鳳梨酵素所含蛋白酶及幾丁質酶之純化與定性

Purification and Characterization of Proteases and Chitinases from Bromelain

陳芊蓓(Chien-Pei Chen)

指導教授 ： 王三郎

淡江大學/理學院/生命科學研究所碩士班/碩士(2006年)

https://doi.org/10.6846/TKU.2006.00094

摘要

本研究主要由鳳梨酵素純化分離幾丁質酶與蛋白酶並探討其生化特性。鳳梨酵素經離子交換及膠體過濾層析，可分離出二種蛋白酶（P1、P2）、二種幾丁質酶（C1、C2）和一個蛋白酶/幾丁質酶（PC1）的酵素。
以酪蛋白為基質所測得P1、PC1、P2之最適反應pH分別為6、7、8，最適溫度分別為40℃、70℃、60 ℃。P1在50℃以下和pH 4∼7，PC1與P2分別在50℃以下與pH 4∼10之間具安定性。基質特異性分析測得三種酵素所含蛋白酶對酪蛋白及偶氮酪蛋白二種基質有較高的水解活性。三種蛋白質酶的活性皆受Cu2+和Zn2+所抑制，此外，PC1和P2會被Fe2+所抑制。P2活性受EDTA和PMSF強烈抑制，而PMSF亦抑制PC1部份活性。以SDS-PAGE分析出來的分子量為26 kDa、28 kDa以及26 kDa之P1、PC1以及P2之蛋白酶。P1蛋白酶的Vmax與Km分別為0.2 U/mL與1.96 mg/mL；PC1蛋白酶的Vmax與Km分別為0.13 U/mL與5.26 mg/mL；P2蛋白酶的Vmax與Km分別為0.16 U/mL與12.7 mg/mL。Lee-S11Me和Lee-S8SO二種化學合成化學品對鳳梨酵素之蛋白酶活性具抑制效果。
C1、PC1和C2三種純化出之幾丁質酶中，PC1和C2之最適pH為4，而C1則為pH 5，最適溫度皆介於50∼60 ℃。安定性方面，三者皆於偏酸，溫度25∼60 ℃間較穩定。PC1和C2之幾丁質酶活性會受EDTA所抑制，此外，PC1亦會受Fe2+和Mn2+所抑制。SDS-PAGE分析C1、PC1以及C2所得的分子量分別為26 kDa、28 kDa以及28 kDa。C1幾丁質酶的Vmax與Km分別為0.04 U/mL與55.56 mM；PC1幾丁質酶的Vmax與Km分別為0.05 U/mL與28.57 mM；C2幾丁質酶的Vmax與Km分別為0.04 U/mL與125 mM。化學合成化學品則以Lee-O12SO、Lee-O7C、Lee-S10C2和Lee-O701C對鳳梨酵素之幾丁質酶活性具抑制效果。

關鍵字

幾丁質酶 ； 蛋白酶 ； 鳳梨酵素

並列摘要

The purification and characterization of chitinases and proteases from the commercial bromelain preparation was studied. Two chitinases (C1 and C2), two proteases (P1 and P2), and a chitinase/protease (PC1) were purified using DEAE-Sepharose ion-exchange chromatography, CM-Sepharose ion-exchange chromatography, and gel filtration on a Sephacryl S-100 column.
The protease of P1, PC1 and P2 was found to have a pH optimum at 6, 7 and 8, and a temperature optimum at 40℃, 70℃, 60℃, respectively, as acting on casein. The protease was stable at 25 ~ 50℃ and pH 4 ~ 7（P1）, pH 4∼10（PC1 and P2）. Among several natural substrates tested, casein and azocasein were the best substrates. The activity of three proteases was inhibited by Cu2+ and Zn2+. In addition, the activity of PC1 and P2 protease was inhibited by Fe2+. The activity of P2 protease was strongly inhibited by EDTA and PMSF, while the activity of PC1 protease was also inhibited by PMSF. The apparent molecular mass based on SDS-PAGE was estimated 26 kDa, 28 kDa and 26 kDa for P1, PC1 and P2 protease enzyme, respectively. The Vmax and Km were 0.2 U/mL and 1.96 mg/mL, respectively, for the P1 protease enzyme, and they were 0.13 U/mL and 5.26 mg/mL, respectively, for the PC1 protease enzyme. The Vmax and Km were 0.16 U/mL and 12.7 mg/mL, respectively, for the P2 protease enzyme. The protease activity on crude enzyme was inhibited by Lee-S10Me and Lee-S8SO.
The chitinase of PC1 and C2 was found to have a pH optimum at 4, but the chitinase of C1 was at 5; all of them were found to have a temperature optimum at 50 ~ 60℃ as acting on suspended chitin. The chitinase was stable at 25 ~ 60℃ and preferential towards acidity. The activity of PC1 and C2 chitinase was inhibited by EDTA; while the activity of PC1 chitinase was inhibited by Fe2+ and Mn2+. The apparent molecular mass based on SDS-PAGE was estimated 26 kDa, 28 kDa and 28 kDa for C1, PC1 and C2 chitinase enzyme, respectively. The Vmax and Km were 0.04 U/mL and 55.56 mM, respectively, for the C1 chitinase enzyme, and they were 0.05 U/mL and 28.57 mM for the PC1 chitinase enzyme. The Vmax and Km were 0.04 U/mL and 125 mM, respectively, for the C2 chitinase enzyme. The chitinase activity on crude enzyme was inhibited by Lee-O12SO、Lee-O7C、Lee-S10C2 and Lee-O701C.

並列關鍵字

chitinase ； protease ； Bromelain

參考文獻

6. 張薏苓，2000。鳳梨酵素粗製品水解幾丁聚醣之研究。靜宜大學食品營養學系研究所碩士論文。

10. 曾惠婷，2005。幾丁聚醣之鳳梨酵素水解產物於抗腫瘤及抗菌之效果探討。淡江大學生命科學系碩士論文。

15. Adler-Nissen, J. 1986. Enzymatic hydrolysis of food proteins. pp. 19-20. Elselier Applied Science Publ. London and New York.

17. Arakane, Y., and D. Koga. 1999. Purification and characterization of a novel chitinase isozyme from yam tuber. Biosci. Biotechnol. Biochem. 63: 1895-1901.

18. Araki, Y., and E. Ito. 1974. A pathway of chitosan formation in Mucor rouxii: enzymatic deacetylation of chitin, Biochem. Biophys. Res.Commun. 56: 669-675.