蛋白酶抑制剂的制备及其抑制狭鳕鱼糜凝胶劣化的研究
|
摘要
鱼糜是一种经粉碎和漂洗后,主要由可溶性肌原纤维蛋白组成的鱼肉制品。鱼糜中残留的蛋白酶可引起鱼糜凝胶结构的破坏,从而导致鱼糜制品品质及商业价值的下降。蛋白酶抑制剂可用于抑制鱼糜凝胶的降解。本研究选取鲑鱼的卵和血浆作为原材料以提取蛋白酶抑制剂。通过酸处理、超滤浓缩和木瓜蛋白酶亲和柱纯化,从鲑鱼卵中纯化出了一种cystatin,其纯化回收率、比活力和纯化倍数分别为0.4%、1.54 U/mg和192.6倍。SDS-PAGE检测发现其分子量为13 kDa。氨基酸测序发现其N-末端为N-Gly-Leu-Ile-Gly-Gly-Pro-Met-Asp-Ala-Asn-C,该序列与鲑鱼其它组织中的cystatin的组成完全一致。通过使用木瓜蛋白酶亲和柱,从鲑鱼血浆中纯化出了一种kiniongen,该纯化的回收率、比活力和纯化倍数分别为0.94%、3.84 U/mg和30.36倍。SDS-PAGE与FPLC的检测表明其分子量为70 kDa。抑制活性染色表明,该抑制剂仅在非还原条件下可检出。PAS染色表明该抑制剂属于糖蛋白。
以木瓜蛋白酶作为目标酶,鲑鱼卵cystatin的抑制能力最强;鲑鱼血浆kiniongen的抑制能力高于玻璃鱼卵抑制剂I、池沼公鱼抑制剂和另一种鲑鱼卵抑制剂;以组织蛋白酶L作为目标酶,鲑鱼卵cystatin的抑制能力略低于玻璃鱼卵抑制剂II,高于其他抑制剂;鲑鱼血浆kininogen的抑制能力高于狭鳕卵抑制剂、玻璃鱼卵抑制剂I和鲑鱼卵抑制剂。鲑鱼卵cystatin和鲑鱼血浆kininogen具有广范围的酸碱和热稳定性。在pH 5–8的范围内,鲑鱼卵cystaitn的活性几乎无损失。在pH 6–9的范围内,鲑鱼血浆kininogen可保留60%以上的抑制活力。鲑鱼卵cystatin在70°C以下处理30 min,可保留大于50%的活性。鲑鱼血浆kininogen在60°C处理30 min,可保留50%的活性。鲑鱼卵cystatin与鲑鱼血浆kininogen主要在20–50°C的范围内表现出抑制活性。Dixon-plot作图发现,鲑鱼卵cystatin属于竞争性抑制剂,其抑制常数为2.12 nM;鲑鱼血浆kininogen属于非竞争性抑制剂,其抑制常数为105.32 nM。
通过合成编码鲑鱼卵cystatin的DNA,采用pYES2NT/C质粒,以啤酒酵母YPH 499为宿主,进行了鲑鱼卵cystatin的重组表达。诱导表达后,分别使用选择性镍亲和柱和乙醇沉淀法进行重组cystatin的纯化,其中亲和纯化的回收率、比活力和纯化倍数分别为61.23%、7.45 U/mg和5.60倍。SDS-PAGE检测发现,重组cystatin的分子量为35 kDa,推测其发生了双体交联。与天然鲑鱼卵cystatin相比,重组cystatin的抑制活力有所下降,在酸性范围内的稳定性也有所下降,但其热稳定性得以增强。采用响应面法进行了摇瓶规模啤酒酵母YPH 499生产重组cystatin的优化研究,发现在培养基pH为5.7,诱导时间为6.7 h,诱导辅助剂的添加量为5.6 g/L时,重组cystatin的产量最大,可达0.57 U/mL。该研究结果与重复实验结果相一致。在摇瓶优化研究的基础上,进行了发酵罐中搅动速率和通气速率影响啤酒酵母YPH 499生产重组cystatin的优化研究,发现在350 rpm的搅动速率结合1.0 vvm的通气速率的发酵条件下,发酵罐中重组cystatin产量达到最大,为0.56 U/mL。该研究结果与重复实验结果相一致。
将纯化的重组cystatin添加到狭鳕鱼糜中,进行了鱼糜凝胶劣化的抑制研究。亲和纯化的重组cystatin,添加量为100μg/g时,其对鱼糜凝胶劣化的抑制率可达到90%以上。在该添加剂量下,凝胶的破碎力和变形量分别增加了4.5倍和30%。同时,其可榨出水分含量由17.85%减至6.27%,白度由49.78增加至52.60。SDS-PAGE检测表明,通过添加重组cystatin,可显著抑制蛋白酶引起的肌球蛋白的降解。重组cystatin的抑制效果显著强于蛋清粉。醇沉纯化的重组cystatin也可有效抑制鱼糜凝胶劣化,通过添加500μg/g的醇沉重组cystatin,可使凝胶的破碎力和变形量增加2.7倍和10%。因此,可将重组cystatin用于抑制狭鳕鱼糜的凝胶劣化。
Surimi is a minced and washed fish muscle consisting of salt-soluble myofibrillar proteins. The endogenous proteases in surimi cause an irreversible destruction of the gel structure of surimi and result in decreasing the quality of surimi-based product and its market value. Protease inhibitors have been used to prevent the degradation of surimi gel. Chum salmon egg and plasma were used as raw materials for preparation of protease inhibitors. A cystatin was purified from chum salmon egg with a series of acidic treatment, ultrafiltration and papain affinity chromatography. The recovery, specific inhibitory activity and purification fold were 0.4%, 1.54 U/mg and 192.6, respectively. Its molecular mass was 13 kDa based on the result of SDS-PAGE. Its N-terminal was determined to be N-Gly-Leu-Ile-Gly-Gly-Pro-Met-Asp-Ala-Asn-C, which was identical to that of chum salmon cystatin. A kininogen was purified from chum salmon plasma with one step of papain affinity chromatography. The recovery, specific inhibitory activity and purification fold were 0.94%, 3.84 U/mg and 30.36, respectively. Its molecular mass was 70 kDa based on SDS-PAGE and FPLC. Only under non reducing condition can this CPI be detected by inhibitory activity staining. The result of Periodic acid-Schiff staining showed that it was a glycoprotein.
Chum salmon egg cystatin showed the highest inhibitory activity against papain. The inhibitory activity of chum salmon plasma kininogen against papain was higher than that of glassfish egg inhibitor I, pond smelt inhibitor and chum salmon egg inhibitor. For cathepsins L, chum salmon egg cystatin showed inhibitory activity only lower than that of glassfish egg inhibitor II. Chum salmon plasma kininogen showed higher activity than did glassfish egg inhibitor I and chum salmon egg inhibitor. Chum salmon egg cystatin and plasma kininogen were stable under a wide range of pH. Nearly no loss of its activity was determined under pH 5-8. Chum salmon plasma kininogen possessed higher than 60% activity at pH 6-9. 50% of chum salmon egg cystatin activity could be retained after heating for 30 min at 70°C, while 50% of that of chum salmon plasma kininogen could be retained. Chum salmon egg cystatin and plasma kininogen showed activity mainly at 20-50°C. The Dixon-plots showed that chum salmon cystatin was a competitive inhibitor with inhibition constant of 2.12 nM. Chum salmon plasma kininogen was a noncompetitive inhibitor with inhibition constant of 105.32 nM.
Recombinant chum salmon cystatin (RC) was overexpressed in Saccharomyces cerevisiae YPH 499 incorporating pYES2NT/C vector. After cultivation and induction, RC could be purified by nickel select affinity chromatography or alcohol precipitation. The recovery, specific inhibitory activity and purification fold for nickel affinity chromatography were 61.23%, 7.45 U/mg and 5.60, respectively. Molecular mass of RC was determined to be 35 kDa based on the result of SDS-PAGE and interlinking of recombinant cystatin was supposed to be taken place for uncertain reason. RC showed lower activity and acidic stability, but higher thermal stability than that of natural chum salmon egg cystatin. The culture condition for the production of RC by S. cerevisiae YPH 499 was optimized in shake flasks using Response Surface Methodology (RSM). The highest amount of RC production in shake flask, 0.57 U/mL, was obtained at 5.7 of medium pH, 6.7 h of inducing time, and 5.6 g/L of inducing assistant, which was consistent with the result of validation experiment. Thereafter, based on the results of shake flask, the effects of agitation and aeration rates on RC production by S. cerevisiae YPH 499 were determined for scale-up in fermentor. The highest RC production in fermentor, 0.56 U/mL, was obtained at 350 rpm of agitation rate and 1.0 vvm of aeration rate, which was consistent with the result of validation experiment.
RC was added to Alaska pollock surimi to determine its inhibition of gel weakening. RC purified by affinity chromatography at 100μg/g showed the highest inhibition rate of 90% against the autolysis of surimi. The addition of RC resulted in increase of 4.5 folds gel strength and 30% of deformation. The expressible drip of surimi gel decreased from 17.85% to 6.27% and whiteness increased from 49.78 to 52.60 at 100μg/g of RC. The degradation of myosin in surimi gel was significantly inhibited after addition of RC based on the result of SDS-PAGE. RC prevented the degradation of proteins in Alaska pollock surimi better than did egg white (EW). Crude RC purified by alcohol precipitation could also inhibit the gel weakening of Alaska pollock surimi. The gel strength and deformation increased 2.7 folds and 10%, respectively, at 500μg/g of crude RC. Therefore, RC could be applied to Alaska pollock surimi to prevent the gel weakening.
以木瓜蛋白酶作为目标酶,鲑鱼卵cystatin的抑制能力最强;鲑鱼血浆kiniongen的抑制能力高于玻璃鱼卵抑制剂I、池沼公鱼抑制剂和另一种鲑鱼卵抑制剂;以组织蛋白酶L作为目标酶,鲑鱼卵cystatin的抑制能力略低于玻璃鱼卵抑制剂II,高于其他抑制剂;鲑鱼血浆kininogen的抑制能力高于狭鳕卵抑制剂、玻璃鱼卵抑制剂I和鲑鱼卵抑制剂。鲑鱼卵cystatin和鲑鱼血浆kininogen具有广范围的酸碱和热稳定性。在pH 5–8的范围内,鲑鱼卵cystaitn的活性几乎无损失。在pH 6–9的范围内,鲑鱼血浆kininogen可保留60%以上的抑制活力。鲑鱼卵cystatin在70°C以下处理30 min,可保留大于50%的活性。鲑鱼血浆kininogen在60°C处理30 min,可保留50%的活性。鲑鱼卵cystatin与鲑鱼血浆kininogen主要在20–50°C的范围内表现出抑制活性。Dixon-plot作图发现,鲑鱼卵cystatin属于竞争性抑制剂,其抑制常数为2.12 nM;鲑鱼血浆kininogen属于非竞争性抑制剂,其抑制常数为105.32 nM。
通过合成编码鲑鱼卵cystatin的DNA,采用pYES2NT/C质粒,以啤酒酵母YPH 499为宿主,进行了鲑鱼卵cystatin的重组表达。诱导表达后,分别使用选择性镍亲和柱和乙醇沉淀法进行重组cystatin的纯化,其中亲和纯化的回收率、比活力和纯化倍数分别为61.23%、7.45 U/mg和5.60倍。SDS-PAGE检测发现,重组cystatin的分子量为35 kDa,推测其发生了双体交联。与天然鲑鱼卵cystatin相比,重组cystatin的抑制活力有所下降,在酸性范围内的稳定性也有所下降,但其热稳定性得以增强。采用响应面法进行了摇瓶规模啤酒酵母YPH 499生产重组cystatin的优化研究,发现在培养基pH为5.7,诱导时间为6.7 h,诱导辅助剂的添加量为5.6 g/L时,重组cystatin的产量最大,可达0.57 U/mL。该研究结果与重复实验结果相一致。在摇瓶优化研究的基础上,进行了发酵罐中搅动速率和通气速率影响啤酒酵母YPH 499生产重组cystatin的优化研究,发现在350 rpm的搅动速率结合1.0 vvm的通气速率的发酵条件下,发酵罐中重组cystatin产量达到最大,为0.56 U/mL。该研究结果与重复实验结果相一致。
将纯化的重组cystatin添加到狭鳕鱼糜中,进行了鱼糜凝胶劣化的抑制研究。亲和纯化的重组cystatin,添加量为100μg/g时,其对鱼糜凝胶劣化的抑制率可达到90%以上。在该添加剂量下,凝胶的破碎力和变形量分别增加了4.5倍和30%。同时,其可榨出水分含量由17.85%减至6.27%,白度由49.78增加至52.60。SDS-PAGE检测表明,通过添加重组cystatin,可显著抑制蛋白酶引起的肌球蛋白的降解。重组cystatin的抑制效果显著强于蛋清粉。醇沉纯化的重组cystatin也可有效抑制鱼糜凝胶劣化,通过添加500μg/g的醇沉重组cystatin,可使凝胶的破碎力和变形量增加2.7倍和10%。因此,可将重组cystatin用于抑制狭鳕鱼糜的凝胶劣化。
Surimi is a minced and washed fish muscle consisting of salt-soluble myofibrillar proteins. The endogenous proteases in surimi cause an irreversible destruction of the gel structure of surimi and result in decreasing the quality of surimi-based product and its market value. Protease inhibitors have been used to prevent the degradation of surimi gel. Chum salmon egg and plasma were used as raw materials for preparation of protease inhibitors. A cystatin was purified from chum salmon egg with a series of acidic treatment, ultrafiltration and papain affinity chromatography. The recovery, specific inhibitory activity and purification fold were 0.4%, 1.54 U/mg and 192.6, respectively. Its molecular mass was 13 kDa based on the result of SDS-PAGE. Its N-terminal was determined to be N-Gly-Leu-Ile-Gly-Gly-Pro-Met-Asp-Ala-Asn-C, which was identical to that of chum salmon cystatin. A kininogen was purified from chum salmon plasma with one step of papain affinity chromatography. The recovery, specific inhibitory activity and purification fold were 0.94%, 3.84 U/mg and 30.36, respectively. Its molecular mass was 70 kDa based on SDS-PAGE and FPLC. Only under non reducing condition can this CPI be detected by inhibitory activity staining. The result of Periodic acid-Schiff staining showed that it was a glycoprotein.
Chum salmon egg cystatin showed the highest inhibitory activity against papain. The inhibitory activity of chum salmon plasma kininogen against papain was higher than that of glassfish egg inhibitor I, pond smelt inhibitor and chum salmon egg inhibitor. For cathepsins L, chum salmon egg cystatin showed inhibitory activity only lower than that of glassfish egg inhibitor II. Chum salmon plasma kininogen showed higher activity than did glassfish egg inhibitor I and chum salmon egg inhibitor. Chum salmon egg cystatin and plasma kininogen were stable under a wide range of pH. Nearly no loss of its activity was determined under pH 5-8. Chum salmon plasma kininogen possessed higher than 60% activity at pH 6-9. 50% of chum salmon egg cystatin activity could be retained after heating for 30 min at 70°C, while 50% of that of chum salmon plasma kininogen could be retained. Chum salmon egg cystatin and plasma kininogen showed activity mainly at 20-50°C. The Dixon-plots showed that chum salmon cystatin was a competitive inhibitor with inhibition constant of 2.12 nM. Chum salmon plasma kininogen was a noncompetitive inhibitor with inhibition constant of 105.32 nM.
Recombinant chum salmon cystatin (RC) was overexpressed in Saccharomyces cerevisiae YPH 499 incorporating pYES2NT/C vector. After cultivation and induction, RC could be purified by nickel select affinity chromatography or alcohol precipitation. The recovery, specific inhibitory activity and purification fold for nickel affinity chromatography were 61.23%, 7.45 U/mg and 5.60, respectively. Molecular mass of RC was determined to be 35 kDa based on the result of SDS-PAGE and interlinking of recombinant cystatin was supposed to be taken place for uncertain reason. RC showed lower activity and acidic stability, but higher thermal stability than that of natural chum salmon egg cystatin. The culture condition for the production of RC by S. cerevisiae YPH 499 was optimized in shake flasks using Response Surface Methodology (RSM). The highest amount of RC production in shake flask, 0.57 U/mL, was obtained at 5.7 of medium pH, 6.7 h of inducing time, and 5.6 g/L of inducing assistant, which was consistent with the result of validation experiment. Thereafter, based on the results of shake flask, the effects of agitation and aeration rates on RC production by S. cerevisiae YPH 499 were determined for scale-up in fermentor. The highest RC production in fermentor, 0.56 U/mL, was obtained at 350 rpm of agitation rate and 1.0 vvm of aeration rate, which was consistent with the result of validation experiment.
RC was added to Alaska pollock surimi to determine its inhibition of gel weakening. RC purified by affinity chromatography at 100μg/g showed the highest inhibition rate of 90% against the autolysis of surimi. The addition of RC resulted in increase of 4.5 folds gel strength and 30% of deformation. The expressible drip of surimi gel decreased from 17.85% to 6.27% and whiteness increased from 49.78 to 52.60 at 100μg/g of RC. The degradation of myosin in surimi gel was significantly inhibited after addition of RC based on the result of SDS-PAGE. RC prevented the degradation of proteins in Alaska pollock surimi better than did egg white (EW). Crude RC purified by alcohol precipitation could also inhibit the gel weakening of Alaska pollock surimi. The gel strength and deformation increased 2.7 folds and 10%, respectively, at 500μg/g of crude RC. Therefore, RC could be applied to Alaska pollock surimi to prevent the gel weakening.
引文
1. An H.,Peters M. Y.,Seymour T. A. Roles of endogenous enzymes in surimi gelation. Trends. Food Sci. Technol. 1996,7,321?327.
2. Lanier T. C.,Lee C. M. Measurement of surimi composition and functional properties. In Surimi Technology; Marcel Dekker: New York,1992; pp 123?l63.
3. Morrissey M. T.,Tan S. M. World resources for surimi. In Surimi and surimi seafood; Park J. W.,ed.,Marcel Dekker: New York,2000; pp 1?22.
4.鸿巢章二,桥本周久编,郭晓风译.水产利用化学,中国农业出版社: 2001,8,pp 51–63。
5.李里特.食品物性学,中国农业出版社: 1998,2,pp 100–154。
6. Motoki M.,Seguro H. Transglutaminase and its use for food processing. Trends. Food Sci. Technol. 1998,9,204?210.
7. Kang I. S.,Lanier T. C. Heat-induced softening of surimi gels by proteinases. In Surimi and surimi seafood; Park J. W.,ed.,Marcel Dekker: New York,2000; pp 445?474.
8.曾广智,谭宁华,贾锐锐,潘蓄林.组织蛋白酶及其抑制剂研究进展.云南植物研究(Acta Botanica Yunnanica),2005,27,337–354.
9.常泓,南庆贤.钙激活酶激活蛋白的研究进展.肉类工业,2001,12,45–48。
10. García-Carreňo,F. L.,Hernándz-Cortés P. Use of protease inhibitors in seafood products. In Seafood enzymes: utilization and influence on postharvest seafood quality; Harrd N. F.,Simpson B. J.,eds.,Marcel Dekker: New York,2000; pp 531–548.
11. Otto H. H.,Schirmeister T. Cysteine proteases and their inhibitors. Chem. Rev. 1997,97,133–171.
12. Sarath G.,Motte R. S.,Wagner F. W. Protease assay methods. In: Proteolytic enzymes: a practical approach. Beynon B. J.,Ed.; Oxford University Press: England,1989; p 28.
13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 1970,227,680–685.
14. García-Carreňo F. L.,Dimes L. E.,Haard N. F. Substrate–gel electrophoresis for compositionand molecular weight of proteinases or proteinaceous proteinase inhibitors. Anal. Biochem. 1993,214,65–69.
15. Zacharius R. M.,Zell T. E.,Morrison J. H.,Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 1969,30,148–152.
16. Pisani F. M.,Rella R.,Raia C. A.,Rozzo C.,Nucci R.,Gambacorta A.,De Rosa M.,Rossi M. Thermostableβ-galactosidase from the Archaebacterium Sulfolobus solfataricus: purification and properties. Eur. J. Biochem. 1990,187,321–328.
17. Tzeng S. S.,Chen G. H.,Jiang S. T. Expression of soluble thioredoxin fused-carp (Cyprinus Carpio) ovarian cystatin in Escherichia coli. J. Food Sci. 2002,67,2309–2316.
18. Robinson H. W.,Hodgen C. G. The biuret reaction in the determination of serum protein. I. A study of the condition necessary for the production of the stable color which bears a quantitative relationship to the protein concentration. J. Biol. Chem. 1940,135,707–725.
19. Dixon M.,Webb E. C. Enzymes; Logman Group Ltd.: London,1979; p 556.
20. Horwitz W. Fish and other marine products. In Official methods of analysis of AOAC international. Tennyson J. M.,Winlers R.S.,Eds.; 2000; Vol. 2.
21. Yamashita M.,Konagaya S. Molecular cloning and gene expression of chum salmon cystatin. J. Biochem. 1996,120,483–487.
22. Yamashita M.,Konagaya S. Cysteine protease inhibitor in egg of chum salmon. J. Biochem. 1991,110,762–766.
23. Tsushima H.,Ueki A.,Mine H.,Nakajima N.,Sumi H.,Hopsu-Havu V. K. Purification and characterization of a cystatin-type cysteine proteinase inhibitor in the human hair shaft. Arch. Dermatol. Res. 1992,284,380–385.
24. Yl?nen A.,Rinne A.,Herttuainen J.,B?gwald J.,J?rvinen M.,Kalkkinen N. Atlantic salmon (Salmo salar L.) skin contains a novel kininogen and another cysteine protease inhibitor. Eur. J. Biochem. 1999,266,1066–1072.
25. Matsuishi M.,Okitani A. Purification and properties of cysteine proteinase inhibitors from rabbit skeletal muscle. Comp. Biochem. Phys. B. 2003,136,309–316.
26. Rassam M.,Laing W. A. Purification and characterization of phytocystatins from kiwifruit cortex and seeds. Phytochem. 2004,65,19–30.
27. Ustadi,Kim K. Y.,Kim S. M. Purification and identification of a protease inhibitor from glassfish (Liparis tanakai) eggs. J. Agric. Food Chem. 2005,53,7667–7672.
28. Kim K. Y.,Ustadi,Kim S. M. Characteristics of the protease inhibitor purified from chum salmon (Oncorhynchus keta) eggs. Food Sci. Biotehnol. 2006,15,28–32.
29. Rawdkuen S. , Benjakul S. , Visessanguan W. , Lanier T. C. Partial purification and characterization of cysteine proteinase inhibitor from chicken plasma. Comp. Biochem. Phys. B. 2006,144,544–552.
30. Zhou W. T.,Fujita M.,Yamamoto S. Effects of ambient temperatures on blood viscosity and plasma protein concentration of broiler chickens. J. Therm. Biol. 1999,24,105–112.
31. Weerasinghe V. C.,Morrissey M. T.,An H. Characterization of active components in food-grade proteinase inhibitors for surimi manufacture. J. Agric. Food Chem. 1996,44,2584–2590.
32. Halliday D. A. Blood–A source of proteins. Proc. Biochem. 1973,8,15–17.
33. Katori M.,Majima M. Role of the renal kallikrein–kinin system in the development of hypertension. Immunopharmacol. 1997,36,237–242.
34. Ustadi,Kim K. Y.,Kim S. M. Protease inhibitor in fish eggs: A comparative study. J. Ocean. Univ. China. 2005,4,198–204.
35. Watson J. D.,Hopkins N. H.,Roberts J. W.,Steitz J. A.,Weiner A. M. Molecular biology of the gene. 4th ed. Park M.,ed.; Benjamin Cummings,1987.
36. K K.,Yu L. L.,Chen J. X. Construction of highly stabilized yeast vector and high expression and secretion of human interferonαA gene in yeast. Sci. Chin. Ser. B. 1992,9: 922–929.
37. Wrenger C,Eschbach ML,Muller IB,Warnecke D,Walter RD. 2005. Analysis of the vitamin B6 biosynthesis pathway in the human malaria parasite Plasmodium falciparum. J. Biol. Chem. 280,5242–5248.
38. Ba? D.,Boyac? ?. H. Modeling and optimization I: Usability of response surface methodology. J. Food Eng. 2007,78,836?845.
39. Chatterjee S., Kumar V., Majumder C.B., Roy P. Screening of some anti-progestin endocrine disruptors using a recombinant yeast based in vitro bioassay. Toxicol. in Vitro. 2008, 22, 788–798.
40. Wang B.D., Strunnikov A. Transcriptional homogenization of rDNA repeats in the episome-based nucleolus induces genome-wide changes in the chromosomal distribution of condensing. Plasmid. 2008, 59, 45–53.
41. Niu M., Li X., Wei J., Cao R., Zhou B., Chen P. The molecular design of a recombinant antimicrobial peptide CP and its in vitro activity. Protein Expression Purif. 2008, 57, 95–100.
42. Kim Y., Hendrickson R., Mosier N.S., Ladisch M.R., Bals B., Balan V., Dale B.E. Enzyme hydrolysis and ethanol fermentation of liquid hot water and AFEX pretreated distillers’grains at high-solids loadings. Bioresour. Technol. 2007.
43. Potath J.,Carlsson J.,Olsson I. Nature,1975,258: 598–599.
44.慕运动.响应面方法及其在食品工业中的应用.郑州工程学院学报(Journal of Zhengzhou Grain College),2001,22,93–96。
45. Medeiros A. B. P.,Pandey A.,Freitas R. J. S.,Christen P.,Soccol C. R. Optimization of the production of aroma compounds by Kluyveromyces marxianus in solid-state fermentation using factorial design and response surface methodology. Biochem. Eng. J. 2000,6,33?39.
46. Wang G.,Mu Y.,Yu H. Q. Response surface analysis to evaluate the influence of pH,temperature and substrate concentration on the acidogenesis of sucrose-rich wastewater. Biochem. Eng. J. 2005,23,175?184.
47. Shih I. L.,Shen M. H. Application of response surface methodology to optimize production of poly-ε-lysine by Streptomyces albulus IFO 14147,Enzyme Microb. Technol. 2006,39,15?21.
48. Sharmaa L. , Singha A. K. , Pandaa B. , Mallick N. Process optimization for poly-β-hydroxybutyrate production in a nitrogen fixing cyanobacterium,Nostoc muscorum using response surface methodology. Bioresour. Technol. 2007,98,987?993.
49. Clark D.,Rowlett R. S.,Coleman J. R.,Klessig D. F. Complementation of the yeast deletion mutantΔNCE103 by members of theβclass of carbonic anhydrases is dependent on carbonicanhydrase activity rather than on antioxidant activity. Biochem. J. 2004,379,609?615.
50. Choi S. U.,Paik H. D.,Lee S. C.,Nihira T.,Hwang Y. I. Enhanced productivity of human lysozyme by pH-controlled batch fermentation of recombinant Saccharomyces cerevisiae. J. Biosci. Bioeng. 2004,98,132?135.
51. Manolov R. J.,Influence of agitation rate on growth and ribonuclease production by free and immobilized Aspergillus clavatus cells. Appl. Biochem. Biotechnol. 1992,33,157?169.
52. Shioya S.,Morikawa M.,Kajihara Y.,Shimizu H. Optimization of agitation and aeration conditions for maximum virginiamycin production,Appl. Microbiol. Biotechnol. 1999,51,164?169.
53.Raman S.B.,Rathinasabapathi B.β-Alanine N-methyltransferase of limonium latifolium. cDNA cloning and functional expression of a novel N-methyltransferase implicated in the synthesis of the osmoprotectantβ-alanine betaine. Plant Physiol. 2003,132,1642?1651.
54. Toma M. K.,Ruklisha M. P.,Vanags J. J.,Zeltina M. O.,Leite M. P.,Galonina N. I.,Viesturs U. F.,Tendergy R. P. Inhibition of microbial growth and metabolism by excess turbulence,Biotechnol. Bioeng. 1991,38,552?556.
55. Morrissey M. T.,Wu J. W.,Lin D. D.,An H. Effect of food grade inhibition autolysis and gel strength of surimi. J. Food Sci. 1993,58,1050–1054.
56. Liu D.,Kanoh S.,Niwa E. Effect of cysteine protease inhibitor on the setting of Alaska pollack surimi paste. Nippon Suisan Gakkaishi 1996,62,275–279.
57. Liu D.,Kanoh S.,Niwa E. Effect of serine protease inhibitor on the setting of Alaska pollack surimi paste. Nippon Suisan Gakkaishi 1996,62,791–795.
58. Park J. W. Ingredient technology and formulation development. In Surimi and surimi seafood,Marcel Dekker: New York,2000; pp 343?391.
59. Park J. W. Functional protein additives in surimi gels. J. Food. Sci. 1994,59,525?527.
60. Ng C. S. Measurement of free and expressible drips. In Laboratory manual on analytical methods and procedure for fish and fish products; Hasegawa H.,Ed; Southeast Asian Fisheries Development Center: Singapore,1987; pp 1?2.
61. Robinson H.W, Hodgen C.G. The biuret reaction in the determination of serum protein. I. A study of the condition necessary for the production of the stable color which bears a quantitative relationship to the protein concentration. J. Biol. Chem. 1940, 135, 707?725.
62. Steel R. G. D.,Torrie J. H. In Principle and procedure of statistics; MacGraw-Hill: New York,1980.
63. Chan J. K.,Gill T. A.,Paulson A. T. Cross-linking of myosin heavy chains from cod,herring and silver hake during thermal setting. J. Food Sci. 1992,57(4),906–912.
64. Benjakul S.,Visessanguan W.,Srivilai C. Porcine plasma proteins as gel enhancer in bigeye snapper (Priacanthus tayenus) surimi. J. Food Biochem. 2001,25(4):285–305.
65. Niwa E. Functional aspect of surimi. In Proceedings of the International Symposium on Engineered Seafood Including Surimi,Martin R. E.; Collete R. L. Eds,p. 141–147. National Fisheries Institute,Washington,DC. 1985.
66. Hamann D. D.,Amato P. M.,Wu M. C.,Foegeding E. A. Inhibition of modori (gel weakening) in surimi by plasma hydrolysate and egg white. J. Food Sci. 1990,55(3),665–669.
67. Lanier T. C.,Lee C. M. Surimi Technology. New York: Marcel Dekker. 1992. p 303–306.
68. Roussel H, Cheflel J.C. Mechanism of gelation of sardine protein influence of thermal processing and various additives on the texture and protein solubility of kamaboko gel. Int. J. Food Sci. Technol. 1990, 25, 260?280.
69. Benjakul S, Visessanguan W. Pig plasma protein: potential use as proteinase inhibitor for surimi manufacture; inhibitory activity and the active components. J. Sci. Food Agric. 2000, 80, 1351–1356.
70. Hossain M.I, Itoh Y, Morioka1 K, Obatake A. Contribution of the polymerization of protein by disulfide bonding to increased gel strength of walleye pollack surimi gel with preheating time. Fish Sci. 2001, 67, 710?717.
2. Lanier T. C.,Lee C. M. Measurement of surimi composition and functional properties. In Surimi Technology; Marcel Dekker: New York,1992; pp 123?l63.
3. Morrissey M. T.,Tan S. M. World resources for surimi. In Surimi and surimi seafood; Park J. W.,ed.,Marcel Dekker: New York,2000; pp 1?22.
4.鸿巢章二,桥本周久编,郭晓风译.水产利用化学,中国农业出版社: 2001,8,pp 51–63。
5.李里特.食品物性学,中国农业出版社: 1998,2,pp 100–154。
6. Motoki M.,Seguro H. Transglutaminase and its use for food processing. Trends. Food Sci. Technol. 1998,9,204?210.
7. Kang I. S.,Lanier T. C. Heat-induced softening of surimi gels by proteinases. In Surimi and surimi seafood; Park J. W.,ed.,Marcel Dekker: New York,2000; pp 445?474.
8.曾广智,谭宁华,贾锐锐,潘蓄林.组织蛋白酶及其抑制剂研究进展.云南植物研究(Acta Botanica Yunnanica),2005,27,337–354.
9.常泓,南庆贤.钙激活酶激活蛋白的研究进展.肉类工业,2001,12,45–48。
10. García-Carreňo,F. L.,Hernándz-Cortés P. Use of protease inhibitors in seafood products. In Seafood enzymes: utilization and influence on postharvest seafood quality; Harrd N. F.,Simpson B. J.,eds.,Marcel Dekker: New York,2000; pp 531–548.
11. Otto H. H.,Schirmeister T. Cysteine proteases and their inhibitors. Chem. Rev. 1997,97,133–171.
12. Sarath G.,Motte R. S.,Wagner F. W. Protease assay methods. In: Proteolytic enzymes: a practical approach. Beynon B. J.,Ed.; Oxford University Press: England,1989; p 28.
13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 1970,227,680–685.
14. García-Carreňo F. L.,Dimes L. E.,Haard N. F. Substrate–gel electrophoresis for compositionand molecular weight of proteinases or proteinaceous proteinase inhibitors. Anal. Biochem. 1993,214,65–69.
15. Zacharius R. M.,Zell T. E.,Morrison J. H.,Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal. Biochem. 1969,30,148–152.
16. Pisani F. M.,Rella R.,Raia C. A.,Rozzo C.,Nucci R.,Gambacorta A.,De Rosa M.,Rossi M. Thermostableβ-galactosidase from the Archaebacterium Sulfolobus solfataricus: purification and properties. Eur. J. Biochem. 1990,187,321–328.
17. Tzeng S. S.,Chen G. H.,Jiang S. T. Expression of soluble thioredoxin fused-carp (Cyprinus Carpio) ovarian cystatin in Escherichia coli. J. Food Sci. 2002,67,2309–2316.
18. Robinson H. W.,Hodgen C. G. The biuret reaction in the determination of serum protein. I. A study of the condition necessary for the production of the stable color which bears a quantitative relationship to the protein concentration. J. Biol. Chem. 1940,135,707–725.
19. Dixon M.,Webb E. C. Enzymes; Logman Group Ltd.: London,1979; p 556.
20. Horwitz W. Fish and other marine products. In Official methods of analysis of AOAC international. Tennyson J. M.,Winlers R.S.,Eds.; 2000; Vol. 2.
21. Yamashita M.,Konagaya S. Molecular cloning and gene expression of chum salmon cystatin. J. Biochem. 1996,120,483–487.
22. Yamashita M.,Konagaya S. Cysteine protease inhibitor in egg of chum salmon. J. Biochem. 1991,110,762–766.
23. Tsushima H.,Ueki A.,Mine H.,Nakajima N.,Sumi H.,Hopsu-Havu V. K. Purification and characterization of a cystatin-type cysteine proteinase inhibitor in the human hair shaft. Arch. Dermatol. Res. 1992,284,380–385.
24. Yl?nen A.,Rinne A.,Herttuainen J.,B?gwald J.,J?rvinen M.,Kalkkinen N. Atlantic salmon (Salmo salar L.) skin contains a novel kininogen and another cysteine protease inhibitor. Eur. J. Biochem. 1999,266,1066–1072.
25. Matsuishi M.,Okitani A. Purification and properties of cysteine proteinase inhibitors from rabbit skeletal muscle. Comp. Biochem. Phys. B. 2003,136,309–316.
26. Rassam M.,Laing W. A. Purification and characterization of phytocystatins from kiwifruit cortex and seeds. Phytochem. 2004,65,19–30.
27. Ustadi,Kim K. Y.,Kim S. M. Purification and identification of a protease inhibitor from glassfish (Liparis tanakai) eggs. J. Agric. Food Chem. 2005,53,7667–7672.
28. Kim K. Y.,Ustadi,Kim S. M. Characteristics of the protease inhibitor purified from chum salmon (Oncorhynchus keta) eggs. Food Sci. Biotehnol. 2006,15,28–32.
29. Rawdkuen S. , Benjakul S. , Visessanguan W. , Lanier T. C. Partial purification and characterization of cysteine proteinase inhibitor from chicken plasma. Comp. Biochem. Phys. B. 2006,144,544–552.
30. Zhou W. T.,Fujita M.,Yamamoto S. Effects of ambient temperatures on blood viscosity and plasma protein concentration of broiler chickens. J. Therm. Biol. 1999,24,105–112.
31. Weerasinghe V. C.,Morrissey M. T.,An H. Characterization of active components in food-grade proteinase inhibitors for surimi manufacture. J. Agric. Food Chem. 1996,44,2584–2590.
32. Halliday D. A. Blood–A source of proteins. Proc. Biochem. 1973,8,15–17.
33. Katori M.,Majima M. Role of the renal kallikrein–kinin system in the development of hypertension. Immunopharmacol. 1997,36,237–242.
34. Ustadi,Kim K. Y.,Kim S. M. Protease inhibitor in fish eggs: A comparative study. J. Ocean. Univ. China. 2005,4,198–204.
35. Watson J. D.,Hopkins N. H.,Roberts J. W.,Steitz J. A.,Weiner A. M. Molecular biology of the gene. 4th ed. Park M.,ed.; Benjamin Cummings,1987.
36. K K.,Yu L. L.,Chen J. X. Construction of highly stabilized yeast vector and high expression and secretion of human interferonαA gene in yeast. Sci. Chin. Ser. B. 1992,9: 922–929.
37. Wrenger C,Eschbach ML,Muller IB,Warnecke D,Walter RD. 2005. Analysis of the vitamin B6 biosynthesis pathway in the human malaria parasite Plasmodium falciparum. J. Biol. Chem. 280,5242–5248.
38. Ba? D.,Boyac? ?. H. Modeling and optimization I: Usability of response surface methodology. J. Food Eng. 2007,78,836?845.
39. Chatterjee S., Kumar V., Majumder C.B., Roy P. Screening of some anti-progestin endocrine disruptors using a recombinant yeast based in vitro bioassay. Toxicol. in Vitro. 2008, 22, 788–798.
40. Wang B.D., Strunnikov A. Transcriptional homogenization of rDNA repeats in the episome-based nucleolus induces genome-wide changes in the chromosomal distribution of condensing. Plasmid. 2008, 59, 45–53.
41. Niu M., Li X., Wei J., Cao R., Zhou B., Chen P. The molecular design of a recombinant antimicrobial peptide CP and its in vitro activity. Protein Expression Purif. 2008, 57, 95–100.
42. Kim Y., Hendrickson R., Mosier N.S., Ladisch M.R., Bals B., Balan V., Dale B.E. Enzyme hydrolysis and ethanol fermentation of liquid hot water and AFEX pretreated distillers’grains at high-solids loadings. Bioresour. Technol. 2007.
43. Potath J.,Carlsson J.,Olsson I. Nature,1975,258: 598–599.
44.慕运动.响应面方法及其在食品工业中的应用.郑州工程学院学报(Journal of Zhengzhou Grain College),2001,22,93–96。
45. Medeiros A. B. P.,Pandey A.,Freitas R. J. S.,Christen P.,Soccol C. R. Optimization of the production of aroma compounds by Kluyveromyces marxianus in solid-state fermentation using factorial design and response surface methodology. Biochem. Eng. J. 2000,6,33?39.
46. Wang G.,Mu Y.,Yu H. Q. Response surface analysis to evaluate the influence of pH,temperature and substrate concentration on the acidogenesis of sucrose-rich wastewater. Biochem. Eng. J. 2005,23,175?184.
47. Shih I. L.,Shen M. H. Application of response surface methodology to optimize production of poly-ε-lysine by Streptomyces albulus IFO 14147,Enzyme Microb. Technol. 2006,39,15?21.
48. Sharmaa L. , Singha A. K. , Pandaa B. , Mallick N. Process optimization for poly-β-hydroxybutyrate production in a nitrogen fixing cyanobacterium,Nostoc muscorum using response surface methodology. Bioresour. Technol. 2007,98,987?993.
49. Clark D.,Rowlett R. S.,Coleman J. R.,Klessig D. F. Complementation of the yeast deletion mutantΔNCE103 by members of theβclass of carbonic anhydrases is dependent on carbonicanhydrase activity rather than on antioxidant activity. Biochem. J. 2004,379,609?615.
50. Choi S. U.,Paik H. D.,Lee S. C.,Nihira T.,Hwang Y. I. Enhanced productivity of human lysozyme by pH-controlled batch fermentation of recombinant Saccharomyces cerevisiae. J. Biosci. Bioeng. 2004,98,132?135.
51. Manolov R. J.,Influence of agitation rate on growth and ribonuclease production by free and immobilized Aspergillus clavatus cells. Appl. Biochem. Biotechnol. 1992,33,157?169.
52. Shioya S.,Morikawa M.,Kajihara Y.,Shimizu H. Optimization of agitation and aeration conditions for maximum virginiamycin production,Appl. Microbiol. Biotechnol. 1999,51,164?169.
53.Raman S.B.,Rathinasabapathi B.β-Alanine N-methyltransferase of limonium latifolium. cDNA cloning and functional expression of a novel N-methyltransferase implicated in the synthesis of the osmoprotectantβ-alanine betaine. Plant Physiol. 2003,132,1642?1651.
54. Toma M. K.,Ruklisha M. P.,Vanags J. J.,Zeltina M. O.,Leite M. P.,Galonina N. I.,Viesturs U. F.,Tendergy R. P. Inhibition of microbial growth and metabolism by excess turbulence,Biotechnol. Bioeng. 1991,38,552?556.
55. Morrissey M. T.,Wu J. W.,Lin D. D.,An H. Effect of food grade inhibition autolysis and gel strength of surimi. J. Food Sci. 1993,58,1050–1054.
56. Liu D.,Kanoh S.,Niwa E. Effect of cysteine protease inhibitor on the setting of Alaska pollack surimi paste. Nippon Suisan Gakkaishi 1996,62,275–279.
57. Liu D.,Kanoh S.,Niwa E. Effect of serine protease inhibitor on the setting of Alaska pollack surimi paste. Nippon Suisan Gakkaishi 1996,62,791–795.
58. Park J. W. Ingredient technology and formulation development. In Surimi and surimi seafood,Marcel Dekker: New York,2000; pp 343?391.
59. Park J. W. Functional protein additives in surimi gels. J. Food. Sci. 1994,59,525?527.
60. Ng C. S. Measurement of free and expressible drips. In Laboratory manual on analytical methods and procedure for fish and fish products; Hasegawa H.,Ed; Southeast Asian Fisheries Development Center: Singapore,1987; pp 1?2.
61. Robinson H.W, Hodgen C.G. The biuret reaction in the determination of serum protein. I. A study of the condition necessary for the production of the stable color which bears a quantitative relationship to the protein concentration. J. Biol. Chem. 1940, 135, 707?725.
62. Steel R. G. D.,Torrie J. H. In Principle and procedure of statistics; MacGraw-Hill: New York,1980.
63. Chan J. K.,Gill T. A.,Paulson A. T. Cross-linking of myosin heavy chains from cod,herring and silver hake during thermal setting. J. Food Sci. 1992,57(4),906–912.
64. Benjakul S.,Visessanguan W.,Srivilai C. Porcine plasma proteins as gel enhancer in bigeye snapper (Priacanthus tayenus) surimi. J. Food Biochem. 2001,25(4):285–305.
65. Niwa E. Functional aspect of surimi. In Proceedings of the International Symposium on Engineered Seafood Including Surimi,Martin R. E.; Collete R. L. Eds,p. 141–147. National Fisheries Institute,Washington,DC. 1985.
66. Hamann D. D.,Amato P. M.,Wu M. C.,Foegeding E. A. Inhibition of modori (gel weakening) in surimi by plasma hydrolysate and egg white. J. Food Sci. 1990,55(3),665–669.
67. Lanier T. C.,Lee C. M. Surimi Technology. New York: Marcel Dekker. 1992. p 303–306.
68. Roussel H, Cheflel J.C. Mechanism of gelation of sardine protein influence of thermal processing and various additives on the texture and protein solubility of kamaboko gel. Int. J. Food Sci. Technol. 1990, 25, 260?280.
69. Benjakul S, Visessanguan W. Pig plasma protein: potential use as proteinase inhibitor for surimi manufacture; inhibitory activity and the active components. J. Sci. Food Agric. 2000, 80, 1351–1356.
70. Hossain M.I, Itoh Y, Morioka1 K, Obatake A. Contribution of the polymerization of protein by disulfide bonding to increased gel strength of walleye pollack surimi gel with preheating time. Fish Sci. 2001, 67, 710?717.