Heterologous Expression, Enzymatic Properties And Structure Study Of Aspergillus Niger GZUF36 Extracellular Lipase

Lipase is a multifunctional enzyme that can catalyze hydrolysis and synthesis reactions.It is considered an excellent biocatalyst and widely used in various industries.Previous studies have found that the activity of sn-1,3 selective extracellular lipase(EXANL1)from Aspergillus niger GZUF36 remains stable in a wide range of pH,and has high sn-1,3 selectivity to substrates,which has good application value.However,the enzyme production of Aspergillus niger GZUF36 is unstable.The culture cycle is long.The study on the enzyme properties is not sufficient,and the selective mechanism of the substrate is not clear from the structure.To solve the above problems,firstly,Aspergillus niger GZUF36 strain sn-1,3selective extracellular lipase(EXANL1)was used as the research object.It achieved the fusion expression of lipase EXANL1 and GST tag in E.coli.Secondly,after the recombinant expressed fusion protein was separated and purified,the enzymatic properties of lipase EXANL1 were studied in detail.Finally,the structure of EXANL1 was characterized based on circular dichroism(CD).The substrate selectivity of EXANL1 was studied by molecular docking,and the possibility of crystallization of EXANL1 was predicted by using the software for predicting the possibility of crystallization.These provide a theoretical basis and foundation for the further industrial application direction and three-dimensional structure research of EXANL1.The main research contents and results are as follows:(1)Cloning and expression,purification and inclusion body renaturation of EXANL1 in Escherichia coliAccording to the Aspergillus niger lipase gene ANL(NCBI accession number:MK592411.1)reported earlier in the laboratory,a GST tag was added to the N-terminus of ANL,and a p GEX-4T-1 vector was used.The gene encoding mature peptide was successfully expressed in E.coli BL21(DE3)at low temperature.The effects of different conditions on the expression of lipase in recombinant Escherichia coli were studied The specific activity of lipase reached 3.74 U/mg,which was 2.7times higher than the initial culture condition when the concentration of IPTG was0.05 mM,OD₆₀₀was 0.6,the post-incubation time was 16 h,and the incubation post temperature was 16?.By glutathione resin and anion exchange chromatography,the electrophoretic pure lipase EXANL1 was obtained.Among them,the pH value of the glutathione resin binding buffer was studied.The pH value of the binding buffer was7.3,and it was found that the contaminated protein was the least.When the GST tag was removed by thrombin cutting,the cutting amount of thrombin was tested.The protein content was 0.03 mg/mL,2 u L(0.1 U)thrombin could completely cut the GST tag.The renaturation of inclusion bodies was carried out by dialysis renaturation,and the active protein was obtained.Compared with the solution of inclusion bodies without renaturation,the activity of EXANL1 after renaturation was 8.66±0.30U/mL.(2)Enzymatic properties study of EXANL1The measured enzymatic properties show that the optimum temperature and optimum pH of recombinant EXANL1 are 40?and 4,respectively.At 60?,the residual activity sharply declined and was inactivated entirely within 15 min.The pH stability test indicated that for long-term(48 h)incubation of the enzyme in pH6.0-9.0 buffer,EXANL1 remained more than 55%of the enzyme activity.These results indicated that the enzyme was not tolerant above the temperature of 50?.The EXANL1 had optimum activity at pH 4 while showing good stability at alkaline pH.At 40%(v/v),the EXANL1 still exhibited high tolerance when incubated with n-hexane,toluene,dimethyl sulfoxide and glycerol.It retained between 75%and104%of its original activity.The residual enzyme activity of EXANL1 was over 85%in the surfactants with low concentration(0.1%m/v).EXANL1 has high tolerance in the surfactants of 5%(w/v)CTAB and polyethylene glycol,maintaining 79.71%and109.73%initial activity of the enzyme,respectively.Both low and high concentrations of PMSF,DTT,and?-mercaptoethanol inhibit the activity of EXANL1.5mM Cu²⁺stimulated lipase activity while 1mM Fe³⁺inhibited enzyme activity.Besides,selectivity of EXANL1 to different substrates experiment indicated EXANL1 had higher specificity for the p-nitrophenyl palmitate than other substrates.(3)Analysis of substrate specificity mechanismThe secondary structure of EXANL1 at different temperatures was studied by circular dichroism.The proportion of alpha-helix decreased significantly at temperatures above 55?.Molecular docking result showed that the binding pockets of lipase EXANL1 were similar to other sn-1,3-selective lipases.Its shape is like a peanut shell and has two pockets.Compared with the catalytic pockets of other lipases with no significant regional selectivity,it is speculated that the differences of pocket size and shape may affect the regional selectivity and the preference of lipases with different carbon chain lengths.Compared with the ester bond at the position of the substrate sn-2,the distance between the hydroxyl oxygen on the triad ser173 of EXANL1 and the carbonyl carbon on the substrates sn-1 and sn-3 is closer,which can initially explain why EXANL1 is an sn-1,3 selective lipase.The molecular docking study of EXANL1 on esters of fatty acids with different chain lengths showed that lipase has the lowest binding energy with p-nitrohexanoate,in contrast,p-nitrohexanoate does not form intermolecular hydrogen bonds with the catalytic triad.Molecular docking showed that the specificity of EXANL1 for p-nitropalmitate was the highest,which was consistent with the experimental results.The crystallization possibility prediction software predicts that exanl1 has the possibility of crystallization,which can be used for further crystallization experiments.