Study on Serine Proteinase in the Muscle of Yesso Scallops (Mizuhopecten yessoensis）
-
摘要: 为了探究虾夷扇贝肌肉冷藏过程中内源蛋白酶对其质构的影响，本文对虾夷扇贝肌肉在4 ℃下冷藏7 d过程中肌肉质构及蛋白变化进行测定，结果表明，整个冷藏过程中扇贝肌肉硬度、弹性、咀嚼性和粘性整体呈下降趋势。SDS-PAGE分析显示肌肉蛋白在第3 d开始出现明显降解。在内源酶活力方面，丝氨酸蛋白酶（Serine proteinase，SP）活力在冷藏2 d后开始急剧下降。通过硫酸铵盐析、离子交换、凝胶过滤和疏水柱层析等从虾夷扇贝肌肉中获得高度纯化的丝氨酸蛋白酶并对其酶学性质进行研究。SDS-PAGE和明胶酶谱结果表明，SP在天然状态下主要以分子量约为52 kDa的二聚体形式存在。SP的最适pH为9.0，最适温度为37 ℃。SP特异性水解羧基侧P1位含有精氨酸或赖氨酸残基的底物。丝氨酸蛋白酶特异性抑制剂PMSF、Leupeptin、Pefabloc SC、Benzamidine分别能抑制其97%、98%、90%和85%的酶活力；金属离子Fe2+、Zn2+、Cu2+也能明显抑制SP的酶活力。37 ℃下SP可有效降解扇贝肌原纤维蛋白，为揭示SP对扇贝肌肉蛋白的品质影响提供参考。Abstract: To investigate the effect of endogenous protease on the texture of Yesso scallop muscle during cold storage, the texture and protein change related indexes of Yesso scallop muscle was measured during 4 ℃ cold storage 7 d with a purpose to identify the functions of proteinases. The results showed that the hardness, springiness, chewiness and gumminess generally decreased during the whole cold storage process. SDS-PAGE analysis revealed that muscular proteins began to degrade obviously on the third day. In terms of enzyme activity, serine proteinase (SP) activity began to decrease sharply after 2 days of cold storage. Serine proteinase was purified from the muscle of Yesso scallop by ammonium sulfate precipitation, ion exchange chromatography, gel filtration and hydrophobic chromatography. SDS-PAGE and gelatin zymogram showed that in native condition SP was in homodimer form with molecular weight of 52 kDa. The optimum pH of SP was 9.0 and the optimum temperature was 37 ℃. SP specifically hydrolyzes substrates containing arginine or lysine residues at P1 position on the carboxyl side. Serine protease specific inhibitors including PMSF, Leupeptin, Pefabloc SC and Benzamidine could inhibit the activity of SP by 97%, 98%, 90% and 85%, respectively. Metal ions Fe2+, Zn2+ and Cu2+ could also significantly suppress the activity of SP. SP could effectively degrade scallop myofibrillar at 37 ℃, which delivered a reference for revealing the effect of SP on the quality of Yesso scallop muscle protein.
-
Key words:
- Mizuhopecten yessoensis /
- serine proteinase /
- texture /
- purification /
- characterization /
- muscle
-
图 1 虾夷扇贝冷藏过程中肌肉蛋白的自身降解（A）和光密度分析（B）
Figure 1. Protein degradation in Yesso scallop muscle during cold storage (A) and densitometry analysis (B)
注：M：标准蛋白。
图 2 虾夷扇贝肌肉冷藏过程中丝氨酸蛋白酶活力的变化
Figure 2. Changes of SP activity in Yesso scallop muscle during cold storage
图 3 丝氨酸蛋白酶分离纯化柱层析图
Figure 3. Chromatography for purification of SP
注：（A） DEAE-Sepharose; （B） Superdex 200; （C） Phenyl-Fast Flow; （D） DEAE-Sepharose。
图 4 虾夷扇贝SP的SDS-PAGE和明胶酶谱分析
Figure 4. SDS-PAGE and gelatin zymography of SP from Yesso scallop
注：M：标准蛋白；1：SP, SDS-PAGE；2：SP＋β-Me，明胶酶谱；3：SP-β-Me，明胶酶谱。
图 5 虾夷扇贝丝氨酸蛋白酶的最适温度及热稳定性分析
Figure 5. Optimum temperature and thermal stability of SP from Yesso scallop
图 6 虾夷扇贝丝氨酸蛋白酶的最适pH及pH稳定性分析
Figure 6. Optimum pH and pH stability of SP from Yesso scallop
图 7 丝氨酸蛋白酶对虾夷扇贝肌肉肌原纤维的分解作用
Figure 7. Effect of SP on myofibrillar proteins of Yesso scallop muscle
表 1 冷藏过程中虾夷扇贝肌肉质构参数变化
Table 1. The change of texture parameters in Yesso callop muscle during cold storage
时间 （d） 硬度 （gf） 咀嚼性（gf） 粘性（gf） 弹性（gf） 0 670±42b 255±18 a 321±2 b 0.34±0.07b 1 736±52a 176±11b 372±17a 0.37±0.04a 2 584±45c 152±8c 276±19c 0.33±0.02b 3 457±38d 132±4d 253±11d 0.32±0.03b 4 330±33e 126±5e 227±12e 0.31±0.02c 5 206±25f 115±2f 176±15f 0.28±0.01d 6 146±27g 102±3g 107±8g 0.27±0.03e 7 68±12h 93±4g 99±7g 0.25±0.02f 注：同列不同小写字母表示差异显著（P<0.05）。 
下载: 导出CSV
表 2 虾夷扇贝丝氨酸蛋白酶的底物特异性分析
Table 2. Substrate characterization of the SP from Yesso scallop
底物（10 µmol/L） 相对活力（%） Boc-Phe-Ser-Arg-MCA 100±0.0 Boc-Gln-Ala-Arg-MCA 123±0.3 Boc-Leu-Lys-Arg-MCA 116±0.4 Boc-Ala-Gly-Pro-Arg-MCA 108±0.2 Boc-Val-Pro-Arg-MCA 82±1.2 Boc-Glu-Lys-Lys-MCA 36±0.4 Z-Phe-Arg-MCA 36±2.3 Z-Arg-Arg-MCA 37±0.9 Suc-Leu-Leu-Val-Tyr-MCA 0 Suc-Ala-Ala-Pro-MCA 0 Arg-MCA 0
下载: 导出CSV
表 3 蛋白酶抑制剂对丝氨酸蛋白酶活力的影响
Table 3. Inhibitory effect of different proteinase inhibitors on the activity of SP
抑制剂 浓度（mmol/L） 相对活力（%） control 0 100±0.0 EGTA 1 47±0.2 EDTA 1 90±0.1 10 93±0.3 1,10-phenanthroline 1 96±0.2 5 84±0.2 PMSF 1 86±0.1 10 3±0.4 E-64 0.015 78±0.1 0.1 80±0.1 Benzazmidine 0.5 15±0.3 5 7±0.2 Pefabloc SC 0.2 11±0.5 2 3±0.1 Leupeptin 0.01 3±0.1 0.03 2±0.6
下载: 导出CSV
表 4 金属离子对丝氨酸蛋白酶活力的影响
Table 4. Effect of metal ions on the activity of SP
金属离子 浓度（mmol/L） 相对活力（%） control 0 100±0.0 Ca2+ 0.01 58±0.4 0.05 72±0.1 0.1 75±0.2 Fe2+ 0.01 78±0.3 0.05 73±0.5 0.1 17±0.4 Zn2+ 0.01 90±0.1 0.05 36±0.3 0.1 16±0.3 Mg2+ 0.01 96±0.3 0.05 95±0.1 0.1 91±0.2 Ba2+ 0.01 98±0.5 0.05 89±0.2 0.1 81±0.2 Cu2+ 0.01 55±0.3 0.05 72±0.1 0.1 11±0.5 Mn2+ 0.01 95±0.3 0.05 97±0.5 0.1 96±0.5
下载: 导出CSV
-
[1] 王丹, 吴反修. 中国渔业统计年鉴[G]. 北京: 中国农业出版社, 2021WANG Dan, WU Fanxiu. China fishery statistics yearbook[G]. Beijing: China Agriculture Press, 2021. [2] 汤俊杰, 段振华, 杨丽娟. 扇贝的加工利用研究进展[J]. 食品工业,2013,34(5):191−194. [TANG Junjie, DUAN Zhenhua, YANG Lijuan. Research progress on processing and utilization of scallop[J]. Food Industry,2013,34(5):191−194.TANG Junjie, DUAN Zhenhua, YANG Lijuan. Research progress on processing and utilization of scallop[J]. Food Industry, 2013, 34(5): 191-194. [3] 高加龙, 章超桦, 秦小明, 等. 虾夷扇贝内脏团对镉的富集特性及生理响应[J]. 广东海洋大学学报,2017,32(3):204−210. [GAO Jialong, ZHANG Chaohua, QIN Xiaoming, et al. Cadmium enrichment characteristics and physiological response of visceral mass of scallop prawn[J]. Journal of Guangdong Ocean University,2017,32(3):204−210.GAO Jialong, ZHANG Chaohua, QIN Xiaoming, et al. Cadmium enrichment characteristics and physiological response of visceral mass of scallop prawn[J]. Journal of Guangdong Ocean University, 2017, 32(3): 204-210. [4] 刘征. 扇贝干燥关键技术研究[D]. 保定: 河北农业大学, 2012LIU Zheng. Study on key drying technology of scallop[D]. Baoding: Agricultural University of Hebei, 2012. [5] 陈文秀. 虾夷扇贝保活品质变化与贮藏条件调控技术研究[D]. 上海: 上海海洋大学, 2020CHEN Wenxiu. Study on quality change and storage condition control technology of scallop shrimp[D]. Shanghai: Shanghai Ocean University, 2020. [6] 姜明慧, 田元勇, 闫丽新, 等. 冷藏过程中虾夷扇贝横纹肌微观结构变化与肌原纤维蛋白稳定性[J]. 食品科学,2020,41(21):175−181. [JIANG Minghui, TIAN Yuanyong, YAN Lixin, et al. Microstructural changes of striated muscle and stability of myofibrin in scallop shrimp during cold storage[J]. Food Science,2020,41(21):175−181. doi: 10.7506/spkx1002-6630-20191016-159JIANG Minghui, TIAN Yuanyong, YAN Lixin, et al. Microstructural changes of striated muscle and stability of myofibrin in scallop shrimp during cold storage[J]. Food Science, 2020, 41(21): 175-181. doi: 10.7506/spkx1002-6630-20191016-159 [7] 徐锦华, 孟泽玲, 葛诗琪, 等. 刺参体壁胰/类胰丝氨酸蛋白酶的性质及其在自溶中的作用[J]. 食品科学,2019,40(18):95−100. [XU Jinhua, MENG Zeling, GE Shiqi, et al. Characterization of trypsin/trypsine-like protease in body wall of Stichopus japonicus and its role in autolysis[J]. Food Science,2019,40(18):95−100. doi: 10.7506/spkx1002-6630-20181004-019XU Jinhua, MENG Zeling, GE Shiqi, et al. Characterization of trypsin/trypsine-like protease in body wall of Stichopus japonicus and its role in autolysis[J]. Food Science, 2019, 40(18): 95-100. doi: 10.7506/spkx1002-6630-20181004-019 [8] CHENG J H, SUN D W, HAN Z, et al. Texture and structure measurements and analyses for evaluation of fish and fillet freshness quality: A review[J]. Comprehensive Reviews in Food Science and Food Safety,2014,13(1):52−61. doi: 10.1111/1541-4337.12043 [9] TANIOKA T, HATTORI A, MIZUTANI S. Regulation of the human leukocyte-derived argine aminopeptidase/endoplasmic reticulum-aminopeptidase 2 gene by interferon-C[J]. FEBS Journal,2005,272:916−928. doi: 10.1111/j.1742-4658.2004.04521.x [10] SINGH A, BENJAKUL S. Proteolysis and its control using protease inhibitors in fish and fish products: A review[J]. Comprehensive Reviews in Food Science and Food Safety,2018,17(2):496−509. doi: 10.1111/1541-4337.12337 [11] YANG F, RUSTAD T, XU Y, et al. Endogenous proteolytic enzymes-a study of their impact on cod (Gadus morhua) muscle proteins and textural properties in a fermented product[J]. Food Chemistry,2015,172:551−558. doi: 10.1016/j.foodchem.2014.09.086 [12] BROWN M R, SIKES A L, ELLIOTT N G, et al. Physicochemical factors of abalone quality: A review[J]. Journal of Shellfish Research,2008,27(4):835−842. doi: 10.2983/0730-8000(2008)27[835:PFOAQA]2.0.CO;2 [13] SRIKET C, BENJAKUL S, VISESSANGUAN W, et al. Collagenolytic serine protease in fresh water prawn (Macrobrachium rosenbergii): Characteristics and its impact on muscle during iced storage[J]. Food chemistry,2011,124(1):29−35. doi: 10.1016/j.foodchem.2010.05.098 [14] LIU J, YOSHIDA A, GAO Y, et al. Purification and characterization of a sarcoplasmic serine proteinase from threadfin bream nemipterus virgatus muscle[J]. Food Chemistry,2019,284:198−204. doi: 10.1016/j.foodchem.2019.01.024 [15] 杜翠红, 曹敏杰. 鱼类肌原纤维结合型丝氨酸蛋白酶研究进展[J]. 食品科学,2013,34(9):336−339. [DU Cuihong, CAO Minjie. Research progress of fish myofibrillary binding serine protease[J]. Food Science,2013,34(9):336−339. doi: 10.7506/spkx1002-6630-201309067DU Cuihong, CAO Minjie. Research progress of fish myofibrillary binding serine protease[J]. Food Science, 2013, 34(9): 336-339. doi: 10.7506/spkx1002-6630-201309067 [16] LIU B, LIU Z Q, LI D Y, et al. Action of endogenous proteases on texture deterioration of the bay scallop (Argopecten irradians) adductor muscle during cold storage and its mechanism[J]. Food Chemistry,2020,323:126790. doi: 10.1016/j.foodchem.2020.126790 [17] KWON T, KIM M, CHOI H, et al. A masquerade-like serine protease homolog is necessary for prophenoloxidase activity in the coleopteran insect, holotrichia diomphalia larvae[J]. European Journal of Biochemistry,2000,267:6188−6196. doi: 10.1046/j.1432-1327.2000.01695.x [18] DELOTTO Y, DELOTTO R. Proteolytic processing of the drosophila spatzle protein by easter generates a dimeric ngf-like molecule with ventralising activity[J]. Mechanisms of Development,1998,72(1-2):141−148. doi: 10.1016/S0925-4773(98)00024-0 [19] KAWABATA S, TOKUNAGA F, KUGI Y, et al. Limulus factor D, a 43-kDa protein isolated from horseshoe crab hemocytes, is a serine protease homologue with antimicrobial activity[J]. FEBS Letters,1996,398(2-3):146−150. doi: 10.1016/S0014-5793(96)01224-0 [20] 游银川. 皱纹盘鲍肌肉质构相关蛋白酶的研究[D]. 厦门: 集美大学, 2016YOU Yinchuan. Study on proteinases related to texture from the muscle of abalone (Haliotis discus hannai)[D]. Xiamen: Jimei University, 2016. [21] 林怡晨, 刘伟峰, 孙小舒, 等. 蓝圆鲹分离蛋白水溶性蛋白酶的鉴定及性质[J]. 集美大学学报(自然科学版),2020,25(2):97−104. [LIN Yichen, LIU Weifeng, SUN Xiaoshu, et al. Identification and characterization of water-soluble protease in scad[J]. Journal of Jimei University (Natural Science Edition),2020,25(2):97−104.LIN Yichen, LIU Weifeng, SUN Xiaoshu, et al. Identification and characterization of water-soluble protease in scad[J]. Journal of Jimei University (Natural Science Edition), 2020, 25(2): 97-104. [22] 麻金花, 翁凌, 颜龙杰, 等. 海参内脏胶原降解酶的纯化及性质研究[J]. 水产学报,2015,39(7):34−37. [MA Jinhua, WENG Ling, YAN Longjie, et al. Purification and characterization of collagen degrading enzymes from sea cucumber viscera[J]. Journal of Fisheries of China,2015,39(7):34−37.MA Jinhua, WENG Ling, YAN Longjie, et al. Purification and characterization of collagen degrading enzymes from sea cucumber viscera[J]. Journal of Fisheries of China, 2015, 39(7): 34-37. [23] CAO M J, SHAO W, LI Y, et al. Identification of a myofibril-bound serine proteinase in the skeletal muscle of silver carp[J]. Journal of Food Biochemistry,2004,28(5):373−386. doi: 10.1111/j.1745-4514.2004.04203.x [24] XIONG X, HE B, JIANG D, et al. Postmortem biochemical and textural changes in the patinopecten yessoensis adductor muscle (Pyam) during iced storage[J]. International Journal of Food Properties,2019,22(1):1024−1034. doi: 10.1080/10942912.2019.1625367 [25] AYALA M D, ABDEL I, SANTAELLA M, et al. Muscle tissue structural changes and texture development in sea bream, Sparus aurata L., during post-mortem storage[J]. LWT-Food Science and Technology,2010,43(3):465−475. doi: 10.1016/j.lwt.2009.08.023 [26] SHARIFIMEHR S, SOLTANIZADEH N, HOSSEIN GOLI S A. Effects of edible coating containing nano-emulsion of aloe vera and eugenol on the physicochemical properties of shrimp during cold storage[J]. Journal of the Science of Food and Agriculture,2019,99(7):3604−3615. doi: 10.1002/jsfa.9581 [27] HULTMANN L, RUSTAD T. Textural changes during iced storage of salmon (Salmo salar) and cod (Gadus morhua)[J]. Journal of Aquatic Food Product Technology,2002,11(3-4):105−123. doi: 10.1300/J030v11n03_09 [28] TAKAHASHI K, KUROSE K, OKAZAKI E, et al. Effect of various protease inhibitors on heat-induced myofibrillar protein degradation and gel-forming ability of red tilefish (Branchiostegus japonicus) meat[J]. LWT-Food Science and Technology,2016,68:717−723. doi: 10.1016/j.lwt.2016.01.022 [29] LIU B, LIU Y X, LIU Z Q, et al. Effects of natural trypsin inhibitor fromssoybean on texture deterioration of the bay scallop (Argopecten irradians) during cold storage and its mechanism[J]. International Journal of Food Science & Technology,2020,55(11):3432−3440. [30] 翁凌, 李腾, 阴利华, 等. 南美白对虾丝氨酸蛋白酶的分离纯化及性质研究[J]. 集美大学学报(自然科学版),2010,15(4):272−278. [WENG Ling, LI Teng, YIN Lihua, et al. Isolation, purification and characterization of serine protease from Penaeus vannamei[J]. Journal of Jimei University (Natural Science Edition),2010,15(4):272−278.WENG Ling, LI Teng, YIN Lihua, et al. Isolation, purification and characterization of serine protease from Penaeus vannamei[J]. Journal of Jimei University (Natural Science Edition), 2010, 15(4): 272-278. [31] 胡健健. 皱纹盘鲍丝氨酸蛋白酶的基因克隆、体外表达及其免疫调控作用[D]. 厦门: 集美大学, 2017HU Jianjian. Gene cloning, in vitro expression and immune regulation of abalone serine protease[D]. Xiamen: Jimei University, 2017. [32] 王梦想, 钟婵, 蔡秋凤, 等. 蓝圆肌肉中丝氨酸蛋白酶的分离纯化及性质研究[J]. 食品工业科技,2012,29(19):58−67. [WANG Mengxiang, ZHONG Chan, CAI Qiufeng, et al. Purification and characterization of serine protease from blue circle muscle[J]. Science and Technology of food Industry,2012,29(19):58−67.WANG Mengxiang, ZHONG Chan, CAI Qiufeng, et al. Purification and characterization of serine protease from blue circle muscle[J]. Science and Technology of food Industry, 2012, 29(19): 58-67. -
点击查看大图
图(7) / 表(4)
计量
- 文章访问数: 7
- HTML全文浏览量: 2
- PDF下载量: 0
- 被引次数: 0
