A New Cold-active And Alkaline Pectate Lyase With High Catalytic Efficiency And Mutations For Stability

Pectate lyase is an enzyme that cleaves polygalacturonic acid alpha-1,4-glycoside bonds.It can hydrolyze pectin and pectin effectively.It has great application value in papermaking,textile,food,feed and washing.At present,most of the pectate lyase used in the industry are belong to the medium-temperature or high-temperature pectinase,and the reaction energy consumption is high.Therefore,it is very meaningful to find a new cold-active pectate lyase.1,Cloning,enzymatic characterization and optimization of fermentation conditions of cold-active alkaline pectate lyase PEL1.Massilia eurypsychrophila is a cryogenic bacterium isolated from the Asman Hills,Antarctica.After genome sequencing,a new pectate lyase gene was found,and its coding product was annotated as pel1.Sequence analysis showed that it belonged to the 6th superfamily of polysaccharide hydrolases and had the highest 66%sequence identity with the reported Pectate lyase(PL_Xc).The recombinant protein was about34.7 KDa.It had high activity to polygalacturonic acid.The optimum reaction temperature was 30?,and the optimum p H was 10.0.The relative activity of PEL1 was over 60%between 10?to 40?.Especially PEL1 had 25%activity at 0?.More than 70%activity was retained after remining for 30 min in 30?,but almost completely lost activity in 10minutes after pretreatment at 40?.The specific enzyme activity of PEL1 was 78.75 U/mg,the kinetic parameters K_mwas 0.930 g/L,V_maxwas 33.6?mol/min,and k_catwas 46.67/s.The specific enzyme activity increased by 1.24 times when the concentration of calcium ion was0.5 m M in the reaction system.By optimizing the fermentation conditions such as induction temperature,IPTG concentration during induction and different fermentation media,it was determined that the best secretory expression effect could be achieved by shaking flask at 18?for 30 hours using TB medium.2,The preliminary exploration of the low temperature mechanism of low temperature alkaline pectin lyase PEL1.First we compared the secondary structure of PEL1 with its highest homologous pectinase(PL_Xc).It was found that irregular curling accounted for more(51%>45%)and beta-folding accounted for less(30%<36%)in PEL1,suggesting that PEL1was more flexible as a cold-active enzyme protein.Therefore,we synthesized the PL_Xcgene and selected four regions for mutation,PL_Xc190-193I,PL_Xc205-219II,PL_Xc279-284III,PL_Xc296-300IV.The reason for the selection of these regions is that there are at least four consecutive amino acids in the amino acid sequence of PEL1 and PL_Xc.The mutation fragments of PL_Xc190-193I and PL_Xc205-219II are close to the active sites,PL_Xc279-284III and PL_Xc296-300IV.The mutant fragments of PL_Xc279-284III and PL_Xc296-300IV are located in the ring region around the protein structure.These sites may be the key factors affecting protein stability.The results showed that the thermal stability of all mutants decreased,and the thermal stability of mutant PL_Xc279-284III decreased the most,indicating that the region had a significant effect on the thermal stability of the enzyme.The optimum p H of mutant PL_Xc279-284 III was further analyzed.The results showed that the optimum p H of PL_Xc279-284 III increased from 8.5 to 9.5,indicating that this region not only affected the optimum reaction temperature of protein,but also affected the optimum reaction p H of protein.The effect of specific sites on the thermal stability of PL_Xcwas further studied by B-factor.Point mutation was carried out at sites with structure less than 10?,relative B-factor greater than 0,irregular curl and amino acid exposure.The mutants were PL_XcD184E/S185K,PL_XcK188L,PL_XcK250R and PL_Xc259-261,respectively.The results showed that the thermal stability of mutant PL_Xc259-261 did not change much,but the thermal stability of mutant PL_XcD184E/S185K,PL_XcK188L and PL_XcK250R decreased sharply at 45?.The activity of mutant PL_XcD184E/S185K decreased to 10%,40%and 30%respectively after 20min treatment.The change of PL_XcD184E/S185K was the most obvious.After 10 min treatment at 45?,the activity of mutant PL_XcD184E/S185K decreased by nearly 50%.After20 min,The activity of wild-type enzymes was almost lost after 30 min treatment at 45?,suggesting that 184 and 185 sites had a great influence on thermal stability.There was researches showed that the half-life of mutant V187I mutated from V187 to I in PL_Xcwas shortened from 54.2 minutes to less than 10 minutes at 45?.The 164I amino acid corresponding to PEL1 was also found to be I by sequence alignment.Therefore,the effect of 164I site on the thermal stability of the enzyme was further studied in this paper.The optimum temperature of mutant V187I,PL_Xcand PEL1 I164V,PL_Xcand PL_XcV187I was determined by sequence alignment.The results show that the optimum temperature of PEL1I164V is raised from 30?to 40?,while the optimum temperature of PL_XcV187I is reduced from 50?to 35?.In addition,PEL1 I164V maintained 44%activity after 10minutes of preheating at 40?,while PEL1 lost most of its activity.This suggests that 164locus may also be one of the important amino acid residues affecting the thermal stability of PEL1.3,Pectate lyase PEL1 thermal stability transformation.Based on the comparison of the structure and sequence of PEL1 and PL_Xcand the analysis of mutation experiments,we selected several sites to mutate on PEL1.PEL1 A50G/S51T,PEL1 L208V,PEL1E184D/K185S,PEL1 L208 were not in PEL1 protein structure.The regular curl position,while PEL1 A8G,PEL1 Y213F and PEL1 A8G/Y213F mutations were based on the reported loci.After mutation,the best mutant is E184D/K185S,which doubles the specific enzyme activity,has little change in the optimum p H.The optimum temperature is 40?,keeps 80%relative activity at 30?,and 20%relative activity at 0?.However,the thermal stability of the mutant is greatly improved compared with PEL1.After 10 minutes at 40?,the relative activity of the mutant is still 80%.Its K_mdecreases to 0.795 g/L and V_maxincreases to 36.9?mol/min,k_catis 112.3/s.The T_mvalue increased from 39.21?to 47.54?.For the experimental analysis of specific enzyme activity and thermal stability of 184 and 185 site mutations,it was determined that both 184 and 185 sites mutations were needed to achieve the best results.This is the cryogenic pectinase with the highest specific enzyme activity reported.In conclusion,this paper discovered a new cold-active pectate lyase gene,which was heterologously expressed,characterized,preliminary investigation of cold-active mechanism and molecular transformation,and we obtained mutants with increased enzyme activity and thermal stability.Making it a better choice for industrial applications.