolved oxygen.