Screening Of Acidic Pectin Lyase Gene And Its High-level Recombinant Expression In Aspergillus Niger

Acidic pectin lyase can effectively degrade the pectin substances via?-elimination reaction.It has been widely used in food industry,especially in fruit-processing industry,which helps to clarify fruit juice and increase its yield.Acidic pectin lyase is mainly produced by fungi,especially Aspergillus niger.However,as for the wild A.niger strain,the expression level and the purity of acidic pectin lyase remains low,so it cannot meet with the industrial demand.This study aimed to achieve the high-level recombinant expression of the acidic pectin lyase in A.niger SH-2 with low-background of secreted proteins,by screening the five acidic pectin lyase genes in A.niger genome?pelA?pelB?pelC?pelD?pelF?.And the enzymatic properties of recombinant acidic pectin lyase were studied,as well as the application of recombinant acidic pectin lyase on the clarification of fruit juices.The main research contents are as follows:?1?In this study,five pectin lyase genes?pelA,pelB,pelC,pelD and pelF?from A.niger pectin lyase gene family were cloned and over-expressed under the control of the A.niger glucoamylase promoter?PglaA?in A.niger strain SH-2,respectively.Enzyme activity assay was used to select the recombinant strain with the highest expression level.Results showed that the pelA and pelD recombinant strain SH2-PelA and SH2-PelD could expressed acid pectin lyase efficiently.In flask fermentation,the acidic pectin lyase activity of SH2-pelA and SH2-pelD achieved 11069.2 U/mL and 8822.6 U/mL.In submerged fermentation,the enzyme activity reached 65148.8 U/mL and 35670.0 U/mL,improved by 5.9 fold and 4.0 fold,respectively.Therefore,the recombinant strain SH2-PelA and SH2-PelD owned the potential of large-scale industrial production of acidic pectin lyase.?2?The recombinant PelA?rePelA?and PelD?rePelD?were purified by Ni-affinity chromatography and specificity of protein was confirmed by western blotting.After one-step purification,rePelA was purified by 5.7-fold,with a 88.4%yield and a specific activity of4618.0 U/mg.rePelD was purified by 4.9-fold,with a 79.4%yield and a specific activity of8522.7 U/mg.The rePelA and rePelD show increasing specific activity as the degree of methyl-esterization of the substrate raises.The maximum activity of rePelA?22423.3 U/mL?and rePelD?31248.0 U/mL?was obtined using citrus pectin??85%esterified?as the substrate.The optimum temperature of rePelA and rePelD were 50?,and they were thermostable in the temperature range from 30?to 50?.The optimum pH of rePelA and rePelD were 4.5 and5.0,respectively,and they were stable under acidic pH conditions.The activity of rePelA was enhanced by Na⁺,Mg²⁺,Zn²⁺,Ni²⁺,Mn²⁺and Ba²⁺,and was inhibited by Ca²⁺,Cu²⁺and SDS.And addition of Zn²⁺and Mn²⁺could increase rePelA activity by 30%.The activity of rePelD was enhanced by Ca²⁺,Mg²⁺,Cu²⁺,Zn²⁺,Ni²⁺,Mn²⁺and Ba²⁺,and was inhibited by SDS.?3?The rePelA and rePelD were applied in the clarification of juice.Results showed that the rePelA and rePelD owned noticeable clarifying effects on orange,apple and grape juice.After the treatment by rePelA,the light transmittance of orange,apple and grape juice was increased 19.2-fold,7.3-fold and 3.8-fold,respectively.And after the treatment by rePelD,the light transmittance of orange,apple and grape juice was increased 17.9-fold,11.5-fold and5.7-fold,respectively.Furthermore,the combination of rePelA with other pectinases achieved a much better clarifying effect on juice with much shorter reaction time.