Isolation, Purification, Characterization Of Polygalacturonase Of Sclerotinia Sclerotiorum And Cloning,Expression Of The Pathogen Polygalacturonase Genes

Five cell wall degrading enzymes such as polygalacturonase (PG), polymethylgalacturonase (PMG), cellulase (Cx), polygalacturonic acid trans-eliminase (PGTE) and pectin methyltrans-eliminase (PMTE) could be produced in the improved Marcus’s medium by Sclerotinia sclerotiorum causing rape stem rot. Among them, PG had the highest enzyme activity with249.11U/mL. The crude PG in the culture filtrate could be precipitated with acetone and obtained by centrifugation (8000r/min) at4℃for15min, and successively purified by DEAE Sepharose Fast Flow ion exchange column, Phenyl-Sepharose6Fast Flow hydrophobic column and Sephadex G-75gel column. The molecular weight of the purified PG was37.17kD by SDS-PAGE and the pI value was6.14by IEF-PAGE. The PG was demonstrated as a kind of glycoprotein that contained2.51%saccharide, but no lipid and aromatic amino acids. The PG activity was observed in the range of pH value from3to11, and the strongest one was at pH5. The PG was not stable in the higher temperature (>40℃) and lost its activity in the treatment of100℃for20min. The PG was also sensitive to ultraviolet radiation, chloroform, trypsin and proteinase K.Two pairs of primers was designed to amplify the complete sequences according to the pathogen PG gene nucleotides in GenBank (AF501307and AY312510) by polymerase chain reaction (PCR). Two PG genes in the pathogen were cloned and sequenced. Of them, Sspg1d consisted of1084bp, coding326deduced amino acids and SspgS consisted of1701bp, coding482deduced amino acids. When being blasted, Sspg1d and Sspg3showed a100%similarity to pg1d and to pg3in the sequences, repectively.The PG genes Sspg1d and Sspg3were constructed in the expression vector pET-28a(+) and resulted in the recombinant plasmids pETSspg1d and pETSspg3, repectively. They were transferred in E. coli BL21(DE3) and expressed by IPTG induction. The PG activity of764.35U/mL was detected when BL21containing pETSspg1d was cultured in LB medium and induced with0.4mM IPTG. When BL21containing pETSspg3was cultured in the same condition, the PG expressed was detected with the activity of346.72U/mL.