添加纳米粒子对脂肪酶交联酶聚集体活性及结构的影响
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摘要
将纳米TiO2、纳米MgO和纳米Cu分别引入假丝酵母脂肪酶(Candida sp.1619)的交联酶聚集体(CLEAs)制备过程,获得相应的纳米粒子-CLEAs,采用傅里叶变换红外光谱仪(FTIR)、扫描电子显微镜(SEM)、激光粒度分析仪(SLP)等分析了纳米粒子对CLEAs活性和结构的影响。结果表明与未加纳米粒子的CLEAs相比,适量纳米TiO2的加入可提高CLEAs的酶活,最大增加15.2%;而不管添加浓度大小,纳米MgO、纳米Cu对CLEAs酶活均有抑制作用,酶活下降63.7%~97.9%。SEM、SLP分析结果表明,与未添加纳米粒子时相比,加入纳米TiO2的CLEAs粒径变小,粒度较均匀,孔道增加;而纳米MgO-CLEAs和纳米Cu-CLEAs则出现粒度不均匀性增加、粒径范围扩大、孔道减少的现象。FTIR分析结果表明,加入3种纳米粒子后CLEAs二级结构中有序结构(α-螺旋、β-折叠)/无序结构(β-转角、无规则卷曲)值显著提高,顺序为纳米TiO2-CLEAs(0.55)>纳米MgO-CLEAs(0.43)>纳米CuCLEAs(0.35)>CLEAs(0.28),这与纳米粒子-CLEAs酶活顺序不一致,表明纳米粒子可能还存在其他影响CLEAs酶活的途径。
Nano-TiO2,nano-Mg O and nano-Cu were added during the preparation of lipase(Candida sp.1619)CLEAs.The activity and structure of the CLEAs prepared in the presence of different nanoparticles were determined by Fourier transform infrared(FTIR) spectroscopy,scanning electron microscopy(SEM) and laser scattering particle(LSP) analysis.The results showed that relative to CLEAs without any added nanoparticles,the activity of CLEAs with nano-TiO2 particle increased by 15.2%,while the activity of CLEAs with nano-Mg O and nano-Cu particles decreased by 63.7% and 97.9%,independent of the concentration added.SEM and LSP showed that relative to CLEAs without added nanoparticles,CLEAs with nano-TiO2 particles had smaller particle size,more uniform particle size and higher porosity,while CLEAs withnano-Mg O and nano-Cu particles had more non-uniform particle size and lower porosity.FTIR showed that in the secondary structures of nanoparticle-CLEAs,the ratio of(α-helix + β-sheet) /(β-turn + random coil) increased in the CLEAs with nanoparticles,and followed the order NTiO2-CLEAs(0.55) > NMg O-CLEAs(0.43) > NCu-CLEAs(0.35) > CLEAs(0.28).However,the variation in the activity of the CLEAs was not consistent with the changesin structure,indicating that nanoparticles may have other effects on the activity of CLEAs.
Nano-TiO2,nano-Mg O and nano-Cu were added during the preparation of lipase(Candida sp.1619)CLEAs.The activity and structure of the CLEAs prepared in the presence of different nanoparticles were determined by Fourier transform infrared(FTIR) spectroscopy,scanning electron microscopy(SEM) and laser scattering particle(LSP) analysis.The results showed that relative to CLEAs without any added nanoparticles,the activity of CLEAs with nano-TiO2 particle increased by 15.2%,while the activity of CLEAs with nano-Mg O and nano-Cu particles decreased by 63.7% and 97.9%,independent of the concentration added.SEM and LSP showed that relative to CLEAs without added nanoparticles,CLEAs with nano-TiO2 particles had smaller particle size,more uniform particle size and higher porosity,while CLEAs withnano-Mg O and nano-Cu particles had more non-uniform particle size and lower porosity.FTIR showed that in the secondary structures of nanoparticle-CLEAs,the ratio of(α-helix + β-sheet) /(β-turn + random coil) increased in the CLEAs with nanoparticles,and followed the order NTiO2-CLEAs(0.55) > NMg O-CLEAs(0.43) > NCu-CLEAs(0.35) > CLEAs(0.28).However,the variation in the activity of the CLEAs was not consistent with the changesin structure,indicating that nanoparticles may have other effects on the activity of CLEAs.
引文
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[3]Cui J D,Jia S R.Optimization protocols and improved strategies of cross-linked enzyme aggregates technology:current development and future challenges[J].Critical Reviews in Biotechnology,2015,35(1):15-28.
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[5]Shah S,Sharma A,Gupta M N.Preparation of crosslinked enzyme aggregates by using bovine serum albumin as a proteic feeder[J].Analytical Biochemistry,2006,351:207-213.
[6]Bhattacharya A,Pletschke B I.Magnetic cross-linked enzyme aggregates(CLEAs):a novel concept towards carrier free immobilization of lignocellulolytic enzymes[J].Enzyme and Microbial Technology,2014,61/62:17-27.
[7]Xun D Y,Yang Y,Yang Z.Activity and stability of cross-linked tyrosinase aggregates in aqueous and nonaqueous media[J].Journal of Biotechnology,2011,152(1/2):30-36.
[8]Ansaria S A,Husain Q.Potential applications of enzymes immobilized on/in nano materials:a review[J].Biotechnology Advances,2012,30(3):512-523.
[9]Park J M,Kim M,Park J Y,et al.Immobilization of the cross-linked para-nitrobenzyl esterase of Bacillus subtilis aggregates onto magnetic beads[J].Process Biochemistry,2010,45(2):259-263.
[10]司士辉,李赛,杨政鹏,等.纳米二氧化钛对尿素酶活性的增强效应[J].中南大学学报,2007,8(4):692-695.Si S H,Li S,Yang Z P,et al.Enhancement of urease activity by Ti O2nanoparticles[J].Journal of Center South University,2007,8(4):692-695.(in Chinese)
[11]任湘菱,唐芳琼.纳米铜颗粒-酶-复合功能敏感膜生物传感器[J].催化学报,2000,21(5):455-458.Ren X L,Tang F Q.Biosensor of Cu nanoparticles-enzyme-composite sensitive film[J].Chinese Journal of Catalysis,2000,21(5):455-458.(in Chinese)
[12]江慧芳,王雅琴,刘春国.三种脂肪酶活力测定方法的比较及改进[J].化学与生物工程,2007,24(8):72-75.Jiang H F,Wang Y Q,Liu C G.Comparison and improvement of three determination methods for lipase activity[J].Chemistry and Bioengineering,2007,24(8):72-75.(in Chinese)
[13]Cui J D,Cui L L,Zhang S P,et al.Hybrid magnetic cross-linked enzymes aggregates of phenylalanine ammonia lyase from Rhodotorula glutinis[J].Plos One,2014,9(5):e97221.
[14]章玲英.纳米氢氧化铝粉体的制备及工艺优化[D].杭州:浙江大学,2006.Zhang L Y.The preparation of the aluminium hydroxide nanoparticles and itsprocess optimization[D].Hangzhou:Zhejiang University,2006.(in Chinese)
[15]Natalello A,Ami D,Brocca S,et al.Secondary structure,conformational stability and glycosylation of a recombinant Candida rugosa lipase studied by Fouriertransform infrared spectroscopy[J].Biochemical Journal,2005,385:511-517.
[16]黄金凤,沈文洁,刘能盛,等.FT-IR光谱法测定蔗糖酶的二级结构[J].广东化工,2010,7(37):254-256.Huang J F,Shen W J,Liu N S,et al.The determination of the secondary structures of invertase by FT-IR spectra[J].Guangdong Chemistry,2010,7(37):254-256.(in Chinese)