Cloning And Characetrization Of Pectinolytic Enzymes

Pectinolytic enzymes have become important industrial application enzymes, sothey are used in many industries. Pectinolytic enzymes including protopectinase、polygalacturonic acid enzyme, pectin lyase, pectin esteras widely exist in plants and avariety of microorganisms. In recent years, a lot of studies of pectinolytic enzymeswere discussed by researchers, such as producing strain selection, breeding,identification, fermentation, separation and purification, enzymatic properties, andtheir application. In-depth study of pectinolytic enzymes will increase theirapplications in the food, textile, environment, medicine and other fields, thus theresources can be recycled, are conducive to human health, protect environmental andslow down energy crisis. This would be of great economic and social value.Pectinmethylesterase catalyzed methoxy pectin, changed the number anddistribution of the main chain ester of pectin, transformed pectin into pectic acid,could increase the next other enzymatic effects..In this study, gene ofPectinmethylesterase was amplified from the genome of the Clostridiumphytofermentans ISDg, and was cloned into the pET-20b vector, then the recombinantplasmids were respectively transferred into E. coli BL21, BLP （DE3）, Origami B（DE3） for protein expression. The result was that the expression of soluble protein inthe Origami B （DE3） was higher than in the other two host bacteria. Usinghistidine-tagged in PET-20b vector, protein purification was done through nickelaffinity chromatography.Pectin lyase directly broke highly esterified glycosidic bond of internal pectinthrough the beta-oxidation, no de-methyl ester role of Pectin esterase, which play animportant role in the pectin degradation processes. In this study, the pectin lyaserecombinant plasmid PL3/pET-20bw saved in our chamber were transferred into the E.coli BL21, BLP （DE3）, Origami B （DE3） for protein expression, The result was thatthe expression of soluble protein in BL21is higher than in the other two hostbacterium. Recombinant pectin lyase gene is derived from thermophilic bacteria.Some hybrid protein can be removed when it was treated in70℃, but a small amountof the target protein losed at the same time. Protein Purification was done throughnickel affinity chromatography. The Characterization of purified protein was studied after dialysis. When recombinant pectin lyase reacted with a different pH-substrate,the pH value for optimum reaction was9.5, the relative activity of recombinant pectinlyase was more than70%in pH8.5～10, which proved that this enzyme was alkalinepectin lyase. After the reaction mixture was incubated in the waters at differenttemperatures, the highest activity of the enzyme was shown at75℃. When therecombinant pectin lyase was incubated for8h in waters at60℃, the relative activitywas stil more than80%, which proved that this enzyme is thermophilic pectin lyase.When the reaction solution was added with Ca²⁺of different concentrations, then wasincubated for10min in the waters at70℃, the activity of recombinant pectin lyasewas the best at0.75mM calcium concentration. But the activity of recombinant pectinlyase was no shown when Ca²⁺was not added into the reaction mixture, whichsuggested that this enzyme is calcium ion dependent type.In this study pectinmethylesterase was successfully constructed, and theproperties of pectin lyase were studied. The results showed that the pectin lyase has agood thermal stability and basophilic, those would provide the new enzyme source forindustrial applications.