由商品果胶酶制剂制备高纯果胶酯酶制剂的研究
|
摘要
果胶酯酶(PE)广泛存在于植物、霉菌和细菌中。在一些水果和蔬菜的加工中,天然存在的果胶酯酶起保护果蔬质构的作用。高纯度的果胶酯酶(PE)酶制剂主要应用于果蔬保藏、制备低甲氧基果胶、研究酶的行为模式以及确定果胶结构和功能性质之间的基本关系等。本研究以商品果胶酶作为原料,探索一种制备高纯度果胶酯酶(PE)制剂的生产工艺。
采用超滤和SP Sepharose(?)Fast Flow强阳离子交换凝胶色谱结合的方法复合果胶酶制剂Pc-3中的果胶酯酶和聚半乳糖醛酸酶进行分离。研究结果表明,超滤后,果胶酯酶活力由11.22U/ml上升至98.12U/ml,总酶活力由1402.5下降到1349.15,活力损失3.8%;聚半乳糖醛酸酶酶活由17.46U/ml上升至156.35U/ml,总活力由2182.5下降到2110.73,活力损失了3.3%;酶活比(PE:PG)没有发生改变,均为0.64。在pH4.2时,粗酶中果胶酯酶的吸附率为43.08%,聚半乳糖醛酸酶的吸附率为71.08%。在0.25ml/min的洗脱流速下得到最高的第一部分酶活比(PE:PG):3.69,但是果胶酯酶的总得率仅为55.97%,而且整个洗脱结果也并未将两种酶完全分离。
研究了PC-3中果胶酯酶(PE)和聚半乳糖醛酸酶(PG)的热稳定性,发现粗酶中果胶酯酶的最适温度为43℃,聚半乳糖醛酸酶有两个最适温度,分别为30℃和50℃。不同温度下两种酶的热失活研究表明,果胶酯酶是热不稳定的,聚半乳糖醛酸酶分为热稳定和热不稳定两个部分。在55℃加热10min后,有78.16%的果胶酯酶酶活力保留下来,聚半乳糖醛酸酶残留酶活为9.98%,两种酶的失活均为不可逆失活。说明通过适当的热处理可以改变粗酶制剂中果胶酯酶和聚半乳糖醛酸酶的酶活比例,从而得到高果胶酯酶酶活的酶制剂。
研究了抑制剂对PC-3中果胶酯酶(PE)和聚半乳糖醛酸酶(PG)抑制效果,发现高氯化铁(FeCl_3),七水合硫酸亚铁(FeSO_4·7H_2O)和L-谷氨酸钠对聚半乳糖醛酸酶酶活均具有明显的抑制作用,甘氨酸并不具有很好的抑制能力。同时上述几种试剂对果胶酯酶都没有抑制作用。而脲对两种酶均有抑制作用。从食品安全和抑制效果两个角度出发,七水合硫酸亚铁(FeSO_4·7H_2O)是一种较好的聚半乳糖醛酸酶酶活抑制剂。在其浓度为1mmol/L的时候,PG酶活迅速下降到原酶活的33.15%,在浓度为5mmol/L时,PE残留酶活仍高达92.7%。
通过正交实验,得到制备高纯果胶酯酶制剂的最优工艺条件是:温度55℃,七水合硫酸亚铁(FeSO_4·7H_2O)浓度1mol/L,时间10min。在此条件下,果胶酯酶酶活残余为80.57%,而聚半乳糖醛酸酶的活力仅仅残留了8.29%。
研究了自制果胶酯酶(PE)制剂对菠萝颗粒硬度的影响,并将实验结果与Rapidase(?)FP Super进行了比较。正交实验表明自制果胶酯酶制剂应用于菠萝果粒的最优条件是果胶酯酶溶液浓度为0.5%,乳酸钙浓度为0.3%,处理时间是25min。在此条件处理,放置一周后,菠萝颗粒硬度较未处理的样品增加了84.29%。在相同果胶酯酶活力的处理条件下,Rapidase(?)FP Super使菠萝颗粒的硬度增加了1.63倍。
Pectinesterase(PE) exists in the plant,fungi and the germ widely.In some fruits and vegetable processing,the natural PE has the function of protecting the texture.The PE production of high pure degree is mainly applied to preserve the fruit and vegetable,product low methyl oxygen pectin,study the basic relationship of PE's behavior pattern and the pectin's structure and function property,and so on.This research investigated a kind of production craft that makes a high pure degree PE production from commercial pectinase preparation.
The PE and Polygalacturonase(PG) of Pc-3 were isolated by ultrafiltration and SP Sepharose(?) Fast Flow—one sort of intense cationic gel filtration chromatography.After ultrafiltration,the PE activity was raised from 11.22U/ml to 98.12U/ml and the total activity was descended from 1402.5 to 1349.15.The PG activity was raised from 17.46U/ml to 156.35U/ml and the total activity was descended from 2182.5 to 2110.73.But the activity ratios(PE:PG) are both 0.64.At pH4.2,the adsorption ratio of PE in the crude enzyme solution was 43.08%and that of PG was 71.08%.The maximal activity ratio(PE:PG) was obtained at 0.25ml/min and the value was 3.69.But the yield of PE was only 55.97%.They were not separated completely.
The thermal Characterization of PE and PG of Pc-3 was studied.The optimal temperature of PE is 43℃.PG has two optimal temperatures:30℃and 50℃.The study on stability of PE and PG at different temperatures declared that PE is thermal unstable and PG has two sorts:thermal stable and thermal unstable.After treated 10min at 55℃,PE activity retained 78.16%and PG activity remained only 9.98%.It proves we can obtain high PE activity production through proper thermal treatment.
The inhibiting effect on PE and PG activity of commercial pectinase preparation by Iron(Ⅲ) chloride hexahydrate,Iron(Ⅱ) sulfate heptahydrate,glycine,L-Glutamic acid sodium salt and urea was studied. Iron(Ⅲ) chloride hexahydrate,Iron(Ⅱ) sulfate heptahydrate and L-Glutamic acid sodium salt have the obvious and similar inhibiting effect:their inhibiting abilities increased companied with their concentration in a certain range and kept unalterable out of the range.Glycine has unstable inhibiting ability.The above four reagents have no inhibiting effect on PE.Urea has the inhibiting effect on two enzymes and the effect on PG is stronger.Considering to safe,FeSO_4·7H_2O is the best inhibitor among them.When it's concentration was 1mmol/L,the PG activity dropped off to 33.15%and When it's concentration was 5mmol/L,the PE activity remained 92.7%.
The optimum processing conditions of high PE activity production was studied and obtained as-following:temperature:55℃,FeSO_4·7H_2O concentration:1mol/L,time:10min.At this condition,the PE residual activity(%) was 80.57%and the PG residual activity(%) was 8.29%respectively。
The influence on the hardness of pineapple grain by the self-made PE production was studied.Under the optimal condition(PE concentration:0.5%,the lactic acid calcium concentration:0.3%,time:25min.), the hardness increased 84.29%after bestowed 1 week.Rapidase(?) FP Super made the hardness raised 163%with the equal PE activity.
采用超滤和SP Sepharose(?)Fast Flow强阳离子交换凝胶色谱结合的方法复合果胶酶制剂Pc-3中的果胶酯酶和聚半乳糖醛酸酶进行分离。研究结果表明,超滤后,果胶酯酶活力由11.22U/ml上升至98.12U/ml,总酶活力由1402.5下降到1349.15,活力损失3.8%;聚半乳糖醛酸酶酶活由17.46U/ml上升至156.35U/ml,总活力由2182.5下降到2110.73,活力损失了3.3%;酶活比(PE:PG)没有发生改变,均为0.64。在pH4.2时,粗酶中果胶酯酶的吸附率为43.08%,聚半乳糖醛酸酶的吸附率为71.08%。在0.25ml/min的洗脱流速下得到最高的第一部分酶活比(PE:PG):3.69,但是果胶酯酶的总得率仅为55.97%,而且整个洗脱结果也并未将两种酶完全分离。
研究了PC-3中果胶酯酶(PE)和聚半乳糖醛酸酶(PG)的热稳定性,发现粗酶中果胶酯酶的最适温度为43℃,聚半乳糖醛酸酶有两个最适温度,分别为30℃和50℃。不同温度下两种酶的热失活研究表明,果胶酯酶是热不稳定的,聚半乳糖醛酸酶分为热稳定和热不稳定两个部分。在55℃加热10min后,有78.16%的果胶酯酶酶活力保留下来,聚半乳糖醛酸酶残留酶活为9.98%,两种酶的失活均为不可逆失活。说明通过适当的热处理可以改变粗酶制剂中果胶酯酶和聚半乳糖醛酸酶的酶活比例,从而得到高果胶酯酶酶活的酶制剂。
研究了抑制剂对PC-3中果胶酯酶(PE)和聚半乳糖醛酸酶(PG)抑制效果,发现高氯化铁(FeCl_3),七水合硫酸亚铁(FeSO_4·7H_2O)和L-谷氨酸钠对聚半乳糖醛酸酶酶活均具有明显的抑制作用,甘氨酸并不具有很好的抑制能力。同时上述几种试剂对果胶酯酶都没有抑制作用。而脲对两种酶均有抑制作用。从食品安全和抑制效果两个角度出发,七水合硫酸亚铁(FeSO_4·7H_2O)是一种较好的聚半乳糖醛酸酶酶活抑制剂。在其浓度为1mmol/L的时候,PG酶活迅速下降到原酶活的33.15%,在浓度为5mmol/L时,PE残留酶活仍高达92.7%。
通过正交实验,得到制备高纯果胶酯酶制剂的最优工艺条件是:温度55℃,七水合硫酸亚铁(FeSO_4·7H_2O)浓度1mol/L,时间10min。在此条件下,果胶酯酶酶活残余为80.57%,而聚半乳糖醛酸酶的活力仅仅残留了8.29%。
研究了自制果胶酯酶(PE)制剂对菠萝颗粒硬度的影响,并将实验结果与Rapidase(?)FP Super进行了比较。正交实验表明自制果胶酯酶制剂应用于菠萝果粒的最优条件是果胶酯酶溶液浓度为0.5%,乳酸钙浓度为0.3%,处理时间是25min。在此条件处理,放置一周后,菠萝颗粒硬度较未处理的样品增加了84.29%。在相同果胶酯酶活力的处理条件下,Rapidase(?)FP Super使菠萝颗粒的硬度增加了1.63倍。
Pectinesterase(PE) exists in the plant,fungi and the germ widely.In some fruits and vegetable processing,the natural PE has the function of protecting the texture.The PE production of high pure degree is mainly applied to preserve the fruit and vegetable,product low methyl oxygen pectin,study the basic relationship of PE's behavior pattern and the pectin's structure and function property,and so on.This research investigated a kind of production craft that makes a high pure degree PE production from commercial pectinase preparation.
The PE and Polygalacturonase(PG) of Pc-3 were isolated by ultrafiltration and SP Sepharose(?) Fast Flow—one sort of intense cationic gel filtration chromatography.After ultrafiltration,the PE activity was raised from 11.22U/ml to 98.12U/ml and the total activity was descended from 1402.5 to 1349.15.The PG activity was raised from 17.46U/ml to 156.35U/ml and the total activity was descended from 2182.5 to 2110.73.But the activity ratios(PE:PG) are both 0.64.At pH4.2,the adsorption ratio of PE in the crude enzyme solution was 43.08%and that of PG was 71.08%.The maximal activity ratio(PE:PG) was obtained at 0.25ml/min and the value was 3.69.But the yield of PE was only 55.97%.They were not separated completely.
The thermal Characterization of PE and PG of Pc-3 was studied.The optimal temperature of PE is 43℃.PG has two optimal temperatures:30℃and 50℃.The study on stability of PE and PG at different temperatures declared that PE is thermal unstable and PG has two sorts:thermal stable and thermal unstable.After treated 10min at 55℃,PE activity retained 78.16%and PG activity remained only 9.98%.It proves we can obtain high PE activity production through proper thermal treatment.
The inhibiting effect on PE and PG activity of commercial pectinase preparation by Iron(Ⅲ) chloride hexahydrate,Iron(Ⅱ) sulfate heptahydrate,glycine,L-Glutamic acid sodium salt and urea was studied. Iron(Ⅲ) chloride hexahydrate,Iron(Ⅱ) sulfate heptahydrate and L-Glutamic acid sodium salt have the obvious and similar inhibiting effect:their inhibiting abilities increased companied with their concentration in a certain range and kept unalterable out of the range.Glycine has unstable inhibiting ability.The above four reagents have no inhibiting effect on PE.Urea has the inhibiting effect on two enzymes and the effect on PG is stronger.Considering to safe,FeSO_4·7H_2O is the best inhibitor among them.When it's concentration was 1mmol/L,the PG activity dropped off to 33.15%and When it's concentration was 5mmol/L,the PE activity remained 92.7%.
The optimum processing conditions of high PE activity production was studied and obtained as-following:temperature:55℃,FeSO_4·7H_2O concentration:1mol/L,time:10min.At this condition,the PE residual activity(%) was 80.57%and the PG residual activity(%) was 8.29%respectively。
The influence on the hardness of pineapple grain by the self-made PE production was studied.Under the optimal condition(PE concentration:0.5%,the lactic acid calcium concentration:0.3%,time:25min.), the hardness increased 84.29%after bestowed 1 week.Rapidase(?) FP Super made the hardness raised 163%with the equal PE activity.
引文
[1].王璋,食品酶学[M].北京,中国轻工业出版社,1997.7
[2].王璋,许时婴,汤坚,食品化学[M].北京,中国轻工业出版社,2002.8
[3].Y.Tierny,M.Bechet,J.-C.Joncquiert,H.-C.Dubourguier and J.-B.Guillaume,Molecular cloning and expression in Escherichia coli of genes encoding pectate lyase and pectin methylesterase activities from Bacteroides thetaiotaomictron[J].Journal of Applied Bacteriology.1994,76:592-602
[4].Maria C.Maldonado,Ana M.Strasser de Saad,and Danley Callieri.Purification and Characterization of pectinesterase Produced by a Strain of Aspergillus niger[J].Current Microbiology,1994,28:193-196
[5].Sridevi Annapurna Singh and S.G.Appu Rao.A simple fractionation protocol for,and a comprehensive study of the molecular properties of,two major endopolygalacturonases from Aspergillus niger[J].Biotechnol.Appl.Biochem.2002,35:115-123.
[6].食品技术国际研讨会Murcin April 2003,unlimited.DSM
[7].多聚半乳糖醛酸酶在食品中的应用[Z],中国食品产业网2003.9.25
[8].Maria C.Maldonado,Ana M.Strasser de Saad,and Danley Callieri.Catabolite Repression of Synthesis of Inducible Polygalacturonase and Pectinesterase by Aspergillus niger sp.[J].Current Microbiology,1989,18:303-306
[9].Laurent,Alain Kotoujansky,and Yves Bertheayu.Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937:One-step purification,biochemical characteriztion,and production of polyclonal antibodies-Franck[J].Can.J.Microbiol.2000,46:474-480
[10].徐伟,马力,袁永俊等,低甲氧基果胶的胶凝机理及防止预凝胶形成的措施[J],中国食品工业,2004,(1)JAN.:52-53
[11].ANAND NIGHOJKAR,SADHANA SRIVASTAVA and ANIL KUMAR.Production of Low Methoxyl Pectin Using Immobilized Pectinesterase Bioreactors[J].Journal of Fermentation and Bioengineering,1995,80(4):346-349
[12].Brett J.Savary.Perfusion Chromatography Separation of the Tomato Fruit-Specific Pectin Methylesterase from a Semipurified Commercial Enzyme Preparation[J].PREP.BIOCHEM.AND BIOTECHNOL,2001,31(3):241-258
[13].Jesus Alonso,Wenceslao Canet and Teresa Rodriguez.Thermal and Calcium Pretreatment Affects Texture,Pectinesterase and Pectic Substances of Frozen Sweet Cherries[J].Journal of Food Science.1997,62(3):511-515
[14].Jesus Alonso,Teresa Todriguez and Wenceslao Caner.Effect of Calcium Pretreatments on the Texture of Frozen Cherries.Role of Pectinesterase in the Changes in the Pectic Materials[J].J.Agric.Food Chem.1995,43:1011-1016
[15].N.Ben-shalom,Rivka Pinto,J.Kanner and Monika Berman.A Model System of Natural Orange Juice Cloud:Effect of Calcium on Hesperidin-Pectin Particles[J].Journal of Food Science.1985,50:1130-1132,1142
[16].B.Ly Nguyen,A.Van Loey,D.Fachin,I.Verlent,I.M.Hendrickx.Purification,Characterzation,Thermal and High-Pressure Inactivation of Pectin Methylesterase from Bananas(cv Cavendish)[J].Biotechnology and bioengineering,2002,78(6):683-691
[17].M.D.Walkinshaw and Struther Arnott.Conformations and Interactions of Pectins Ⅱ.Models for Junction Zones in Pectinic Acid and Calcium Pectate Gels[J].J.Mol.Biol.1981,153:1075-1085
[18].D.A.Powell,E.R.Morris,M.J.Gidley and D.A.Rees.Conformations and Interactions of Pectins Ⅱ.Influence of Residue Sequence on Chain Association in Calcium Pectate Gels[J].J.Mol.Biol.1982,155:517-531
[19].M Dolores.Alvarez,M Jose Morillo and Wenceslao Canet.Optimisation of freezing Process with Pressure steaming of potato tissues(cv Monalisa)[J].Journal of the Science of Food and Agriculture,1999,79:1237-1248
[20].Johan E.Hoff.Starch "Swelling Pressure" of Cooked Potatoes[J].J.Agr.Food Chem.,1972,20(6):1283-1284
[21].Kozo Komae,Yoshiaki Sone,Mariko Kakuta and Akira Misaki.Isolation of Pectinesterase from Ficus awkeotsang Seeds and Its Implication in Gel-formation of the Awkeotsang Polygalacturonide[J].Agric.Biol.Chem..1989,53(5):1247-1254.
[22].叶华,马力,高秀容,果胶酯酶的研究进展[J].生物学杂志,2005,22(6):5-7
[23].Franck Laurent,Alain Kotoujansky,and Yves Bertheau,Overproduction in Escherichia coli of the pectin methylestease A from Erwinia chrysanthemi3937:One-step purification,biochemical characterization,and production of polyclonal antibodies[J].Can.J.Microbiol,2000,46:474-480
[24].Osamu Yoshihara,Takaharu Matsuo and Akira Kaji,Purification and Properties of Acid Pectinesterase from Corticium rolfsii[J].Agric.Biol.Chem.,1977,41(12):2335-2341
[25].Abel Schejter and Lionel Marcus,Isozymes of Pectinesterase and Polygalacturonase from Botrytis cinerea Pers.[M].Methods in Enzymology,Academic Press Inc.1988:161,366-373
[26].Helga Forster,Pectinesterase from Phytophthora Infestans[M].Methods in Enzymology,Academic Press Inc.1988,161:355-361
[27].Alexander K.Frittrang,Holger Deising and Kurt Mendgen,Characterization and partial purification of pectinesterase,a differentiation-specific enzyme of Uromyces viciae-fabae[J].Journal of General Microbiology,1992,138:2213-2218
[28].张红霞,江晓路,牟海津,胡晓珂,微生物果胶酶的研究进展[J].生物技术,2005,115(15):92-95
[29].Rosane F.Schwan,Richard M.Cooper,and Alan E.Wheals,Endopolygalacturonase sectetion by Kluyveromyces Marxianus and other Cocoa Pulp-degrading Yeasts[J].Enzyme and Microbial Technology,1997,21:234-244
[30].Manuel Serrate,Rose Catalina Bermudez,and Tomas Gonzales Villa,Production,Purification,and Characterization of a Polygalacturonase from a new strain of Kluyveromyces Marxianus Isolated from Coffee Wet-Processing Wastewater[J].Applied Biochemistry and Biotechnology,2002,97:193-208
[31].汤鸣强,谢必峰,吴松刚,黑曲霉SL22111聚半乳糖醛酸酶的纯化和特性[J].药物生物技术,2005,12(1):10-14
[32].N.Anjana Devi and A.G.Appu Rao.Fractionation,purification,and preliminary characterization of polygalacteronases by Aspergillus carbonarius[J].Enzyme and Microbial Technology,1996,18.59-65
[33].Shigetaka Ishii and Tamotsu Yokotsuka.Purifacation and Properties of Endo-polygalacturonase from Aspergillus japonicus[J].Agr.Bio.Chem.,1972,36(11):1885-1893
[34].汤鸣强,黑曲霉SL2-111固体发酵生产果胶酯酶的研究[J],生物技术,2006,16(2):65
[35].焦云鹏,黑曲霉果胶酯酶的研究,食品工业科技[J],2004,25(8):126-127,137
[36].Lilizna Ceci and Jorge Lozano.Deternation of enzymatic activities of commercial pectinases for the clarification of apple juice[J].Food Chemistry,1998,61(1/2):237-241
[37].G.Cameron and Karel Grohmann.Partial Purification and Thermal Characterization of Pectinmethylesterase from Red Grapefruit Finisher Pulp-Randall[J].J.Food Science,1995,60(4):821-825
[38].B.Ly Nguyen.A.Van Loey.D.Fachin.I.Verlent.I.M.Hendrickx.Purification,Characterization,Thermal,and High-pressure inactivation of Pectin Methylesterase from Bananas(Cv cavendish)[J].Biotechnology and Bioengineering,2002,78(6):683-691
[39].Euclides J.Lourenco and Adelaide T.Catutani.Purifacation and Proterties of Pectinesterase fuom Papaya[J].J.Sci.FoodAgric.,1984,35:1120-1127
[40].Sidney P.Colowick and Nathan O.Kaplan.,Methods in Enzymology(I)[M].Academic Press Inc.1995:159-166
[41].L.Miller and J.D.Macmillan,Mode of Action of Pectic Enzymes Ⅱ.Further Purification of Exopolygalacturonate Lyase and Pectinesterase from Clostridium multifermntans[J].Journal of Bacteriology,1970,102(1):72-78
[42].Helen M.Macdonald and Roger Evans,Purification and Properties of Apple Pectinesterase[J].J Sci Food Agric,1996,70:321-326
[43].Brett J.Savary,Arland T.Hotchkiss,and Randall G.Cameron,Characterization of a Salt-Independent Pectin Methylesterase Purified from Valencia Orange Peel[J].Journal of Agricultural and Food Chemistry,2002,50:3553-3558
[44].Jesus Alonso,Nazlin Howell and Wenceslao Caner,Purification and Characterization of Two Pectinmethtylesterase from Persimmon(Diospyros kaki)[J].J Sci Food Agric,1997,75:352-358
[45].Helen M.Macdonald,Roger Evans and Wendy J Spencer,Purification and Properties of the Major Pectinesterases in Lemon Fruits[J].J Sci Food Agric,1993,62:163-168
[46].Graham Ben-Hod,Nurit Bar Nun,Salit Tzaban and A.M.Mayer,Inhibitors of Polygalacturonase in Calli of Orobanche Aegyptiaca[J].Phytochemistry,1997,45(6):1115-1121
[47].Neelam Pathak,Sanjay Mishra,G.G.Sanwal,Purification and characterization of polygalacturonase from banana fruit[J].Phytochemistry,2000,54:147-152
[48].Abel Schejter and Lionel Marcus,Isozymes of Pectinesterase and Polygalacturonase from Botrytis cinerea Pers[M].Academic Press Inc.1988:161,366-373
[491.G.Eldon Brown a,*,Jacqueline K.Bums b,Enhanced activity of abscission enzymes predisposes oranges to invasion by Diplodia natalensis during ethylene degreening[J].Postharvest Biology and Technology,1998,14:217-227
[50].Xuqiao Feng,Akiva Apelbaum,Edward C.sisler,Raphael Goren,Control of ethylene responses in avocado fruit with 1-methylcyclopropene[J].Postharvest Biology and Technology,2000,20:143-150
[51].李远方,撑起冷链物流尚需“联盟”发力[Z],中国商报网站
[52].王莉等,番茄辣椒豆角贮运保鲜实用技术[M],中国农业科学出版社,2004-08
[53].俞中,果胶酯酶在果粒生产中的应用[Z]
[54].Arun Behere,V.Satyanarayan and S.R.Padwal-Desai,Separation and Limited Characterization of three Polygalacturonases of Aspergillus niger[J].Enzyme Microb.Technol.,1993,15:158-161
[55].RANDALL G.CAMERON and KAREL GROHMANN,Partial Purification and Thermal Characterization of Pectinmethylesterase from Red Grapefruit Finisher Pulp[J].JOURNAL OF FOOF SCIENCE,1995,60(4):821-825
[56].S.M.Arbaisah,B.A.Asbi,A.H.Junainah and B.Jamilah,Purification and properties of pectinesterase from soursop(Anona muricata) pulp[J].Food Chemistry,1997,59,(1):33-40
[57].酶学实验讲义 江南大学
[58].A.Kilara,Enzymes and their uses in the processed apple industry:A Review[J].Process Biochemistry,1982,6:35-41
[59].陈来同,徐德昌,生化工艺学[M].北京大学出版社,1997.4
[60].Liu,Y.K.and Luh,B.S.,Purifacation and characterization of endo-polygalacturonase from Rhizopus arrhizus[J].JOURNAL OF FOOF SCIENCE,1978,43:721-726
[61].Sakai T.,Sakarnoto T.,Hallaert,J.and Vandamme,E.J.,Pectin,pectinase,and protopectinase:production,proterties,and applications[J].Advances in Applied Microbiology,1993,39:213-294
[62].Ishii S,Kho K,Sugiyama S et al.,Low methoxyl pectin prepared by pectinesterase from Aspergillus japonicus[J].Food Sci.,1979,44(2):611-614.
[63].叶华,马力,高秀容,果胶酯酶的研究进展[J],生物学杂志.2005 22(6)
[64].魏宝东,姜炳义,冯辉,番茄果实货架期硬度变化及其影响因素的研究[J],食品科学,2005,26(3),249-252
[65].吴彩娥,王文生,寇晓虹.果实成熟软化机理研究进展[J],果树学报,2001,18(6):365-369
[66].Knee M,Bartely IM.Rescent Advances in the Biochemisty of Fruits and Vegetables[M].London:Acad Press,1981
[67].王晓明,佘小平,黄辰等.乙烯和Ca~(2+)在莴苣种子萌发中的作用及其相互关系[J].植物学报,1992,34(11):856-861
[68].闫瑞香,王仁才.果实软化衰老的生理生化机制[J].湖南农业大学学报,2000,26(3):230-235
[69].宗迪.果肉悬浮饮料的研制--结冷胶的流变学性质及其在果肉悬浮饮料中的应用[D]:[硕士学位论文].无锡:江南大学食品学院,2004
[2].王璋,许时婴,汤坚,食品化学[M].北京,中国轻工业出版社,2002.8
[3].Y.Tierny,M.Bechet,J.-C.Joncquiert,H.-C.Dubourguier and J.-B.Guillaume,Molecular cloning and expression in Escherichia coli of genes encoding pectate lyase and pectin methylesterase activities from Bacteroides thetaiotaomictron[J].Journal of Applied Bacteriology.1994,76:592-602
[4].Maria C.Maldonado,Ana M.Strasser de Saad,and Danley Callieri.Purification and Characterization of pectinesterase Produced by a Strain of Aspergillus niger[J].Current Microbiology,1994,28:193-196
[5].Sridevi Annapurna Singh and S.G.Appu Rao.A simple fractionation protocol for,and a comprehensive study of the molecular properties of,two major endopolygalacturonases from Aspergillus niger[J].Biotechnol.Appl.Biochem.2002,35:115-123.
[6].食品技术国际研讨会Murcin April 2003,unlimited.DSM
[7].多聚半乳糖醛酸酶在食品中的应用[Z],中国食品产业网2003.9.25
[8].Maria C.Maldonado,Ana M.Strasser de Saad,and Danley Callieri.Catabolite Repression of Synthesis of Inducible Polygalacturonase and Pectinesterase by Aspergillus niger sp.[J].Current Microbiology,1989,18:303-306
[9].Laurent,Alain Kotoujansky,and Yves Bertheayu.Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937:One-step purification,biochemical characteriztion,and production of polyclonal antibodies-Franck[J].Can.J.Microbiol.2000,46:474-480
[10].徐伟,马力,袁永俊等,低甲氧基果胶的胶凝机理及防止预凝胶形成的措施[J],中国食品工业,2004,(1)JAN.:52-53
[11].ANAND NIGHOJKAR,SADHANA SRIVASTAVA and ANIL KUMAR.Production of Low Methoxyl Pectin Using Immobilized Pectinesterase Bioreactors[J].Journal of Fermentation and Bioengineering,1995,80(4):346-349
[12].Brett J.Savary.Perfusion Chromatography Separation of the Tomato Fruit-Specific Pectin Methylesterase from a Semipurified Commercial Enzyme Preparation[J].PREP.BIOCHEM.AND BIOTECHNOL,2001,31(3):241-258
[13].Jesus Alonso,Wenceslao Canet and Teresa Rodriguez.Thermal and Calcium Pretreatment Affects Texture,Pectinesterase and Pectic Substances of Frozen Sweet Cherries[J].Journal of Food Science.1997,62(3):511-515
[14].Jesus Alonso,Teresa Todriguez and Wenceslao Caner.Effect of Calcium Pretreatments on the Texture of Frozen Cherries.Role of Pectinesterase in the Changes in the Pectic Materials[J].J.Agric.Food Chem.1995,43:1011-1016
[15].N.Ben-shalom,Rivka Pinto,J.Kanner and Monika Berman.A Model System of Natural Orange Juice Cloud:Effect of Calcium on Hesperidin-Pectin Particles[J].Journal of Food Science.1985,50:1130-1132,1142
[16].B.Ly Nguyen,A.Van Loey,D.Fachin,I.Verlent,I.M.Hendrickx.Purification,Characterzation,Thermal and High-Pressure Inactivation of Pectin Methylesterase from Bananas(cv Cavendish)[J].Biotechnology and bioengineering,2002,78(6):683-691
[17].M.D.Walkinshaw and Struther Arnott.Conformations and Interactions of Pectins Ⅱ.Models for Junction Zones in Pectinic Acid and Calcium Pectate Gels[J].J.Mol.Biol.1981,153:1075-1085
[18].D.A.Powell,E.R.Morris,M.J.Gidley and D.A.Rees.Conformations and Interactions of Pectins Ⅱ.Influence of Residue Sequence on Chain Association in Calcium Pectate Gels[J].J.Mol.Biol.1982,155:517-531
[19].M Dolores.Alvarez,M Jose Morillo and Wenceslao Canet.Optimisation of freezing Process with Pressure steaming of potato tissues(cv Monalisa)[J].Journal of the Science of Food and Agriculture,1999,79:1237-1248
[20].Johan E.Hoff.Starch "Swelling Pressure" of Cooked Potatoes[J].J.Agr.Food Chem.,1972,20(6):1283-1284
[21].Kozo Komae,Yoshiaki Sone,Mariko Kakuta and Akira Misaki.Isolation of Pectinesterase from Ficus awkeotsang Seeds and Its Implication in Gel-formation of the Awkeotsang Polygalacturonide[J].Agric.Biol.Chem..1989,53(5):1247-1254.
[22].叶华,马力,高秀容,果胶酯酶的研究进展[J].生物学杂志,2005,22(6):5-7
[23].Franck Laurent,Alain Kotoujansky,and Yves Bertheau,Overproduction in Escherichia coli of the pectin methylestease A from Erwinia chrysanthemi3937:One-step purification,biochemical characterization,and production of polyclonal antibodies[J].Can.J.Microbiol,2000,46:474-480
[24].Osamu Yoshihara,Takaharu Matsuo and Akira Kaji,Purification and Properties of Acid Pectinesterase from Corticium rolfsii[J].Agric.Biol.Chem.,1977,41(12):2335-2341
[25].Abel Schejter and Lionel Marcus,Isozymes of Pectinesterase and Polygalacturonase from Botrytis cinerea Pers.[M].Methods in Enzymology,Academic Press Inc.1988:161,366-373
[26].Helga Forster,Pectinesterase from Phytophthora Infestans[M].Methods in Enzymology,Academic Press Inc.1988,161:355-361
[27].Alexander K.Frittrang,Holger Deising and Kurt Mendgen,Characterization and partial purification of pectinesterase,a differentiation-specific enzyme of Uromyces viciae-fabae[J].Journal of General Microbiology,1992,138:2213-2218
[28].张红霞,江晓路,牟海津,胡晓珂,微生物果胶酶的研究进展[J].生物技术,2005,115(15):92-95
[29].Rosane F.Schwan,Richard M.Cooper,and Alan E.Wheals,Endopolygalacturonase sectetion by Kluyveromyces Marxianus and other Cocoa Pulp-degrading Yeasts[J].Enzyme and Microbial Technology,1997,21:234-244
[30].Manuel Serrate,Rose Catalina Bermudez,and Tomas Gonzales Villa,Production,Purification,and Characterization of a Polygalacturonase from a new strain of Kluyveromyces Marxianus Isolated from Coffee Wet-Processing Wastewater[J].Applied Biochemistry and Biotechnology,2002,97:193-208
[31].汤鸣强,谢必峰,吴松刚,黑曲霉SL22111聚半乳糖醛酸酶的纯化和特性[J].药物生物技术,2005,12(1):10-14
[32].N.Anjana Devi and A.G.Appu Rao.Fractionation,purification,and preliminary characterization of polygalacteronases by Aspergillus carbonarius[J].Enzyme and Microbial Technology,1996,18.59-65
[33].Shigetaka Ishii and Tamotsu Yokotsuka.Purifacation and Properties of Endo-polygalacturonase from Aspergillus japonicus[J].Agr.Bio.Chem.,1972,36(11):1885-1893
[34].汤鸣强,黑曲霉SL2-111固体发酵生产果胶酯酶的研究[J],生物技术,2006,16(2):65
[35].焦云鹏,黑曲霉果胶酯酶的研究,食品工业科技[J],2004,25(8):126-127,137
[36].Lilizna Ceci and Jorge Lozano.Deternation of enzymatic activities of commercial pectinases for the clarification of apple juice[J].Food Chemistry,1998,61(1/2):237-241
[37].G.Cameron and Karel Grohmann.Partial Purification and Thermal Characterization of Pectinmethylesterase from Red Grapefruit Finisher Pulp-Randall[J].J.Food Science,1995,60(4):821-825
[38].B.Ly Nguyen.A.Van Loey.D.Fachin.I.Verlent.I.M.Hendrickx.Purification,Characterization,Thermal,and High-pressure inactivation of Pectin Methylesterase from Bananas(Cv cavendish)[J].Biotechnology and Bioengineering,2002,78(6):683-691
[39].Euclides J.Lourenco and Adelaide T.Catutani.Purifacation and Proterties of Pectinesterase fuom Papaya[J].J.Sci.FoodAgric.,1984,35:1120-1127
[40].Sidney P.Colowick and Nathan O.Kaplan.,Methods in Enzymology(I)[M].Academic Press Inc.1995:159-166
[41].L.Miller and J.D.Macmillan,Mode of Action of Pectic Enzymes Ⅱ.Further Purification of Exopolygalacturonate Lyase and Pectinesterase from Clostridium multifermntans[J].Journal of Bacteriology,1970,102(1):72-78
[42].Helen M.Macdonald and Roger Evans,Purification and Properties of Apple Pectinesterase[J].J Sci Food Agric,1996,70:321-326
[43].Brett J.Savary,Arland T.Hotchkiss,and Randall G.Cameron,Characterization of a Salt-Independent Pectin Methylesterase Purified from Valencia Orange Peel[J].Journal of Agricultural and Food Chemistry,2002,50:3553-3558
[44].Jesus Alonso,Nazlin Howell and Wenceslao Caner,Purification and Characterization of Two Pectinmethtylesterase from Persimmon(Diospyros kaki)[J].J Sci Food Agric,1997,75:352-358
[45].Helen M.Macdonald,Roger Evans and Wendy J Spencer,Purification and Properties of the Major Pectinesterases in Lemon Fruits[J].J Sci Food Agric,1993,62:163-168
[46].Graham Ben-Hod,Nurit Bar Nun,Salit Tzaban and A.M.Mayer,Inhibitors of Polygalacturonase in Calli of Orobanche Aegyptiaca[J].Phytochemistry,1997,45(6):1115-1121
[47].Neelam Pathak,Sanjay Mishra,G.G.Sanwal,Purification and characterization of polygalacturonase from banana fruit[J].Phytochemistry,2000,54:147-152
[48].Abel Schejter and Lionel Marcus,Isozymes of Pectinesterase and Polygalacturonase from Botrytis cinerea Pers[M].Academic Press Inc.1988:161,366-373
[491.G.Eldon Brown a,*,Jacqueline K.Bums b,Enhanced activity of abscission enzymes predisposes oranges to invasion by Diplodia natalensis during ethylene degreening[J].Postharvest Biology and Technology,1998,14:217-227
[50].Xuqiao Feng,Akiva Apelbaum,Edward C.sisler,Raphael Goren,Control of ethylene responses in avocado fruit with 1-methylcyclopropene[J].Postharvest Biology and Technology,2000,20:143-150
[51].李远方,撑起冷链物流尚需“联盟”发力[Z],中国商报网站
[52].王莉等,番茄辣椒豆角贮运保鲜实用技术[M],中国农业科学出版社,2004-08
[53].俞中,果胶酯酶在果粒生产中的应用[Z]
[54].Arun Behere,V.Satyanarayan and S.R.Padwal-Desai,Separation and Limited Characterization of three Polygalacturonases of Aspergillus niger[J].Enzyme Microb.Technol.,1993,15:158-161
[55].RANDALL G.CAMERON and KAREL GROHMANN,Partial Purification and Thermal Characterization of Pectinmethylesterase from Red Grapefruit Finisher Pulp[J].JOURNAL OF FOOF SCIENCE,1995,60(4):821-825
[56].S.M.Arbaisah,B.A.Asbi,A.H.Junainah and B.Jamilah,Purification and properties of pectinesterase from soursop(Anona muricata) pulp[J].Food Chemistry,1997,59,(1):33-40
[57].酶学实验讲义 江南大学
[58].A.Kilara,Enzymes and their uses in the processed apple industry:A Review[J].Process Biochemistry,1982,6:35-41
[59].陈来同,徐德昌,生化工艺学[M].北京大学出版社,1997.4
[60].Liu,Y.K.and Luh,B.S.,Purifacation and characterization of endo-polygalacturonase from Rhizopus arrhizus[J].JOURNAL OF FOOF SCIENCE,1978,43:721-726
[61].Sakai T.,Sakarnoto T.,Hallaert,J.and Vandamme,E.J.,Pectin,pectinase,and protopectinase:production,proterties,and applications[J].Advances in Applied Microbiology,1993,39:213-294
[62].Ishii S,Kho K,Sugiyama S et al.,Low methoxyl pectin prepared by pectinesterase from Aspergillus japonicus[J].Food Sci.,1979,44(2):611-614.
[63].叶华,马力,高秀容,果胶酯酶的研究进展[J],生物学杂志.2005 22(6)
[64].魏宝东,姜炳义,冯辉,番茄果实货架期硬度变化及其影响因素的研究[J],食品科学,2005,26(3),249-252
[65].吴彩娥,王文生,寇晓虹.果实成熟软化机理研究进展[J],果树学报,2001,18(6):365-369
[66].Knee M,Bartely IM.Rescent Advances in the Biochemisty of Fruits and Vegetables[M].London:Acad Press,1981
[67].王晓明,佘小平,黄辰等.乙烯和Ca~(2+)在莴苣种子萌发中的作用及其相互关系[J].植物学报,1992,34(11):856-861
[68].闫瑞香,王仁才.果实软化衰老的生理生化机制[J].湖南农业大学学报,2000,26(3):230-235
[69].宗迪.果肉悬浮饮料的研制--结冷胶的流变学性质及其在果肉悬浮饮料中的应用[D]:[硕士学位论文].无锡:江南大学食品学院,2004