• 关键词：白腐真菌  黄孢原毛平革菌  锰过氧化物酶  稳定性  蛋白酶

• 基金项目：国家自然科学基金(No.50478010)

• 熊小平
• 清华大学环境科学与工程系, 环境模拟与污染控制国家重点实验室, 北京 100084

• 文湘华
• 清华大学环境科学与工程系, 环境模拟与污染控制国家重点实验室, 北京 100084

• 白亚楠
• 清华大学环境科学与工程系, 环境模拟与污染控制国家重点实验室, 北京 100084

• 徐康宁
• 清华大学环境科学与工程系, 环境模拟与污染控制国家重点实验室, 北京 100084

• 钱易
• 清华大学环境科学与工程系, 环境模拟与污染控制国家重点实验室, 北京 100084

• 摘要：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PepstatinA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PepstatinA可使MnP酶活提高且稳定性增强;培养过程中投加HgCl₂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HgCl₂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PepstatinA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.

• Abstract：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 Aaddition 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.PMSFand Pepstatin Aaddition on the 4^th day helped stabilize MnP by retaining its activity on a higher level.HgCl₂ addition on the 1^st and 4^th 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 MnPemergence 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 Aaddition helped keep MnP stable to some extent.These results indicated: ①some component of primary protease favors MnPproduction during the culture;②both primary and protease destabilize existing MnP;③MnPdestabilization is caused by serine protease and aspartic protease in both primary and secondary proteases.

• Key words：white rot fungus  Phanerochaete chrysosporium  manganese peroxidase  enzyme stability  protease

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