翡翠贻贝副肌球蛋白的特性及在模拟胃肠液中的消化
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摘要
为探究贝类副肌球蛋白(paramyosin,PM)的热稳定性、pH值稳定性及其在模拟胃肠液中的消化特性,以翡翠贻贝(Perna viridis)为对象,利用硫酸铵盐析和羟基磷灰石柱层析等方法,从肌肉中纯化PM,采用肽质量指纹图谱法对其进行鉴定,利用圆二色谱测定其二级结构及热变性温度。结果显示,翡翠贻贝PM分子质量为99.5 kDa;肽质量指纹图谱分析获得26个肽段,共330个氨基酸残基,与地中海贻贝(Mytilusgalloprovincialis)PM的序列一致性达到100%,表明纯化的蛋白为PM。圆二色谱结果显示,PM呈现典型的α-螺旋结构,其热变性温度为(56.3±0.2)℃。在30～100℃热处理30 min,PM未出现沉淀聚集现象,在pH 6～11范围内也较稳定,但当pH≤5时稳定性差,出现沉淀聚集现象。与胃蛋白酶、胰蛋白酶及胰凝乳蛋白酶对PM的单独消化作用相比,3种酶连续作用可使PM有效降解,但仍有分子质量30 kDa的片段未被完全消化。本研究表明,翡翠贻贝PM具有较好的耐热性及耐消化性,为贻贝深加工及PM的后续研究提供一定理论参考。
In order to investigate the thermal stability, pH stability and digestion characteristics of paramyosin(PM) in simulated gastrointestinal fluids, we purified PM to homogeneity from the muscle of Perna viridis by consecutive ammonium sulfate fractionation and hydroxyapatite chromatography, and we further identified it by peptide mass fingerprinting(PMF). Circular dichroism(CD) spectroscopy was employed to measure its secondary structure and thermal denaturation temperature. SDS-PAGE was carried out to explore its thermal stability, pH stability and digestion characteristics in simulated gastrointestinal fluids. PM showed a single band corresponding to 99.5 kDa on SDS-PAGE. Using peptide mass fingerprinting, a total of 26 peptide fragments, including 330 amino acid residues, were obtained from purified PM, which revealed 100% identity to PM from Mytilus galloprovincialis, indicating that the purified protein is PM. CD spectral analysis demonstrated that PM had a typical α-helix structure with thermal denaturation temperature(Td) of(56.3 ± 0.2) ℃. No obvious precipitate was observed when PM was heated in the temperature range from 30 to 100 ℃ for 30 min. PM was stable between pH 6.0–11.0. However, it was unstable below pH 5.0 and aggregated. PM could only be partially hydrolyzed by pepsin, trypsin or chymotrypsin individually, while it was effectively degraded by continuous hydrolysis with the three proteinases. However, protein bands with molecular mass of approximately 30 kDa remained undigested. In conclusion, PM from Perna viridis is thermally stable and resistant to gastrointestinal digestion. This work provides a valuable theoretical basis for mussel processing and further study on PM.
In order to investigate the thermal stability, pH stability and digestion characteristics of paramyosin(PM) in simulated gastrointestinal fluids, we purified PM to homogeneity from the muscle of Perna viridis by consecutive ammonium sulfate fractionation and hydroxyapatite chromatography, and we further identified it by peptide mass fingerprinting(PMF). Circular dichroism(CD) spectroscopy was employed to measure its secondary structure and thermal denaturation temperature. SDS-PAGE was carried out to explore its thermal stability, pH stability and digestion characteristics in simulated gastrointestinal fluids. PM showed a single band corresponding to 99.5 kDa on SDS-PAGE. Using peptide mass fingerprinting, a total of 26 peptide fragments, including 330 amino acid residues, were obtained from purified PM, which revealed 100% identity to PM from Mytilus galloprovincialis, indicating that the purified protein is PM. CD spectral analysis demonstrated that PM had a typical α-helix structure with thermal denaturation temperature(Td) of(56.3 ± 0.2) ℃. No obvious precipitate was observed when PM was heated in the temperature range from 30 to 100 ℃ for 30 min. PM was stable between pH 6.0–11.0. However, it was unstable below pH 5.0 and aggregated. PM could only be partially hydrolyzed by pepsin, trypsin or chymotrypsin individually, while it was effectively degraded by continuous hydrolysis with the three proteinases. However, protein bands with molecular mass of approximately 30 kDa remained undigested. In conclusion, PM from Perna viridis is thermally stable and resistant to gastrointestinal digestion. This work provides a valuable theoretical basis for mussel processing and further study on PM.
引文
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[12]游银川,张凌晶,颜龙杰,等.皱纹盘鲍副肌球蛋白的分离纯化及初步性质研究[J].水产学报, 2017, 41(6):836-844. DOI:10.11964/jfc.20160810521.
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[14]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.
[15]US Food and Drug Administration. The national formulary:simulated gastricfluidandsimulatedintestinalfluid,TS[M].Washington:Rockville, 1995:2053.
[16]万楚君,游银川,翁凌,等.四种加工方式对皱纹盘鲍制品消化特性的影响[J].水产学报, 2017, 41(6):928-936. DOI:10.11964/jfc.20161010574.
[17]HUANG Y Y, LIU G M, CAI Q F, et al. Stability of major allergen tropomyosin and other food proteins of mud crab(Scylla serrata)by in vitro gastrointestinal digestion[J]. Food and Chemical Toxicology,2010, 48(5):1196-1201. DOI:10.1016/j.fct.2010.02.010.
[18]WATABE S, IWASAKI K, FUNABARA D, et al. Complete amino acid sequence of Mytilus anterior byssus retractor paramyosin and its putativephosphorylationsite[J].JournalofExperimentalZoology,2000, 286(1):24-35. DOI:10.1002/(SICI)1097-010X(20000101).
[19]ZHANG G, FANG X, GUO X, et al. The oyster genome reveals stress adaptationandcomplexityofshellformation[J].Nature,2012,490:49-54. DOI:10.1038/nature11413.
[20]SUZUKI M, SHIMIZU K, KOBAYASHI Y, et al. Paramyosin from the disc abalone(Haliotis discus discus)[J]. Journal of Food Biochemistry,2014, 38(4):444-451. DOI:10.1111/jfbc.12072.
[21]周茹,倪渠峰,林伟伟,等.肌原纤维蛋白溶解度对盐离子浓度的依赖性[J].中国食品学报, 2015, 15(3):32-39. DOI:10.16429/j.1009-7848.2015.03.005.
[22]ZHU B W, DONG X P, SUN L M, et al. Effect of thermal treatment on the texture and microstructure of abalone muscle(Haliotis discus)[J].Food Science&Biotechnology, 2011, 20(6):1467-1473. DOI:10.1007/s10068-011-0203-6.
[23]SANO T, NOGUCHI S F, TSUCHIYA T, et al. Paramyosin-myosinactin interactions in gel formation of invertebrate muscle[J]. Journal of Food Science, 2010, 54(4):796-799. DOI:10.1111/j.1365-2621.1989.tb07885.x.
[24]EPSTEIN H F, ARONOW B J, HARRIS H E. Myosin-paramyosin cofilaments:enzymatic interactions with F-actin[J]. Proceedings of the National Academy of Sciences, 1976, 73(9):3015-3019. DOI:10.1073/pnas.73.9.3015.
[25]COHEN C, PARRY D A D. A conserved C-terminal assembly region in paramyosin and myosin rods[J]. Journal of Structural Biology, 1998,122(1/2):180-187. DOI:10.1006/jsbi.1998.3983.
[26]张凌晶,蔡秋凤,刘光明,等.太平洋牡蛎肌肉蛋白的模拟胃肠液消化研究[J].集美大学学报(自然科学版), 2011, 16(2):81-86.DOI:10.3969/j.issn.1007-7405.2011.02.001.
[27]REESE G, AYUSO R, LEHRER S B. Tropomyosin:an invertebrate pan-allergen[J].International Archivesof AllergyandImmunology,1999, 119(4):247-258. DOI:10.1159/000024201.
[28]SHANTI K N, MARTIN B M, NAGPAL S, et al. Identification of tropomyosin as the major shrimp allergen and characterization of its IgEbinding epitopes[J]. Journal of Immunology, 1993, 151(10):5354-5363.
[29]YU H L, CAO M J, CAI Q F, et al. Effects of different processing methodsondigestibilityofScyllaparamamosain,allergen(tropomyosin)[J]. Food and Chemical Toxicology, 2011, 49(4):791-798. DOI:10.1016/j.fct.2010.11.046.
[30]张晴晴,吴子健,胡志和,等.凡纳滨对虾过敏原结构与性质的研究进展[J].食品科学, 2014, 35(9):285-290. DOI:10.7506/spkx1002-6630-201409056.
[31]胡志和,王星璇,张晴青,等.高压处理诱发虾原肌球蛋白结构变化与致敏性的关系[J].食品科学, 2017, 38(11):33-39. DOI:10.7506/spkx1002-6630-201711006.
[32]HIROSHI Q, OLGA M, YOHEI M, et al. The SH3 domain of UNC-89(obscurin)interacts with paramyosin, a coiled-coil protein, in Caenorhabditiselegans muscle[J]. Molecular Biology of the Cell,2016, 27(10):1606-1620. DOI:10.1091/mbc.E15-09-0675.
[33]蔺海鑫,林洪,王晓斐,等.美拉德反应对菲律宾蛤仔原肌球蛋白结构及免疫活性的影响[J].食品科学, 2016, 37(3):22-26.DOI:10.7506/spkx1002-6630-201603005.
[2]SONOBE H, OBINATA T, MINOKAWA T, et al. Characterization of paramyosin and thin?laments in the smooth muscle of acorn worm, a member of hemichordates[J]. Journal of Biochemistry, 2016, 160(6):369-379. DOI:10.1093/jb/mvw047.
[3]COHENC,SZENT-GYORGYIAG,KENDRICK-JONESJ.Paramyosinandthefilamentsofmolluscan“catch”muscles.I.Paramyosin:structure and assembly[J]. Journal of Molecular Biology,1971, 56(2):223-237. DOI:10.1016/0022-2836(71)90462-1.
[4]EPSTEIN H F, RD M D, ORTIZ I, et al. Myosin and paramyosin are organized about a newly identi?ed core structure[J]. Journal of Cell Biology, 1985, 100(3):904-915. DOI:10.1083/jcb.100.3.904.
[5]CASTELLANIL,VIBERTP,COHENC.Structureofmyosin/paramyosinfilamentsfromamolluscansmoothmuscle[J].Journal ofMolecularBiology,1983,167(4):853-872.DOI:10.1016/S0022-2836(83)80115-6.
[6]FUKUDA N, FUJIURA M, KIMURA M, et al. Thermally induced gelation of paramyosin from scallop adductor muscle[J]. Fisheries Science, 2006,72(6):1261-1268. DOI:10.1111/j.1444-2906.2006.01284.x.
[7]NAZ S, DESCLOZEAUX M, MOUNSEY K E, et al. Characterization of Sarcoptes scabiei tropomyosinand paramyosin:immunoreactive allergensinscabies[J].American Journal of Tropical Medicine&Hygiene, 2017, 97(3):851-860. DOI:10.4269/ajtmh.16-0976.
[8]WU H W, FU Z Q, LU K, et al. Vaccination with recombinant paramyosininMontanideISA206protectsagainstSchistosoma japonicum infection in water buffalo[J]. Vaccine, 2017, 35(26):3409-3415. DOI:10.1016/j.vaccine.2017.05.007.
[9]CONTIA,BURASTEROGJ,SULIC,etal.Identificationby serological proteomeanalysis of paramyosinas prominen tallergen industmiteallergy[J].Journal of Proteomics,2017,166:19-26.DOI:10.1016/j.jprot.2017.06.024.
[10]SUZUKI M, KOBAYASHI Y, HIRAKI Y, et al. Paramyosin of the disc abalone Haliotis discus discus:identification as a new allergen and cross-reactivity with tropomyosin[J]. Food Chemistry, 2011,124(3):921-926. DOI:10.1016/j.jprot.2017.06.024.
[11]EHARA T, NAKAGAWA K, TAMIYA T, et al. Effect of paramyosin on invertebrate natural actomyosin gel formation[J]. Fisheries Science,2004, 70(2):306-313. DOI:10.1016/j.jprot.2017.06.024.
[12]游银川,张凌晶,颜龙杰,等.皱纹盘鲍副肌球蛋白的分离纯化及初步性质研究[J].水产学报, 2017, 41(6):836-844. DOI:10.11964/jfc.20160810521.
[13]BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Analytical Biochemistry, 1976, 72:248-254. DOI:10.1006/abio.1976.9999.
[14]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.
[15]US Food and Drug Administration. The national formulary:simulated gastricfluidandsimulatedintestinalfluid,TS[M].Washington:Rockville, 1995:2053.
[16]万楚君,游银川,翁凌,等.四种加工方式对皱纹盘鲍制品消化特性的影响[J].水产学报, 2017, 41(6):928-936. DOI:10.11964/jfc.20161010574.
[17]HUANG Y Y, LIU G M, CAI Q F, et al. Stability of major allergen tropomyosin and other food proteins of mud crab(Scylla serrata)by in vitro gastrointestinal digestion[J]. Food and Chemical Toxicology,2010, 48(5):1196-1201. DOI:10.1016/j.fct.2010.02.010.
[18]WATABE S, IWASAKI K, FUNABARA D, et al. Complete amino acid sequence of Mytilus anterior byssus retractor paramyosin and its putativephosphorylationsite[J].JournalofExperimentalZoology,2000, 286(1):24-35. DOI:10.1002/(SICI)1097-010X(20000101).
[19]ZHANG G, FANG X, GUO X, et al. The oyster genome reveals stress adaptationandcomplexityofshellformation[J].Nature,2012,490:49-54. DOI:10.1038/nature11413.
[20]SUZUKI M, SHIMIZU K, KOBAYASHI Y, et al. Paramyosin from the disc abalone(Haliotis discus discus)[J]. Journal of Food Biochemistry,2014, 38(4):444-451. DOI:10.1111/jfbc.12072.
[21]周茹,倪渠峰,林伟伟,等.肌原纤维蛋白溶解度对盐离子浓度的依赖性[J].中国食品学报, 2015, 15(3):32-39. DOI:10.16429/j.1009-7848.2015.03.005.
[22]ZHU B W, DONG X P, SUN L M, et al. Effect of thermal treatment on the texture and microstructure of abalone muscle(Haliotis discus)[J].Food Science&Biotechnology, 2011, 20(6):1467-1473. DOI:10.1007/s10068-011-0203-6.
[23]SANO T, NOGUCHI S F, TSUCHIYA T, et al. Paramyosin-myosinactin interactions in gel formation of invertebrate muscle[J]. Journal of Food Science, 2010, 54(4):796-799. DOI:10.1111/j.1365-2621.1989.tb07885.x.
[24]EPSTEIN H F, ARONOW B J, HARRIS H E. Myosin-paramyosin cofilaments:enzymatic interactions with F-actin[J]. Proceedings of the National Academy of Sciences, 1976, 73(9):3015-3019. DOI:10.1073/pnas.73.9.3015.
[25]COHEN C, PARRY D A D. A conserved C-terminal assembly region in paramyosin and myosin rods[J]. Journal of Structural Biology, 1998,122(1/2):180-187. DOI:10.1006/jsbi.1998.3983.
[26]张凌晶,蔡秋凤,刘光明,等.太平洋牡蛎肌肉蛋白的模拟胃肠液消化研究[J].集美大学学报(自然科学版), 2011, 16(2):81-86.DOI:10.3969/j.issn.1007-7405.2011.02.001.
[27]REESE G, AYUSO R, LEHRER S B. Tropomyosin:an invertebrate pan-allergen[J].International Archivesof AllergyandImmunology,1999, 119(4):247-258. DOI:10.1159/000024201.
[28]SHANTI K N, MARTIN B M, NAGPAL S, et al. Identification of tropomyosin as the major shrimp allergen and characterization of its IgEbinding epitopes[J]. Journal of Immunology, 1993, 151(10):5354-5363.
[29]YU H L, CAO M J, CAI Q F, et al. Effects of different processing methodsondigestibilityofScyllaparamamosain,allergen(tropomyosin)[J]. Food and Chemical Toxicology, 2011, 49(4):791-798. DOI:10.1016/j.fct.2010.11.046.
[30]张晴晴,吴子健,胡志和,等.凡纳滨对虾过敏原结构与性质的研究进展[J].食品科学, 2014, 35(9):285-290. DOI:10.7506/spkx1002-6630-201409056.
[31]胡志和,王星璇,张晴青,等.高压处理诱发虾原肌球蛋白结构变化与致敏性的关系[J].食品科学, 2017, 38(11):33-39. DOI:10.7506/spkx1002-6630-201711006.
[32]HIROSHI Q, OLGA M, YOHEI M, et al. The SH3 domain of UNC-89(obscurin)interacts with paramyosin, a coiled-coil protein, in Caenorhabditiselegans muscle[J]. Molecular Biology of the Cell,2016, 27(10):1606-1620. DOI:10.1091/mbc.E15-09-0675.
[33]蔺海鑫,林洪,王晓斐,等.美拉德反应对菲律宾蛤仔原肌球蛋白结构及免疫活性的影响[J].食品科学, 2016, 37(3):22-26.DOI:10.7506/spkx1002-6630-201603005.