Discovery And Function Of Pectate Lyase From Paenibacillus Polymyxa KF-1

Pectate lyase?EC 4.2.2.2?catalyzes the cleavage of?-1,4-glycosidic bonds of pectin polymers and has potential applications in the textile industry.In this study,the secreted enzyme extract of Paenibacillus polymyxa KF-1 was identified by LC-MS/MS analysis,four pectate lyases were identified,which belonged to different polysaccharide lyase?PL?families,10,9,3 and 1.In this study,the polysaccharide lyase family 10?PL10?member from P.polymyxa KF-1,named Pp Pel10a,was selected for further study.The gene encoding PpPel10a was amplified from the genome of P.polymyxa KF-1,ligated with pET-28a?+?vector,and transformed into Escherichia coli BL21?DE3?competent cells for recombinant expression.The recombinant enzyme PpPel10a with an N-terminal His6-tag was purified by Ni-NTA agarose column.The recombinant enzyme PpPel10a had a predicted Mr of 45.2 kDa and p I of 9.41.Using polygalacturonic acid?PGA?as the substrate,the optimum reaction conditions of PpPel10a were determined.The optimum pH of PpPel10a was determined to be pH 9.0,and the enzyme remains more than 70%of its activity in the pH range of 6.0-11.0,indicated that PpPel10a was an alkaline resistant pectate lyase.The optimum temperature is 50?,and it remained more than 50%activity in the range of 20-50? after 30 min incubation,which suggested that Pp Pel10a had excellent thermal stability.Besides,Ca²⁺is found to be a critical activator of the enzyme activity.The effect of Ca²⁺on the enzyme activity assay showed that the PpPel10a exhibited the highest activity with 2.5 mM Ca²⁺.The Km,Vmax,and k_cat values of PpPel10a with PGA as substrate were 0.12 g/L,289?mol/min/mg,and 202.3 s^-1,respectively.The application of recombinant PpPel10a was studied.PpPel10a degraded the pectin from orange peel to produce unsaturated monogalacturonic acid and galactooligosaccharide.PpPel10a reduced the viscosity of PGA.Besides,the application of PpPel10a on degumming of ramie was studied.The weight loss of ramie fibers treated with enzyme alone or with enzyme combined with alkali was significantly lower than that treated with 1 U/m L PpPel10a combined with 0.5%NaOH.In this process,the use of alkali reduced by half,which showed excellent environmental compatibility.The alkali-resistant pectate lyase PpPel10a may have potential application value in plant fiber processing.Furthermore,to improve the stability and reuse rate,the recombinant enzyme PpPel10a was immobilized.The single factor of the immobilized enzyme was optimized,and three factors,including chitosan concentration,glutaraldehyde concentration,and enzyme concentration,were selected for response surface analysis.The results showed that when the concentration of chitosan,glutaraldehyde,and enzyme was 3%,4%,and 0.8 mg/g respectively,the activity of immobilized enzyme reached the highest.The immobilized rate of the immobilized enzyme was 87.3%.The feasibility of immobilized PpPel10a observed by SEM and FTIR indicated that the enzyme was successfully immobilized.The stability of the immobilized enzyme was compared with that of free enzyme.It was found that the residual activity of the immobilized enzyme was higher than that of free enzyme.At pH 11.0,the residual activity of the immobilized enzyme was approximately 70%,while that of free enzyme is only about 50%.Also,immobilized PpPel10a was more stable in a wider pH range than the free enzyme.After incubated at 80? for 30 min,almost all of the free enzymes were inactivated,while the activity of the immobilized enzyme remained about 50%.These results would be useful for the application of PpPel10a in the textile industry.