Study On Alkaline Tolerance Mechanism Of Pectate Lyase And Efficient Expression System

Pectate lyase(EC 4.2.2.2)can degrade pectin-like substances,by β-elimination to break the α-1,4 glycosidic bond of the polygalacturonic acid chain and form unsaturated double bonds at the C5 position of galacturonic acid.Pectate lyase is widely used in papermaking,ramie degumming,biorefining,and treatment of pectin-containing wastewater.It is widely derived from various plants or microorganisms.At present,alkaline pectate lyase in various strains has achieved heterologous expression,but there are not many studies on the mechanism of alkaline resistance of enzymes.In this thesis,the optimal p H value and enzyme activity of the recombinant enzymes were obtained by substituting fragment of the pectate lyase BspPel,and the key regions and sites affecting alkali heat resistance were analyzed.The results obtained were as follows:1.Analysis of using the Signal P 5.0 peptide online prediction site.The signal peptide sequence of the pel gene of Bacillus sp.RN1 was removed to construct the pET28a-pel plasmid.Pel4-N which obtained a sequence similarity of >60 % and strong alkali resistance,was selected for homologous fragment replacement with the K250-P261 loop region of pectate lyase BspPel and the engineering plasmid pET28a-pel-th was constructed.It was heterologous expressed in Escherichia coli BL21(DE3)to obtain the recombinant mutant protein BspPel-th with a molecular weight of 34.3 k Da.Its specific enzyme activity was 27057.5 U/mg,which was 4.4 times higher than that of wild type,and the optimal p H was increased from 10 to 11.Kinetic simulations show that the replaced loop region has greater flexibility and exhibits better substrate binding ability.2.The carbohydrate-binding domain Ye CBM32 that specifically binds to pectin is fused with segments A and B of yellow fluorescent protein,respectively,Plasmids pET20b-eyfp A-ye CBM32 and pET20b-ye CBM32-eyfp B were constructed and converted to Heterologous expression in E.coil BL21(DE3)to achieve recombinant protein expression.The ratio of recombinant fluorescent proteins Eyfp A-ye CBM32 and Ye CBM32-eyfp B bound to pectin was optimized,and a rapid detection system for pectate lyase activity was established.The system had good substrate specificity and shows an inverse relationship between enzyme activity and the fluorescence intensity of the screening system.The mutant BspPel-th/T250 N and BspPel-th/T250 R were obtained and the enzymatic properties showed that the optimal temperature of the recombinase was increased by 5 °C and the thermal stability was improved,among which the optimal p H of BspPel-th/T250 R was raised from 11 to 11.5.3.Through sequence alignment and three-dimensional structural analysis.Based on the reverting mutation of BspPel-th’s L253 and G254,single mutants BspPel-th/L253 I and BspPel-th/G254 V were obtained.The residual enzyme activities after 1 h incubation at 60 °C were 71 % and 66.4 %,which were 54.8 % and 50.2 % higher than BspPel-th,respectively,and the thermal stability of both was improved.BspPel-th/L253I-G254 V did not obtain thermal stability after the two-site mutation,which indicated that the two-site mutation did not obtain the functional superposition effect.The optimal p H of the mutant BspPe-th/R260 S decreased from 11 to 10.5,indicating that 260 arginine,as an alkaline amino acid,plays an important role in the alkaline resistance of enzyme molecules.