聚丙烯腈膜固定化海藻糖合成酶的研究
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摘要
以聚丙烯腈膜(PAN)为载体,采用吸附法固定化海藻糖合成酶粗酶液。通过单因素法探讨最佳固定化条件,并分析固定化酶酶学性质。结果表明,最佳固定化条件为:pH 7.6、30℃、加酶量0.1 mg/cm~2条件下震荡吸附3 h;该条件下制备的固定化酶最适反应条件为:温度40℃、pH 7.4、初始麦芽糖底物浓度200 g/L。与游离酶相比,固定化酶热稳定性提高10℃、酸碱稳定性由pH 6.6～7.4扩展至pH 5.4～8.0;反应达到平衡时,反应液中海藻糖含量为50.9%,副产物葡萄糖含量为9.1%,较游离酶降低6个百分点,在目前已报道的固定化海藻糖合成酶催化反应中副产物含量最低;在40℃、pH 7.4、麦芽糖底物浓度200 g/L条件下,重复使用累计72 h后,固定化酶活仍保留在初始酶活的64.4%。
The crude trehalose synthase is immobilized on polyacrylonitrile membrane by adsorbtion. The optimal conditions of immobilization is research and the properties of immobilized enzyme by single factor method is analyzied. The results show that the optimum immobilization conditions are:p H 7.6,30 ℃ for 3 h with 0.1 mg trehalose synthase per square centimeter PAN membrane. The optimal reaction conditions of the immobilized trehalose synthase are 7.4,40 ℃ and initial substrate concentration 200 g/L. Compared with the free trehalose synthase,the thermal stability increases by 10 ℃,pH stability expande from pH 6.6 ～ 7.4 to pH 5.4 ～ 8.0. The max trehalose content is 50.9% when the reaction reached equilibrium,the by-product of glucose content is 9.1%,which reduced by 6 percentage points compared with the free enzyme and is the lowest byproduct content of the reported immobilization trehalose synthase. Under the optimum reaction conditions of p H 7.4,40 ℃,substrate concentration 200 g/L,the immobilized enzyme remains64.4% of the initial enzyme activity after repeating used for 72 h.
The crude trehalose synthase is immobilized on polyacrylonitrile membrane by adsorbtion. The optimal conditions of immobilization is research and the properties of immobilized enzyme by single factor method is analyzied. The results show that the optimum immobilization conditions are:p H 7.6,30 ℃ for 3 h with 0.1 mg trehalose synthase per square centimeter PAN membrane. The optimal reaction conditions of the immobilized trehalose synthase are 7.4,40 ℃ and initial substrate concentration 200 g/L. Compared with the free trehalose synthase,the thermal stability increases by 10 ℃,pH stability expande from pH 6.6 ～ 7.4 to pH 5.4 ～ 8.0. The max trehalose content is 50.9% when the reaction reached equilibrium,the by-product of glucose content is 9.1%,which reduced by 6 percentage points compared with the free enzyme and is the lowest byproduct content of the reported immobilization trehalose synthase. Under the optimum reaction conditions of p H 7.4,40 ℃,substrate concentration 200 g/L,the immobilized enzyme remains64.4% of the initial enzyme activity after repeating used for 72 h.
引文
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[10]WANG Jun. Studies on immobilization of trehalose synthesizing to chitosan as carrier[J]. Chemical Industry and Engineering Progress,2004,23(10):1117-1120.(in Chinese)
[11]PANEK A,PIETROW O,SYNOWIECKI J,et al. Immobilization on magnetic nanoparticles of the recombinant trehalose synthase from Deinococcus geothermalis[J]. Food and Bioproducts Processing,2013,91(4):632-637.
[12]CHO Y J,PARK O J,SHIN H J. Immobilization of thermostable trehalose synthase for the production of trehalose[J]. Enzyme and Microbial Technology,2006,39(1):108-113.
[13]WU T T,LIN S C,SHAW J F. Integrated process for the purification and immobilization of recombinant trehalose synthase for trehalose production[J]. Process Biochemistry,2011,46(7):1481-1485.
[14]李光.高分子材料加工工艺学[M].北京:中国纺织出版社,2010.
[15]WANG Chunmei,JIA Mengli,YU Jingyu. Dyeing of waste polyacrylonitrile-based carbon fiber precursor[J]. Dyeing and Finishing,2015(3):33-35.(in Chinese)
[16]WU Qingyun,WAN Lingshu,XU Zhikang. Enzyme immobilization on polyacrylonitrile-based membranes[J]. University Chemistry,2010,25(5):1-7.(in Chinese)
[17]宁正祥.食品成分分析手册[M].北京:中国轻工业出版社,1998:78.
[18]黄日波.海藻糖:21世纪的新型糖类[M].北京:化学工业出版社,2010:48.
[2]CHEN Pei,ZHAO Bing,PANG Yuchen,et al. Research on the effects of trehalose on cake quality[J]. Guangdong Agricultural Sciences,2015,42(3):94-98.(in Chinese)
[3]CHEN Yan,LU Zhigang,BAI Hai,et al. Effect of intra/extra-cellular trehalose on the preservation of red blood cells by lyophilization[J]. Chinese Journal of Blood Transfusion,2012,25(3):236-239.(in Chinese)
[4]MA Lukai,ZHANG Bing,WANG Xiaoling,et al. Comparison of the cryoprotective effects of trehalose,alginate and alginatederived oligosaccharides on cooked shrimp(Litopenaeus vannamei)[J]. Food Science,2015,36(16):266-271.(in Chinese)
[5]NIE Linhong,NING Zhengxiang. Trehalose in Biopreservation[J]. Chemisfry Flife,2001,21(3):206-209.(in Chinese)
[6]QIN Jiamin,LUO Shudong,HE Shaoyu,et al. Researching in characters and functions of trehalose and trehalase in insects[J].Natural Enemies of Insects,2015(1):163-169.(in Chinese)
[7]ZHANG Yuhua,LING Peixue,JI Baoping. Current status of research for trehalose and its prospective applications[J]. Food and Drug,2005,7(3):8-13.(in Chinese)
[8]SHANG Hongli,GU Ying. Research on immobilization of trehalose synthase on mesoporous molecular sieve[J]. Journal of Anhui Agricultural Sciences,2012,40(10):6173-6176.(in Chinese)
[9]DING Zhen,LIU Jianlong,WANG Ruiming. Immobilization of trehalose synthase on bacterial cellusose[J]. China Brewing,2006(9):19-23.(in Chinese)
[10]WANG Jun. Studies on immobilization of trehalose synthesizing to chitosan as carrier[J]. Chemical Industry and Engineering Progress,2004,23(10):1117-1120.(in Chinese)
[11]PANEK A,PIETROW O,SYNOWIECKI J,et al. Immobilization on magnetic nanoparticles of the recombinant trehalose synthase from Deinococcus geothermalis[J]. Food and Bioproducts Processing,2013,91(4):632-637.
[12]CHO Y J,PARK O J,SHIN H J. Immobilization of thermostable trehalose synthase for the production of trehalose[J]. Enzyme and Microbial Technology,2006,39(1):108-113.
[13]WU T T,LIN S C,SHAW J F. Integrated process for the purification and immobilization of recombinant trehalose synthase for trehalose production[J]. Process Biochemistry,2011,46(7):1481-1485.
[14]李光.高分子材料加工工艺学[M].北京:中国纺织出版社,2010.
[15]WANG Chunmei,JIA Mengli,YU Jingyu. Dyeing of waste polyacrylonitrile-based carbon fiber precursor[J]. Dyeing and Finishing,2015(3):33-35.(in Chinese)
[16]WU Qingyun,WAN Lingshu,XU Zhikang. Enzyme immobilization on polyacrylonitrile-based membranes[J]. University Chemistry,2010,25(5):1-7.(in Chinese)
[17]宁正祥.食品成分分析手册[M].北京:中国轻工业出版社,1998:78.
[18]黄日波.海藻糖:21世纪的新型糖类[M].北京:化学工业出版社,2010:48.
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