Purification And Characterization Of A Cold-Adapted Pectinase

A strain producing cold-adapted pectinase was isolated from Jiaozi Snow Mountain which lies in the county of Yi and Miao nationality. It was idengtified as Paenibacillus amylolyticus based on morphological, physiological characteristics and phylogenetic analysis of 16S rRNA. The strain was named as P. amylolyticus P17 (GenBank:EU523226, the number of spawn preservation of invention patent application:CGMCC No.2640).The conditons for enzyme production from strain P17 was optimized with carbon resource, nitrogen resource, pH, teperature and time. The total enzyme activity could be improved by 4.7 times under the optimum conditions. The strain was cultivated at 25℃for 24 hours. The cold-adapted pectinase was purified to homogeneity with 1.1% yield from fermentation broth by centrifugation, ultrafiltration, ammonium sulfate precipitation, DEAE-Sepharose and CM-Sepharose ion exchange chromatography (named as PNL-17). The specific activity of PNL-17 increased from 38.1 U-mg-1 to 451.4 U·mg-1.PNL-17 showed high activity at the temperature range of 0℃-50℃and maximum activity at 40℃. Thermal stability of the enzyme was poor, approximately 80% of the activity was lost at 50℃for 10 min. PNL-17 was stable at pH 6.0-10.0 and showed maximum activity at pH 8.0. Its activity was activated slightly with mental ion such Mg2+,Zn2+,Cd2+ however was inhibited slightly by Ca2+,Ba2+,Pb2+ etc, Hg2+ significantly inhibited the activity of the enzyme. EDTA obviously inhibitly the activity of PNL-17, which indicated that the enzyme exhibited some dependence on mental ion.PNL-17 was stable in many organic solvents even at 95% methanol, ethanol and acetone, the enzyme could keep 61%,83% and 86% residual activity respectively. SDS-PAGE and gel filtration analysis indicated that the molecular mass of PNL-17 was 22.4 KDa and the enzyme was inferred monomeric protein.PNL-17 could not be inhibited by PMSF. It indicated that serine residues were not involved in the active site of this enzyme. The activity of PNL-17 toward substrate was dependent on methyl esterification degree of substrate. The enzyme showed maximum activity on 90% methylated pectin, and can be classified as a polymethylgalacturonate lyase. The Km value of PNL-17 was found to be 1.362 mg·mL-1 at 40℃, however it was 0.233 mg·mL-1 at 4℃, which showed that the enzyme had high affinity with pectin at low temperature. The Vmax and Kcat value of PNL-17 was 54.505 mmol·min-1·L-1 and 7.44×102 S respectively. Compared to other polymethylgalacturonate lyases, PNL-17 had higher activity toward pectin. The Ea value of PNL-17 was calculated to be 33.9 KJ·mol-1, which was higher than only one cold-adapted pectinase (Ea=28.7 KJ·mol-1).PNL-17 not only had a high catalytic activity at low temperature but also good tolerance to metal ions and organic solvents. The enzyme had good specificity toward pectin with high degree of esterification. Based on these characteristics, the enzyme should have potential value in fruit processing industries, cold washing industries, textile industries, biological pharmacy, green chemical industries, wastewater treatment of heavy metal pollution and so on.