The Purification And Fundamental Characterization Of Pectolytic Enzyme From Erwinia Carotovora CXJZU-120

Pectolytic enzyme can be widely used in food, fodder, textile, medical and other industries. A high-yielding pectolytic enzyme strain named CXJZU-120 is selected from more than 300 strain resources by using optional culture medium. Seed culture medium and enzyme-yielding culture medium is modified by investigateing the growth and enzyme-yielding characteristic of CXJZU-120. When cultured 6 hours in seed culture medium and 9 hours in enzyme-yield culture medium, the crude pectolytic enzyme activity of CXJZU-120 reached 158 U/ml, and it is 66% higher than which fermented by strain CXJZ95-198. DNS method was adopted to systematically analyse the effect of different reaction conditions on crude pectolytic enzyme activity of CXJZU-120, the optimum reaction conditions run as follow: temperature 50℃, concentration of pectine from citrus fruits (Sigma) 0.2%, pH 5.2 (1/15mol·L-1KH2PO4 -Na2HPO4 buffer). Investigation on the synthesis mode is shown that the pectolytic enzyme of CXJZU-120 is continuous synthesis mode. Pectolytic enzyme were separated from fermentation liquid on different ultrafiltrating conditions. The optimum ultrafiltration condition runs as follow: DTT 0.5mmol·L-1 to 1.0mmol·L-1, pressure≤1.0bar, the relative activity raised 20.28%, the recovery ratio raised from 30.91% to 83.90%, and the multiple of purification raised from 4.73 to 5.72 under this condition. The result of Sephadex G-100 gel permeation chromatography and SDS-PAGE shows that the type of protein can be decreased effectively by the process of ultrafiltration. The pectolytic enzyme activity of ultrafiltration condensation liquid can be activated by the ion K+, Ca²⁺, Ba²⁺, Mn²⁺,Co²⁺ and Ag+ while restrained by the ion Cu²⁺, Hg²⁺ and Fe3+, among which the activity can be raised 1.7 times of constrasting by Mn²⁺ while decreased 67% of constrasting by Fe3+.