| 摘要: | 以蝦殼粉為唯一碳/氮源，自台灣土壤篩選到Chryseobacterium indologenes TKU014此株蛋白酶生產菌。
C. indologenes TKU014在含有0.5% 蝦殼粉、0.05 % MgSO4．7H2O及0.1 % K2HPO4(pH6)的液體培養基，於30℃培養一天，可得到較高的蛋白酶活性(0.44U/mL)。
所得發酵液之離心上清液經過硫酸銨沉澱、離子交換樹脂層析法及疏水性層析分離等純化步驟，得到三種蛋白酶P1、P2和P3，經SDS-PAGE測得分子量分別為56kDa、40kDa和40kDa。最適反應溫度分別為30~50℃、40℃和40~50℃，最適pH均偏鹼性分別為pH10、pH8和pH9，其pH安定性為pH5~11、pH6~8和pH8~10，熱安定性則為<50℃、<40℃和<40℃。
三種蛋白酶的活性均受Mn2+、Cu2+、Fe2+的抑制。P1、P2、P3均受EDTA和1,10-phenanthroline的抑制，判定，P1、P2和P3皆屬Zn-金屬型蛋白酶。
基質特異性方面，對酪蛋白、彈性蛋白和角蛋白為基質時，蛋白酶P1、P2和P3具有較佳的活性，對於白蛋白、纖維蛋白、血球蛋白、偶氮白蛋白、偶氮酪蛋白的活性不佳。
Chryseobacterium indologenes TKU014, a protease-producing strain, was isolated from the soil in Taiwan, by using shrimp shell powder (SSP) as the sole carbon/nitrogen source.
The optimized conditions for protease production was found when the culture was shaken at 30℃ for one day in 50mL of medium (pH6 ) containing 0.5% SSP, 0.1% K2HPO4, 0.05% MgSO4．7H2O
Three proteases (P1, P2, and P3) were purified from culture supernatant by ammonium sulfate precipitation, ionic exchange of DEAE-sepharose CL-6B chromatography and Phenyl Sepharose hydrophobic interaction. The molecular mass of TKU014 proteases (P1, P2, and P3) determined by SDS-PAGE was approximately 56 kDa, 40 kDa, and 40 kDa, respectively. The three proteases (P1, P2, and P3) were found to have optimum temperature at30~50℃,40℃,and40~50℃; optimum pH at 10, 8 and 9; thermal stability lower then 50℃, 40℃ and 40℃ ; pH stability at pH 5~11, pH 6~8 and pH 8~10, respectively.
The activity of three proteases (P1, P2, and P3) was completely inactivated by EDTA and 1,10-phenanthroline, so the three proteases (P1, P2, and P3) were Zn-metalloprotease.
As for substrate specificity, these three proteases showed good activity toward casein, elastin and keratin azure as substrates, low activity with hemoglobin, and poor activity with albumin, fibrin, hemoglobin, azocasein, albumin. |
