Expression And Acid Resistance Modification Of Pectin Lyase Of Aspergillus Niger

Acid pectin lyase（EC 4.2.2.2,pelA）breaks theα-1,4 glycosidic bond in pectin to form oligomeric unsaturated galacturonic acids under acidic conditions byβ-trans elimination.Currently,pelA is used in a wide range of applications such as fruit and vegetable juice processing,brewing material treatment,feed efficiency,and natural product extraction.In the above application,the pH of the reaction system is low（in fruit and vegetable juice processing the reaction pH system is as low as 3.0）.Acid resistance is therefore key to the efficient application of pelA.In this study,we achieved soluble expression of Aspergillus niger（A.niger）AG11-derived pelA in Escherichia coli（E.coli）,based on which we modified its acid tolerance and constructed a recombinant A.niger producing an acid-tolerant pelA mutant.The main findings are as follows:（1）Analysis of the expression and enzymatic properties of pelA in E.coliTo promote soluble expression of pelA in E.coli,protein tags such as xylanase Carbohydrate-binding module（CBM）,Maltose-binding protein（MBP）,Thioredoxins（TrxA）,and Glutathione S-Transferase（GST）were fused at the N-terminus,respectively.Among them,the fusion enzyme CBM-pelA was significantly more soluble after expression,with intracellular pelA activity reaching 3.25 U·mL^-1,a 4.12-fold increase compared to wild-type pelA enzyme activity.Wild-type pelA and CBM-pelA were purified and their enzymatic properties and kinetic parameters of the enzymatic reaction were determined using citrus pectin as a substrate,respectively.The results showed that the specific enzyme activity of CBM-pelA was 29%higher than that of pelA,reaching 15.82 U·mg^-1;the optimum reaction temperature was 50℃ for the former and 40℃ for the latter.Notably,the optimum reaction pH of CBM-pelA was 6.0,1 unit higher than that of pelA;at pH 5.7,CBM-pelA maintained 80.25%activity after heat treatment（2 h incubation at 60℃）compared to 47.59%for pelA.At 40℃,CBM-pelA maintained 84.9%activity after acid treatment（pH 4.0 incubation for 2 h）,while pelA only had 62.87%.Compared to pelA,CBM-pelA had a lower K_m(2.68 mmol·L^-1)and a higher k_cat(37.07 s^-1).（2）Surface charge design improves the acid resistance of pelATo improve the acid resistance of the enzyme,11 sites on the surface of the pelA molecule were replaced with L-aspartic acid and L-glutamic acid,which bind protons in low pH environments,based on Rosetta Supercharge.Four mutants,S118D,S295E,S317D,and S341E,with a fold-free energy change of less than-1 kj-mol-1,were selected for expression and acid resistance analysis（treatment at pH 3.0 and 40℃ for 2 h）.The results showed that the residual enzyme activities of mutants S118D,S295E,S317D,and S341E constructed on the basis of CBM-pelA were increased by 29.11%,19.12%,35.21%,and 44.56%,respectively,compared with CBM-pelA.Different combination mutants were constructed based on the above single point mutations,in which the residual enzyme activity of S317D/S341E was increased by 47.9%compared to CBM-pelA at pH 3.0 and the optimum reaction pH decreased from 6.0 to 4.0.Under the optimum reaction conditions for both,the catalytic efficiency(k_cat/K_m)of S317D/S341E was 1.78 times higher than that of CBM-pelA,although the specific enzyme activity of the former was similar to that of the latter.Analysis by fluorescence spectroscopy revealed an increase in the unfolding of the protein structure in an acidic environment.Molecular dynamics analysis was performed at pH 4.0.The results show that S317D/S341E has a higher flexibility of C-terminal residues near the substrate pocket and a more rigid overall protein structure than CBM-pelA.（3）S317D/S341E expression and property analysis in A.nigerThe pMD-19 vector was used to construct the CBM-pelA and S317D/S341E gene expression frames with P_bglA as the promoter.Using the above vector as a template,a gene fragment containing the CBM-pelA or S317D/S341E gene expression frame and the thaumatin resistance gene（hyg B）was obtained by PCR amplification.The above gene fragments were transformed into A.niger AG11 to obtain recombinant bacteria with genomes randomly integrated with the CBM-pelA or S317D/S341E genes.The extracellular enzyme activities of the recombinant bacteria expressing CBM-pelA and S317D/S341E were 39394 U·L^-1 and41576 U·L^-1,respectively,after 72 h of fermentation at 30℃.In particular,the residual enzyme activity of S317D/S341E after heat treatment（2 h incubation at 40℃）was increased by 38.02%compared to CBM-pelA at pH 3.0.The optimum reaction pH for S317D/S341E is 4.0,a decrease of 1 unit compared to CBM-pelA.Under standard pH conditions（pH 5.7）,S317D/S341E has similar thermal stability to CBM-pelA（over 80%activity at 30-50℃ for 2h）.The specific enzyme activities of S317D/S341E and CBM-pelA were similar,but the catalytic efficiency of the former(k_cat/K_m)was 1.19 times higher than that of the latter.