Rapid monitoring of autolysis process of proteases by capillary electrophoresis.
A protease, MCP-01, produced by a deep-sea psychrotrophic strain of Pseudoaltermonas sp. SM9913 was purified and its autolysis reaction at 20 degrees C-50 degrees C was monitored by capillary electrophoresis. Capillary electrophoresis provides a rapid assay because the degree and state of autolysis of protease MCP-01 could be observed within 6 min. The autolysis rate increased as the temperature rose in the tested range. After 30 min incubation at 30 degrees C, 77% of MCP-01 autolyzed into peptides. However, its activity for the hydrolysis of casein was reduced by only 4%. The rate of loss of activity of MCP-01 was thus slower than that of autolysis of MCP-01 at 30 degrees C. Similar results were obtained when MCP-01 was incubated at 20 degrees C, 40 degrees C and 50 degrees C. Large peptides produced by autolysis of MCP-01 therefore still have catalytic activity. When these large peptides autolyzed further into smaller peptides, the enzyme conformation that retained its catalytic activity was destroyed and activity was lost.
Chen
Xiu-Lan
XL
State Key Lab of Microbial Technology, Shandong University, Jinan 250100, PR China.
Shun
Cai-Yun
CY
Zhang
Yu-Zhong
YZ
Gao
Pei-Ji
PJ
D001329:Autolysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D010450:Endopeptidases / Q000032:analysis* / Q000737:chemistry*; D004789:Enzyme Activation; D011487:Protein Conformation; D011489:Protein Denaturation; D011786:Quality Control; D013696:Temperature
D010450:Endopeptidases; C477696:protease MCP-01, Pseudoaltermonas