Property Chatacerization, Purification And Biocontrol Effect Of Extracellular Protease Produced By Coniothyrium Minitans

Coniothyrium minitans is an important biocontrol agent of Sclerotinia sclerotiorum. During the process that C. minitans parasited SI sclerotiorum, C. minitans would secret many extracellular enzymes including glucanase, chitinase and protease. The biocontrol function of glucanase and chitinase during interaction between C. minitans and S. sclerotiorum had been well studied in previous researches, however, the bicocontrol function of protease remain to be determined. In order to clarify the role of protease during interaction between the two organisms, A study was carried out on the protease production conditions, protease properties, protease purification and the effect of the crude extraction of protease on S.sclerotiorum mycelium and protoplasts.Five media, namely potato dextrose broth (PDB), potato broth (PB), S. sclerotiorum extracts (Ss), potato broth S. sclerotiorum extracts (PBSs) and potato dextrose broth S. sclerotiorum extracts (PDBSs) were used for comparing the yield of the protease. Results showed that the medium Ss was the most suitable medium for protease production by C. minitans. In addition, the result also showed that glucose inhibited the production of protease.The activity of protease produced by C. minitans were measured quantitatively by Folin phenol method and the factors affected the protease activity were also determined. The results were:the optimum temperature of the crude protease was 60℃, the optimum pH was 7, and the protease was stable bellow 40℃.5 mM Na+, K+, Li+, Mg2+, Zn2+, Ca2+, Cu2+, Mn2+ had no effect on protease activity,5 mM Fe2+ could enhance the protease activity, whereas 250mM would inhibit the the protease activity. The protease was highly sensitive to PMSF (phenylmethyl sulfony fluoride) indicating it belonged to the serine protease family.The proteinase was purified by ammonium sulfate, dialysis, Q sepharose Fast Flow chromatography. Its purity was checked to be about four bands by SDS-PAGE. The protease activity could be detected. Then, using ultrafiltration and HiPrep 16/60 sephocryl s-100 High Resolution chromatography, it was checked to be a single band by SDS-PAGE, but the protease activity could not be detected. This problem needed to be resolved in further study. After a Q sepharose Fast Flow chromatography, S. sclerotiorum mycelium and protoplasts we're treated with the colletion that showed protease activity, the result showed that the colletion could degrade S. sclerotiorum mycelium and protoplasts. All the rusults indicate that the protease produced by C. minitans may play an important role in the infection of S. sclerotiorum by C. minitans.