海参肠组织蛋白酶D的提取及酶学特性研究

郭晓坤; 李傲婷; 韩佳润; 杜椅楠; 郭天民; 于翠平; 唐越; 吴海涛

doi:10.13386/j.issn1002-0306.2017.18.026

海参肠组织蛋白酶D的提取及酶学特性研究

大连工业大学食品学院国家海洋食品工程技术研究中心

基金项目:

国家自然科学基金资助项目(31370037)；辽宁省教育厅科学研究一般项目(L2015050)；大连市高层次人才创新支持计划项目(2015R083)；

详细信息

作者简介:
郭晓坤 (1990-) , 男, 硕士研究生, 研究方向:食品生物技术, E-mail:gxkzy2016@126.com。;

吴海涛 (1980-) , 女, 博士, 副教授, 研究方向:食品生物技术, E-mail:wht205@163.com。;

中图分类号: TQ914.2
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出版历程
- 收稿日期: 2017-02-12

Extraction and characterization of cathepsin D from sea cucumber (Stichopus japonicus) guts

School of Food Science and Technology, Dalian Polytechnic University, National Engineering Research Center of Seafood

摘要

摘要: 本研究以海参肠为原料,采用p H3.0 50 mmol/L甘氨酸-盐酸缓冲体系,按1∶6（w/v）的料液比,在4℃浸提1 h,获得海参肠组织蛋白酶D的粗酶液。以酸变性牛血红蛋白为底物,进行酶活测定,并研究了该酶的酶学性质。结果表明,海参肠组织蛋白酶D粗酶的最适p H为3.0,在p H3.0⁷.0之间稳定性较好;最适反应温度为50℃,在4⁴0℃具有较高稳定性。5 mmol/L的金属离子Mg²⁺、Ca²⁺、Ni²⁺、Zn²⁺、Cd²⁺和K⁺对该酶有激活作用,而Fe³⁺和Fe²⁺则对其有抑制作用。天冬氨酸蛋白酶抑制剂Pepstatin A对该酶活性有较强的抑制作用。上述结果说明,在本研究条件下提取获得的海参肠组织蛋白酶D粗酶是一种酸性蛋白酶,其组成以天冬氨酸蛋白酶为主。
- 海参肠 /
- 组织蛋白酶D /
- 提取 /
- 酶学特性
Abstract: The crude cathepsin D was obtained from sea cucumber ( Stichopus japonicus) guts in this paper.The crude cathepsin D was extracted by using 50 mmol/L Glycine-HCl buffer ( p H3.0) with material to solvent ratio of 1∶ 6 ( w/v) at 4 ℃ for 1 h.The activity and characterization of the enzyme were investigated by using acid denatured hemoglobin as a substrate.The results showed that the optimum p H of the crude cathepsin D was p H3.0 and was stable between p H3.0 ~ 7.0. The crude cathepsin D exhibited its optimal temperature at 50 ℃ and was stable at 4 ~ 40 ℃. The crude cathepsin D was activated by metal ions of Mg²⁺, Ca²⁺, Ni²⁺, Zn²⁺, Cd²⁺and K⁺, while inhibited by Fe³⁺ and Fe²⁺at concentration of 5 mmol/L.The crude cathepsin D was strongly inhibited by aspartic protease inhibitor pepstatin A. These results suggested that the crude cathepsin D obtained from S.japonicus guts was acid protease maily composed of aspartic protease under the condition of present study.
- sea cucumber gut /
- cathepsin D /
- extraction /
- enzyme characterization

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参考文献(29)

[1]	朱蓓薇.海珍品加工理论与技术的研究[M].北京:科学出版社, 2010:3-4.
[2]	Wu H T, Li D M, Zhu B W, et al.Proteolysis of noncollagenous proteins in sea cucumber, Stichopus japonicus, body wall:characterization and the effects of cysteine protease inhibitors[J].Food Chemistry, 2013, 141 (2) :1287-1294.
[3]	Qi H, Dong X P, Cong L N, et al.Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber Stichopus japonicus[J].Fish Physiology and Biochemistry, 2007, 33 (2) :181-188.
[4]	Zhu B W, Zhao L L, Sun L M, et al.Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus[J].Bioscience, Biotechnology, and Biochemistry, 2008, 72 (6) :1430-1437.
[5]	Zhu B W, Yu J W, Zhang Z S, et al.Purification and partial characterization of an acid phosphatase from the body wall of sea cucumber Stichopus japonicus[J].Process Biochemistry, 2009, 44 (8) :875-879.
[6]	Sun L M, Zhu B W, Wu H T, et al.Purification and characterization of cathepsin B from the gut of the sea cucumber (Stichopus japonicas) [J].Food Science and Biotechnology, 2011, 20 (4) :919-925.
[7]	Wu H T, Li D M, Zhu B W, et al.Characterization of acetylcholinesterase from the gut of sea cucumber Stichopus japonicus[J].Fisheries Science, 2013, 79 (2) :303-311.
[8]	Wu H T, Li D M, Zhu B W, et al.Purification and characterization of alkaline phosphatase from the gut of sea cucumber Stichopus japonicus[J].Fisheries Science, 2013, 79 (3) :477-485.
[9]	张志林, 肖蓉, 李庆伟.组织蛋白酶D的功能多样性[J].中国生物化学与分子生物学报, 2014 (7) :647-654.
[10]	Wright L M, Levy E S, Patel N P, et al.Purification and characterization of cathepsin D from normal human breast tissue[J].The Protein Journal, 1997, 16 (3) :171-181.
[11]	Gubensek F, Barstow L, Kregar I, et al.Rapid isolation of cathepsin D by affinity chromatography on the immobilized synthetic inhibitor[J].FEBS Letters, 1976, 71 (1) :42-44.
[12]	Canduri F, Ward R J, Wf D A J, et al.Purification and partial characterization of cathepsin D from porcine (Sus scrofa) liver using affinity chromatography[J].IUBMB Life, 1998, 45 (4) :797-803.
[13]	Jiang S T, Wang J H, Chen C S.Purification and some properties of calpain II from tilapia muscle (Tilapia nilotica X Tilapia aurea) [J].Journal of Agricultural&Food Chemistry, 1991, 39 (2) :237-241.
[14]	Wang P A, Stenvik J, Larsen R, et al.Cathepsin D from Atlantic cod (Gadus morhua, L.) liver.Isolation and comparative studies[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology, 2007, 147 (3) :504-511.
[15]	Balti R, Hmidet N, Jellouli K, et al.Cathepsin D from the Hepatopancreas of the Cuttlefish (Sepia officinalis) :Purification and Characterization[J].Planta, 1973, 109 (4) :293-306.
[16]	Merino V, Kumar N S.Isolation, affinity purification and biochemical characterization of a lysosomal cathepsin D from the deuterostome Asterias rubens[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology, 2012, 161 (3) :240-246.
[17]	Anson M L.The estimation of pepsin, trypsin, papain, and the cathepsin with hemoglobin[J].Journal of General Physiology, 1938, 22 (1) :79-89.
[18]	Lowry O H, Rosebrough N J, Farr A L, et al.Protein measurement with the Folin phenol reagent[J].Journal of Biological Chemistry, 1951, 193 (1) :265-275.
[19]	杜英.海参肠乙酰胆碱酯酶的提取、纯化及其特性研究[D].大连:大连工业大学, 2011:14-15.
[20]	Komai T, Kawabata C, Amano M, et al.Todarepsin, a new cathepsin D from hepatopancreas of Japanese common squid (Todarodes pacificus) [J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology, 2004, 137 (3) :373-382.
[21]	Stoknes I, Rustad T.Purification and characterization of a multicatalytic proteinase from Atlantic salmon (Salmo salar) muscle[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology, 1995, 111 (4) :587-596.
[22]	Doke S N, Ninjoor V, Nadkami G B.Characterization of Cathepsin D from the Skeletal Muscle of Fresh Water Fish[J].Bioscience, Biotechnology, and Biochemistry, 1980, 44 (7) :1521-1528.
[23]	Jiang S T, Nei F P, Chen H C, et al.Comparative study on the cathepsin D from banded shrimp (Penaeus japonicus) and grass shrimp (Penaeus monodon) [J].Journal of Agricultural&Food Chemistry, 1992, 40 (6) :961-966.
[24]	Goldman-Levkovitz S, Rimon A, Rimon S.Purification properties and specificity of cathepsin D from Cyprinus carpio[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology, 1995, 112 (1) :147-151.
[25]	Krause J, Tshidino S C, Ogawa T, et al.Purification and partial characterization of ostrich skeletal muscle cathepsin D and its activity during meat maturation[J].Meat Science, 2011, 87 (3) :196-201.
[26]	Venugopal A, Kumar N S.Biochemical characterization of cathepsin D from the mussel Lamellidens corrianus[J].Comparative Biochemistry&Physiology Part B Biochemistry&Molecular Biology, 2014, 169 (1) :25-30.
[27]	Jamdar S N, Harikumar P.Purification, Identification and Characterization of Aspartic Proteases of Chicken Intestine[J].Journal of Food Biochemistry, 2015, 40 (4) :451-462.
[28]	Draper A M, Zeece M G.Thermal Stability of Cathepsin D[J].Journal of Food Science, 1989, 54 (6) :1651-1652.
[29]	Jiang S T, Her Y H, Lee J J, et al.Comparison of the Cathepsin D from Mackerel (Scomber australasicus) and Milkfish (Chanos chanos) Muscle[J].Bioscience Biotechnology&Biochemistry, 1993, 57 (4) :571-577.

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