果糖基转移酶及过氧化氢酶的固定化
摘要
本文主要研究了果糖基转移酶及过氧化氢酶的固定化,并在此基础上分别对两种固定化酶的酶学性质进行了研究。
从18种离子交换树脂和吸附树脂中,筛选出固定化效果较好的大孔弱碱性苯乙烯系阴离子交换树脂D380为载体、以戊二醛为交联剂,采用先吸附后交联的方法对果糖基转移酶的固定化进行分析,并对固定化条件进行了优化,结果表明,最佳固定化条件为:加酶量为200U/g树脂,吸附pH值为6.0,吸附时间为6h,吸附温度为30℃,交联剂戊二醛浓度为0. 01% ,交联时间为6h ,交联温度为4℃,固定化酶活回收率最高可达87.6%以上。
固定化果糖基转移酶的的最适温度为50～55℃,比游离酶提高了5℃;热稳定性与游离酶基本相同;最适pH6.0比游离酶提高了一个单位;pH稳定性也稍有提高;固定化酶的Km值为0.60离酶的大;固定化酶重复使用10次后残余酶活为10%左右,固定化酶和游离酶均具有较好的贮存稳定性。
以一种无载体固定化技术-交联酶聚集体技术固定化过氧化氢酶,考察了交联剂浓度、交联时间、交联温度对固定化效果的影响。优化的固定化条件为:交联剂戊二醛浓度为1%、交联温度为4℃、交联时间为4h,在此条件下可达到较好的固定化效果。
固定化过氧化氢酶的最适温度为40℃,与游离酶相比提高了5℃;热稳定性比游离酶稍有提高,在25～35℃范围内保温30min后,二者都可保留90%以上的酶活;游离酶与固定化酶的最适pH相同都为8;pH稳定性也稍有提高。
在低聚果糖的生产中,大量副产物葡萄糖的产生阻遏了底物的转化,可通过葡萄糖氧化酶与过氧化氢酶的协调作用,消除普通低聚果糖中的葡萄糖,因此,果糖基转移酶与过氧化氢酶的固定化,为得到高纯度的低聚果糖奠定了一定基础。
This paper studied immobilization of the fructosyltransferase and catalase, and then studied the properties of the immobilized enzyme respectively.
Fructosyltransferase was immobilized on eighteen kinds of ion-exchange and adsorption resins, among them D380 showed the excellent result for immobilization. Then the enzyme was immobilized through cross-linkage of glutaraldehyde. The optimum conditions for the immobilization were as follows: 200U of enzyme per g wet resin, pH 6.0, 30℃, and 6 hours, respectively. The crosslinking temperature and time were 4℃and 6 h respectively, and the concentration of the crosslinking agent (glutaraldehyde) was 0.01%. The activity yield of immobilized enzyme was above 87.6%.
The optimum temperature and pH of the immobilized fructosyltransferase were 50～55℃and 6.0,respectively, whereas for free enzyme,were 45～50℃and 5.0. Thermal and pH stability were both improved by immobilization comparing with the free enzyme.The Km value was 0.60M(for sucrose),which was higher than that(0.29 M)of the free enzyme.This indicated that the affinity between enzyme and substrate decreased significantly.
A kind of carrier-free technology was used in the immobilization of catalase, the enzyme was aggregated first then cross-linked by glutaraldehyde. The optimum conditions for the immobilization were as follows: the concentration of the crosslinking agent (glutaraldehyde) was 1%, and the crosslinking temperature and time were 4℃and 4h respectively.
The optimum temperature of free and immobilized catalase were 40℃and 35℃,respectively, optimum pH and pH stability had no significant change, the thermal stability was better than that of free catlase.
High-purity of fructooligosaccharides was prepared after glucose was removed in normal products by glucose oxidase(GOD) and catalase(CAT). So the study of the immobilization of Fructooligosaccharides and Catalase will make a foundation for the production.
从18种离子交换树脂和吸附树脂中,筛选出固定化效果较好的大孔弱碱性苯乙烯系阴离子交换树脂D380为载体、以戊二醛为交联剂,采用先吸附后交联的方法对果糖基转移酶的固定化进行分析,并对固定化条件进行了优化,结果表明,最佳固定化条件为:加酶量为200U/g树脂,吸附pH值为6.0,吸附时间为6h,吸附温度为30℃,交联剂戊二醛浓度为0. 01% ,交联时间为6h ,交联温度为4℃,固定化酶活回收率最高可达87.6%以上。
固定化果糖基转移酶的的最适温度为50～55℃,比游离酶提高了5℃;热稳定性与游离酶基本相同;最适pH6.0比游离酶提高了一个单位;pH稳定性也稍有提高;固定化酶的Km值为0.60离酶的大;固定化酶重复使用10次后残余酶活为10%左右,固定化酶和游离酶均具有较好的贮存稳定性。
以一种无载体固定化技术-交联酶聚集体技术固定化过氧化氢酶,考察了交联剂浓度、交联时间、交联温度对固定化效果的影响。优化的固定化条件为:交联剂戊二醛浓度为1%、交联温度为4℃、交联时间为4h,在此条件下可达到较好的固定化效果。
固定化过氧化氢酶的最适温度为40℃,与游离酶相比提高了5℃;热稳定性比游离酶稍有提高,在25～35℃范围内保温30min后,二者都可保留90%以上的酶活;游离酶与固定化酶的最适pH相同都为8;pH稳定性也稍有提高。
在低聚果糖的生产中,大量副产物葡萄糖的产生阻遏了底物的转化,可通过葡萄糖氧化酶与过氧化氢酶的协调作用,消除普通低聚果糖中的葡萄糖,因此,果糖基转移酶与过氧化氢酶的固定化,为得到高纯度的低聚果糖奠定了一定基础。
This paper studied immobilization of the fructosyltransferase and catalase, and then studied the properties of the immobilized enzyme respectively.
Fructosyltransferase was immobilized on eighteen kinds of ion-exchange and adsorption resins, among them D380 showed the excellent result for immobilization. Then the enzyme was immobilized through cross-linkage of glutaraldehyde. The optimum conditions for the immobilization were as follows: 200U of enzyme per g wet resin, pH 6.0, 30℃, and 6 hours, respectively. The crosslinking temperature and time were 4℃and 6 h respectively, and the concentration of the crosslinking agent (glutaraldehyde) was 0.01%. The activity yield of immobilized enzyme was above 87.6%.
The optimum temperature and pH of the immobilized fructosyltransferase were 50～55℃and 6.0,respectively, whereas for free enzyme,were 45～50℃and 5.0. Thermal and pH stability were both improved by immobilization comparing with the free enzyme.The Km value was 0.60M(for sucrose),which was higher than that(0.29 M)of the free enzyme.This indicated that the affinity between enzyme and substrate decreased significantly.
A kind of carrier-free technology was used in the immobilization of catalase, the enzyme was aggregated first then cross-linked by glutaraldehyde. The optimum conditions for the immobilization were as follows: the concentration of the crosslinking agent (glutaraldehyde) was 1%, and the crosslinking temperature and time were 4℃and 4h respectively.
The optimum temperature of free and immobilized catalase were 40℃and 35℃,respectively, optimum pH and pH stability had no significant change, the thermal stability was better than that of free catlase.
High-purity of fructooligosaccharides was prepared after glucose was removed in normal products by glucose oxidase(GOD) and catalase(CAT). So the study of the immobilization of Fructooligosaccharides and Catalase will make a foundation for the production.
引文
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[2]王璋.食品酶学[M].北京轻工业出版社,1991
[3]张伟,杨秀山.酶的固定化技术及其应用[J].自然杂志,2000,22(5), 282-286.
[4]刘秀伟,司芳,郭林.酶固定化研究进展[J].化工技术经济,2003,21(4),12-17.
[5]黄秀玲.过氧化氢酶及其模拟酶的电化学研究[D]:[硕士学位论文].武汉:华中师范大学化学学院,2005
[6]陈淘声.固定化酶理论与应用[M].北京:中国轻工业出版社,1987:86-88
[7] Tiseher W, Kasche V. Immobilized enzymes: crystals or carries? Trends Biotechnol.1999, 17:326-335
[8] Bryjak J, Kolarz BN. Ilnmobilisation of trypsin on acrylic copolymers proeess. Biochemistry, 1998,33:409-417
[9] Janssen MHA, Van Langen LM, Pereira SRM, et al. Evaluation of the Performance of immobilised penieillin G acylase using aetive一site titration. Biotechnol Bioeng,2002,78:425-432
[10] Linqiu Cao,Langen Luuk van,Sheldon RA. Immobilised enzymes:carrier-bound or carrier-free? Current Opinion in Biotechnology, 2003, 14:387-394
[11]魏甲乾,周剑平,张文齐.无载体固定化酶的研究进展[J].甘肃科学学报,2006,1(18):66-70
[12] Quiocho FA,Richards FM. Intermolecular cross-linking of a protein in the crystalline state: carboxypeptidase A.Proc Natl Acid Sci USA,1964,52:833-839.
[13] St Clair NL,Navia MA. Cross-linked enzyme crystals as robust biocatalysts.Jam Chem Soc, 1992, 114:7314-7316
[14]董晓毅,夏仕文.交联脲酶聚集体的制备和初步应用[J].生物工程学报, 2003 ,19(3) :332 - 336.
[15] Linqiu Cao , Fred van Rantwijk. Cross - linked enzyme aggregates: A simple and effective method for the immobilization of Penicillin acylase [J]. Org. Lett. ,2000, 2 (10):1361 - 1364.
[16] Lopez - Serrano P, Cao L, Rantwijk. F V et al. Cross - linked enzyme aggregates with enhanced activity: application to lipases [J]. Biotechnology Letters, 2002, 24:1379 - 1383.
[17] Mateo C, Palomo JM,van Langen LM. A new mild cross - linking methodology to prepare cross - linked enzyme aggregates [J]. Biotechnol Bioeng, 2004, 86 (3):273 - 2761
[18] Wilson L, Illanes A, Abian O. Co - aggregation of penicillin acylase and polyionic polymers :an easy methodology to prepare enzyme biocatalysts stabe in organic media[J].Biomacromolecules ,2004 ,5(3) :852 - 8571
[19] Manecke G. Immmobilization of enzymes by various synthetic polymers. Biotechnol Biotechnol Bioeng Symp, 1972, 3:185-187.
[20] Lee KM, Blaghen M, Samama JP, et al. Cross-linked crystalline horse liver alcohol dehydrogenase as a redox catalyst: activity and stability towards organic solvent.Bioorg Chem, 1986, 14:202-210
[21]武仙山,何立千,叶磊.交联酶聚集体—一种无载体酶固定化方法.生物技术,2005,15(2):91-92
[22] Lopez.Serrano P, Cao L , van Rantw ijk F, et al. Croos2linked Enzyme Aggregates with Enhanced Activity: Application to Lipases [J]. Biotechnol Lett, 2002, 24: 137921383.
[23]魏远安,姚评佳,谢庆武,梁锦添.固定化果糖基转移酶的理化性质及稳定性研究[M].广西大学学报.2002.3.27,1001-7445(2002)01-0001-04.
[24]Bacon,J.S.D.and Edelman,J.The action of invertase preparations. J. Arch. Biochem.1950, 28:467
[25]Blanchard, P.H. and Albon, N.The inversion of sucrose. J. Arch.Biochem. 1950,29:220
[26]Bacon,J.S.D.and Bell,D.J.A new trisaccharide produced from sucrose by mould invertase.J.Chem.Soc.1953:2528-2530
[27]Gross,D.,Blanchard,P.H.and Bell,D.J.A trisaccharide formed from sucrose by Yeast invertase.J.Chem.Soc.1954,1927-1730
[28] Hayashi S, Hayashi T , Kiyohisa J , et al. Immobilizat ion ofβ-fructofuranosidase from Aureobasidium sp. ATCC 20524 on porous silica [J]. J IndM icrobio, 1992, 15 (9) : 247-250.
[29] Yun J , Jung K, OH J. Cont inuous p roduct ion of fructoo ligo saccharides from sucrose by immobilized fructosyltransferase [J]. Biotech Techniques, 1995, 9 (11) : 8052808.
[30] Chiang C , L ee C , Sheu D , et al . Immobilization ofβ-froctofuranosidase from Aspergillius on methacryl amide-based polymeric beads for production of fructooligosaccharides [J]. Biotechnol Prog, 1997, 13 (4) : 5772582.
[31]江波,王璋.固定化黑曲酶增殖细胞生产低聚果糖的研究[J].无锡轻工大学学报.1996,1-15
[32] Yun, J.W., Jung, K.H., Oh, J.W. and Lee, J.H. (1990). Appl. Biochem. Biotechnol. 4/25, 299-308.
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