Culture Optimization, Immobilization And Preliminary Crystal Screening Of Recombinant Aspergillus Niger GZUF36 Extracellular Lipase

Lipase is a multi-purpose hydrolytic enzyme with excellent activity in hydrophobic environment.It is mainly screened from animals,plants and microorganisms,and can be widely used in food processing,fine chemical industry,cosmetics,pharmaceutical industry,biodiesel production and detergent industry.Aspergillus niger GZUF36 extracellular lipase(EXANL1)was successfully expressed in Pichia pastoris by our laboratory.In this paper,the cultivate conditions of recombinant Pichia pastoris EXANL1(PEXANL1)were optimized to improve the activity of the enzyme,which is conducive to subsequent immobilization,purification,crystallization of PEXANL1.Then,the effects of different immobilization conditions on the activity and secondary structure of PEXANL1,the enzymatic properties of PEXANL1 before and after immobilization were studied.A preliminary crystal study of PEXANL1 was conducted,which laid a foundation for further exploration of the structure-activity relationship of PEXANL1.The main research contents and results are as follows:(1)The effect of different culture conditions on the activity of PEXANL1 were investigated,and the optimal conditions were exhibited as follows:under the condition of 3.2%of inoculum amount,BMMY medium pH of 6,culture time of 4 d,methanol content of 1%,the activity of PEXANL1 was 33.80±0.57 U/mL.Then optimized the culture conditions by response surface design,the optimal conditions were as follows:inoculum amount was 3.2%,BMMY medium pH was 6,culture time was 4 d,and methanol content was 1%,under which the maximum enzyme activity of PEXANL1was 92.51±0.03 U/mL,which was six times higher than the activity of original PEXANL1.(2)Using chitosan-Fe₃O₄ magnetic nanoparticles(Fe₃O₄@CS MNPs)as the support,PEXANL1 was efficiently immobilized by adsorption-precipitation-crosslinking strategy to obtain the immobilized lipase(PEXANL1-Fe₃O₄@CS MNPs).Scanning electron microscopy(SEM),Fourier transform infrared spectroscopy(FTIR),X-ray diffraction(XRD),thermogravimetric analysis(TGA)and vibration sample magnetometer(VSM)were used to characterize the synthesis of Fe₃O₄@CS MNPs and the combination of Fe₃O₄@CS MNPs and PEXANL1.The results exhibited that the Fe₃O₄@CS MNPs is successfully binding with PEXANL1.The effects of different immobilization conditions on the activity and secondary structure of PEXANL1 were investigated.Under the conditions of equal volume mixture of acetonitrile and acetone as precipitator,support mass was 125 mg,the ratio of precipitator to enzyme solution was 4:1,glutaraldehyde concentration was 15 mM,and cross-linking time was 1 h,PEXANL1-Fe₃O₄@CS MNPs obtained the highest activity,and the activity recovery was 153.73±2.00%.The secondary structure of the PEXANL1-Fe₃O₄@CS MNPs was investigated.The relative contents of?-helix,?-folding,?-corner and irregular coil were 31.29%,18.54%,20.33%and 29.85%respectively,which were significantly different from those of free PEXANL1.Compared with free PEXANL1,the content of irregular crimp increased by 29.85%,and the relative content of?-helical,?-folding and?-corner decreased by 5.95%,1.66%and 20.44%,respectively.(3)The enzymatic properties of the free PEXANL1 and PEXANL1-Fe₃O₄@CS MNPs were investigated.The results exhibited that the optimal pH of the free PEXANL1 was 4,and the optimal pH of the PEXANL1-Fe₃O₄@CS MNPs was 3,which was shifted to the acidic range.The immobilization strategy improved the pH stability of the PEXANL1,and the activity of the PEXANL1-Fe₃O₄@CS MNPs remained above 65%in the pH range of 2–10.The optimum temperature of PEXANL1-Fe₃O₄@CS MNPs was 45?,which was 5?higher than that of free PEXANL1.The temperature stability of the PEXANL1-Fe₃O₄@CS MNPs was higher than that of the free PEXANL1.The metal ions tolerance of the PEXANL1-Fe₃O₄@CS MNPs to Zn²⁺,Fe²⁺,Fe³⁺,Na⁺,K⁺,Cu²⁺,Mn²⁺,Ca²⁺,Mg²⁺was higher than that of the free PEXANL1.The organic solvent tolerance of the PEXANL1-Fe₃O₄@CS MNPs improved greatly,and the PEXANL1-Fe₃O₄@CS MNPs after treatment with tetrahydrofuran,acetonitrile,tert-butanol and diethyl ether all showed super activity.In terms of storage stability,the PEXANL1-Fe₃O₄@CS MNPs was significantly higher than the free PEXANL1.By measuring the kinetic parameters of free and immobilized PEXANL1,it was found that the substrate affinity and the maximum reaction speed of the immobilized enzyme were improved compared with the free enzyme.The highest reaction rate(Vmax)of free PEXANL1 was 10.64 mmol/L/min,and the highest reaction rate(Vmax)of PEXANL1-Fe₃O₄@CS MNPs was 18.45 mmol/L/min,higher than that of free PEXANL1.The Km value of the PEXANL1-Fe₃O₄@CS MNPs was lower than that of the free PEXANL1,indicating that the affinity of lipase to substrate was enhanced after immobilization.The reusability test for the PEXANL1-Fe₃O₄@CS MNPs maintained a relative activity of about 50%after eight cycles.(4)A total of 98 crystallization conditions were screened with 8 mg/mL protein concentration of PEXANL1.After 21 days of culture,some crystals suspected to be Aspergillus niger lipase crystals appeared.