Heterologous Expression Of Pectin Lyases From Aspergillus Niger ZJ5 In Pichia Pastoris

Pectin is one of important components of plant cell wall,and mainly consists of a backbone of galacturonic acid residues with methyl-esterified to some extent and interspersed with different side chains linked by a-1,4 glycosidic linkages.There are many kinds of enzymes that can degrading pectin,these enzymes are named as pectinases.Pectin lyase is kind of pectinase and can randomly cleaving glycosidic bond to produce unsaturated galacturonide.Aspergillus niger ZJ5 which can produce pectinases was selected in our research.Through 18s rDNA sequence alignment,the deduced amino acid sequence of A.niger ZJ5 shared the highest identity(99%)with that of A.kawachii IFO 4308.Besides,through PF00544 family of pectinase conservative sequence alignment,the deduced amino acid sequence of A.niger ZJ5 shared the highest identity(97%)with that of A.niger CBS 513.88 and A.kawachii IFO 4308.But the whole genome sequences from A.kawachii IFO 4308 has been published in GenBank.According to A.kawachii IFO 4308 genome sequences in GenBank,we designed the primers for pectin lyase genes of A.niger ZJ5 to obtain eight pectin lyase genes and studied the transcription level of these pectin lyase genes.The results of quantitative PCR showed that the transcriptional level of two pectin lyase genes pnl-zj5a and pnl-zj5b,which are closely related to the pectin hydrolysate,were much higher.The pnl-zj5a and pnl-zj5b were heterologously expressed in P.pastoris respectively.Enzymatic characterisation and application of these two pectin lyases in clarification of fruit juices were studied.Pectin lyase genes pnl-zj5a and pnl-zj5b without signal peptide sequence were successfully amplified using A.niger ZJ5 cDNA as template and ligated into the pPIC9 vector.Then,the resulting plasmids were transformed into competent cells of P.pastoris GS115 for heterologous expression respectively.pnl-zj5a and pnl-zj5b were successfully expressed in Pichia pastoris,respectively.Two pectin lyases showed the maximum activity of 4.64 U/ml and 108.9 U/ml in the culture supernatant in a 3-1 fermentor,respectively.The enzyme expression of PNL-ZJ5B is 7000 folds than that of A.niger ZJ5.The pH optima and temperature optima of the recombinant PNL-ZJ5A were 5.0 and 43?,respectively.While the pH optima and temperature optima of the recombinant PNL-ZJ5B were 4.5 and 58?,respectively.The Km and Vmax values of PNL-ZJ5A for pectin from citrus peel were 0.66 mg/ml and 32.6 ?mol·min-1·mg-1,respectively.To PNL-ZJ5B,the Km and Vmax values for pectin from citrus peel were 0.81 mg/ml and 372.8 ?mol·min-1·mg-1,respectively.After treatment with glycosidic enzyme Endo H,the glycosylation degrees of PNL-ZJ5A decreased,while that of PNL-ZJ5B remained same.The substrate specificity analysis of PNL-ZJ5A and PNL-ZJ5B,two pectin lyases showed a better catalytic action on high-esterase pectin.In addition,two pectin lyases were applied to apple juice extracting experiment.They can effectively decrease juice viscosity,and increased the yield of juice and the transmittance.In a word,they showed good application prospect in the fruit juice processing.