Serratia ureilytica TKU013所生產蛋白酶及幾丁質酶之純化與定性

Purification and Characterization of Proteases and Chitinases from Serratia ureilytica TKU013

林金霖(Cing-Lin Lin)

指導教授 ： 王三郎

共同指導教授 ： 李世元

淡江大學/理學院/生命科學研究所碩士班/碩士(2007年)

https://doi.org/10.6846/TKU.2007.00447

摘要

TKU013係以烏賊軟骨粉為唯一碳/氮源，篩選自台灣北部土壤之一株蛋白酶及幾丁質酶生產菌，經鑑定為Serratia ureilytica。TKU013生產蛋白酶和幾丁質酶之較適培養條件為，於含有1.5 %烏賊軟骨粉、0.1 % K2HPO4及0.05 %MgSO4．7H2O之50 mL液體培養基(pH 9)，於25℃搖瓶培養3天，可同時獲得較高蛋白酶活性(0.19U/ml)和幾丁質酶活性(0.021U/mL)。將發酵所得上清液經硫酸銨沉澱、DEAE- Sepharose、Sephacryl S-100和Phenyl-Sepharose等層析步驟，可純化出二種蛋白酶(P1、P2)和一種幾丁質酶(C1)，經SDS-PAGE測得分子量分別為50 kDa、 50 kDa及60 kDa。P1、P2、C1之最適反應溫度、最適pH、熱安定性、pH安定性分別為(40℃、pH 10、<50℃、pH 7∼11)，(50℃、pH 10、<40℃、pH 8∼11)，(50℃、pH 6、<50℃、pH 5∼8)。幾丁質酶活性受Zn2+、Cu2+完全抑制；而蛋白酶在Mg2+的存在下，約剩原活性的1/3。在2% (v/v) Tween20、Tween40或Triton X-100的存在下，P2、C1幾乎不受影響，P1則受到抑制；第4天的發酵上清液，具有較佳的DPPH自由基清除力。

關鍵字

Serratia ureilytic ； 烏賊軟骨粉 ； 幾丁質酶 ； 蛋白酶 ； DPPH

並列摘要

Serratia ureilytia TKU013, a protease and chitinase producing strain, was isolated from the soil in the north Taiwan. The optimized condition for protease and chitinase production were found when the culture was shaken at 25℃ for 3 days in 50 mL of medium containing 1.5% squid pen powder(SPP), 0.1% K2HPO4 and 0.05% MgSO4•7H2O at pH 9. Two proteases(P1,P2) and a chitinase(C1) were purified from the culture supernatant by chromatography procedures of DEAE-Sepharose, Sephacryl S-100 and Phenyl-Sepharose. The molecular mass of P1, P2 and C1 determined by SDS-PAGE was approximately 50 kDa , 50 kDa and 60 kDa, respectively.
The optimum temperature, optimum pH, thermal stabilities and pH stabilities of P1, P2 and C1 were (40℃, pH 10 <50℃, pH7~ 11),( 50℃, pH 10, <40℃,pH 8~11)and( 50℃,pH 6, <50℃,pH 5~8), respectively. The chitinase(C1)was completely inactivated by Zn2+, Cu 2+. Both proteases retained 35% of its original activity in the presence of Mg2+. C1 was activated in the presence of 2% (v/v) Tween 20, 2% (v/v) Tween 40 or 2% (v/v) Triton X-100. The protease P2 retained 80% of its original activity in the presence of 2% (v/v) Tween 20 and 2% (v/v)Tween 40, respectively. The supernatant of the fourth day showed better DPPH radical scavenging activity.

並列關鍵字

Serratia ureilytia ； protease ； chitinase ； squid pen powder

參考文獻

3.Anwar A, Saleemuddin M (1998) Alkaline protease：a review. Biore Technol. 64：175-183.

4.Allan CR , Hadiger LA (1979) The fungicidal effect of chitosan on fungi of varying cell wall composition. Experi mycol. 3：285-287.

5.Andrew TG, Michael GH, Adrienne H (2005) Production of chitinolytic enzymes by Serratia marcescens QMB1466 using various chitinous substrates. J Chem Technol Biotechnol. 80：28-34.

6.Baumann U, Bauer M, Létoffé S, Delepelaire P, Wandersman C (1995) Crystal Structure of a Complex Between Serratia marcescens Metallo-protease and an Inhibitor from Erwinia chrysanthemi. J. Mol. Biol. 248：653-661.

7.Burd W, Yourkevich O, Voskoboev AJ, K.-H. van Pée (1995) Purification and properties of a non-heam chloroperoxidase from Serratia marcescens. FEMS Microbiol. Lett. 129：255-260.