Purification And Enzymatic Characterization Of Aschersonia Esterase And Lipase

Aschersonia is an entomopathogenic fungus that is widely used in many fields.During its infectious process,it is able to produce some vital virulence factors such as esterase and lipase which can facilitate degradation of host cuticle.In this thesis,a research project on esterase and lipase from Aschersonia was carried out and the research results were summarized as follows.1.Higher enzyme-producing strains were screened from twenty-three isolates of entomopathogenic funngi,with the method of enzyme activity measurement of culture filtrate.The results showed that Aschersonia WYTY-03 was confirmed the highest esterase-producing strain and the highest lipase-producing one was Aschersonia WYTY-21.2.The effects of the carbon source,nitrogen source,metal ions and vitamins on production of enzyme were studied by using single-factor analysis.Optimum medium for enzyme production was obtained by orthogonal optimization and response surface method.The best esterase-producing culture medium consisted of 10.0 g/L L-arabinose,2.5 g/LYeast extract,0.01 g/L Fe3+,2.28 g/L Mg²⁺,5.37 g/L?NH4?2SO4,0.112 g/L vitaminB2 and pH6.7 and the optimum conditions for producing enzyme were 20%of inoculation amount,50 mL of liquid volume,mycelial age 7 days,culture time 2 days.Under this culture conditions,maximum esterse activity reached 29.52± 0.39 U/mL which was increased about 5-fold than the non-optimized basal media.The esterase was puried by using the combination of various chromatographic steps including DEAE-cellulose and Sephadex G-100.The puried esterase had one band with molecular weight of 79 kDa measured by SDS-PAGE.According to the results of esterase characteristics,it showed that the optimum temperature for esterase activity was 40? following its stability at 40? and pH for its activity was 7.0 following its stability at pH 6.5-8.0 with the corresponding Km and Vmax of 0.802 mg/mL and 40.32 ?mol/min,respectively.Almost all metal ions activated esterase activity expect for Al3+ while the vast majority of organic solvents inhibited its activity.Similarly,the highest lipase-producing culture media were composed of 51.5 g/L emulsified olive oil,2.5 g/L a-Lactose,0.35 g/L troptone,1.45 g/L Na+,0.01 g/L Zn²⁺,0.1 g/L VB4 and pH7.1.The best conditions for producing enzyme were 10%of inoculation amount,75 mL of liquid volume,mycelial age 1 day,culture time 4 days.Under this culture conditions,maximum lipase activity reached 39.48 ± 0.52 U/mL which was increased about 7.6-fold than the non-optimized basal media.The puried lipase also had one band with molecular weight of 23 kDa measured by SDS-PAGE.The results of lipase characteristics revealed that the optimum temperature and pH were 50? and 8.0,respectively,following its temperature stability at 50? and its pH stability at 7.5-8.5 with the corresponding Km and Vmax of 1.252 mg/mL and 28.01 ?mol/min respectively.Only Na+and K+ inhibited lipase activity among all metal ions tested.In addition,formaldehyde had an obvious inhibitory effect on lipase.3.According to the results of scanning electron microscopy?SEM?analysis,esterase and lipase had an effect of degradation on the wings of whiteflies that revealed Aschersonia could be applied to biological control.Meanwhile,the parmeters of culture in 5L fermentor also provided some scientific data for industrial production of esterase and lipase.