白腐真菌发酵中胞外蛋白酶对MnP的产生及其稳定性的影响

The influence of protease on manganese peroxidase(MnP) stability during white rot fungus culture

在摇瓶试验中通过投加4种对应不同蛋白酶的抑制剂,研究了白腐真菌Phanerochaete chrysosporium固定化发酵过程中所产的胞外蛋白酶对其MnP的产生以及稳定性的影响.在培养1d后投加Pepstatin A可使MnP峰值提高且提前1d出现,而投加PMSF、EDTA后MnP峰值出现时间不变但峰值皆下降;培养4d后投加PMSF和Pepstatin A可使MnP酶活提高且稳定性增强;培养过程中投加HgC l2均会抑制菌体生长,使MnP酶活水平较低.在培养1d后直接补入初级蛋白酶浓缩液,MnP峰值酶活稍有提高,且第3d补入初级蛋白酶浓缩液会使MnP提前1d出现.在离体条件下,EDTA和HgC l2均可抑制MnP酶活;初级蛋白酶和次级蛋白酶均会导致MnP不稳定,但投加PMSF和Pepstatin A后,MnP稳定性增强.以上结果表明,在培养过程中初级蛋白酶部分组分对MnP的产生过程具有促进作用;初级蛋白酶和次级蛋白酶会导致离体MnP迅速失活,且证实其中2种组分——丝氨酸蛋白酶和天冬氨酸蛋白酶——可导致离体MnP不稳定.

Effect of extracellular protease on MnP production and stability was investigated through addition of 4 protease inhibitors corresponding to 4 different proteases. Tests were performed on immobilized cultures of Phanerochaete chrysosporium in agitated Erlenmeyer flasks. Pepstatin A addition on the 1^st day of culture increased the maximum MnP value, which also moved up the MnP peak moment; However, PMSF and EDTA addition decreased the maximum MnP activity while keeping the same peak moment. PMSF and Pepstatin A addition on the 4th day helped stabilize MnP by retaining its activity on a higher level. HgCl2 addition on the 1^st and 4th days both showed inhibition of biomass growth, which subsequently caused low MnP activity. When concentrated primary protease was added, a peak of higher MnP activity appeared. Particularly, addition of concentrated primary protease on the 3^rd day caused MnP emergence 2 days earlier. Of all the protease inhibitors, EDTA and HgCl2 inhibited MnP activity in vitro. Both primary and secondary proteases destabilized MnP but PMSF and Pepstatin A addition helped keep MnP stable to some extent. These results indicated： （1）some component of primary protease favors MnP production during the culture; （2）both primary and protease destabilize existing MnP; （3）MnP destabilization is caused by serine protease and aspartic protease in both primary and secondary proteases.