化学修饰固定化漆酶合成纳米花催化剂
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摘要
通过对漆酶进行丁二酸酐化学修饰,利用Cu~(2+)与修饰后的漆酶通过配位作用合成纳米花生物催化材料,并应用于催化白藜芦醇转化为二聚体。通过扫描电镜(SEM)研究了漆酶浓度、Cu~(2+)浓度以及反应时间对制备纳米花的影响;相对活性检测纳米花对温度及pH值的稳定性;利用质谱(MS)及氢核磁共振(~1HNMR)分析催化合成白藜芦醇二聚体的结构。结果表明,选用0.2mg/mL漆酶溶液、120mmol/L CuS O_4浓液、反应时间为24h作为较优制备纳米花的条件;所合成纳米花的稳定性相比于天然漆酶得到显著的提高;质谱及核磁结果表明,纳米花可催化白藜芦醇转化为二聚体,即纳米花催化剂可以应用于生物催化领域。
Laccase was chemically modified by succinic anhydride. Then, Cu~(2+) and modified laccase were used to synthesize biocatalytic materials of nanoflowers through coordination. Finally, nanoflowers were used to catalyze resveratrol to synthesize viniferin. The effects of laccase concentration, Cu~(2+) concentration and reaction time on the preparation of nanoflowers were studied by Scanning Electron Microscope(SEM). The relative activity was used to detect the stability of nanoflowers to temperature and pH. The structure of viniferin was analysed by Mass Spectrometry(MS) and Hydrogen Nuclear Magnetic Resonance(~1 H NMR) analysis. The results showed that the selection of 0.2 mg/mL laccase solution, 120 mmol/L CuSO4 concentration, and reaction time of 24 h were the better prepared conditions for nanoflowers. The stability of nanoflowers obtained was significantly improved compared to that of free laccase. MS and ~1 H NMR studies indicated that nanoflowers catalyzed resveratrol to convert to viniferin and nanoflower catalysts could be used in the field of biocatalysis.
Laccase was chemically modified by succinic anhydride. Then, Cu~(2+) and modified laccase were used to synthesize biocatalytic materials of nanoflowers through coordination. Finally, nanoflowers were used to catalyze resveratrol to synthesize viniferin. The effects of laccase concentration, Cu~(2+) concentration and reaction time on the preparation of nanoflowers were studied by Scanning Electron Microscope(SEM). The relative activity was used to detect the stability of nanoflowers to temperature and pH. The structure of viniferin was analysed by Mass Spectrometry(MS) and Hydrogen Nuclear Magnetic Resonance(~1 H NMR) analysis. The results showed that the selection of 0.2 mg/mL laccase solution, 120 mmol/L CuSO4 concentration, and reaction time of 24 h were the better prepared conditions for nanoflowers. The stability of nanoflowers obtained was significantly improved compared to that of free laccase. MS and ~1 H NMR studies indicated that nanoflowers catalyzed resveratrol to convert to viniferin and nanoflower catalysts could be used in the field of biocatalysis.
引文
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[2]Desantis G,Jones J B.Chemical modification of enzymes for enhances functionality.Current Opinion in Biotechnology[J].1999,10(4):3242-3301.
[3]胡平平,付时雨.漆酶催化活性中心结构与其特性研究进展.林产化学与工业[J].2001,21(3):69-75.
[4]冯晓静,谢益民,洪卫.漆酶的特性及其在制浆造纸工业中的应用.湖北造纸[J].2007,(4):29-32.
[5]Shin-Ya Y,Aye H N,Hong K J,et al.Efficacy of amphiphile-modified laccase in enzymatic oxidation and removal of phenolics in aqueous solution.Enzyme&Microbial Technology[J].2005,36(1):147-152.
[6]Tischer W,Wedekind F.Immobilized enzymes:Methods and applications[M].Berlin Heidelberg:Biocatalysis-From Discovery to Application,1999,p95-126.
[7]D'Souza S F.Immobilized Enzyme in Bioprocess.Current Science[J].1999,77(1):69-79.
[8]张永帅,李元召,孙俊良.酶修饰研究进展.食品工业科技[J].2014,35(2):350-353.
[9]柯彩霞,范艳利,苏枫,等.酶的固定化技术最新研究进展.生物工程学报[J].2018,34(2):188-203.
[10]Ge J,Lei J D,Zare D R N.Protein-inorganic hybrid nanoflowers.Nat Nanotechnol[J].2012,7(7):428-432.
[11]Zhang B,Li P,Zhang H,et al.Preparation of lipase/Zn3(PO4)2,hybrid nanoflower and its catalytic performance as an immobilized enzyme.Chemical Engineering Journal[J].2016,291:287-297.
[12]熊亚红,高敬忠,郑坚鹏.提高漆酶稳定性的化学修饰方法的研究.化学研究与应用[J].2011,23(8):985-990.
[13]李燕.白藜芦醇二聚体-cis-δ-viniferin的酶促合成及其功能研究[D].西安:西北大学,2009.
[14]Lee S W,Cheon S A,Kim M I,et al.Organic-inorganic hybrid nanoflowers:types,characteristics,and future prospects.Journal of Nanobiotechnology[J].2015,13(1):54.
[15]Lin Z,Xiao Y,Yin Y,et al.Facile synthesis of enzyme-inorganic hybrid nanoflowers and its application as a colorimetric platform for visual detection of hydrogen peroxide and phenol.Acs Appl Mater Interfaces[J].2014,6(13):10775-10782.
[16]Yu Y,Fei X,Tian J,et al.Self-assembled enzyme-inorganic hybrid nanoflowers and their application to enzyme purification.Colloids&Surfaces B Biointerfaces[J].2015,130:299-304.
[17]戈钧,卢滇楠,朱晶莹,等.纳米酶催化剂制备方法研究进展.化工学报[J].2014,65(7):2668-2675.
[18]Cui J,Zhao Y,Liu R,et al.Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance.Sci Rep[J].2016,6:27928.
[19]马丁,胡凯.白藜芦醇脱氢二聚体viniferin的研究进展.科技信息[J].2011,(29):55-56.
[20]周攀登.真菌漆酶在非水介质中的催化特性及其用于偶联聚合的研究[D].广州:华南理工大学,2014.
[21]Wu Z,Li H,Zhu X J,et al.Using laccases in the nanoflower to synthesize viniferin.Catalysts[J].2017,7(6):188.