Zniszczoł Aurelia, Herman Artur P, Szymańska Katarzyna, Mrowiec-Białoń Julita, Walczak Krzysztof Z, Jarzębski Andrzej, Boncel Sławomir
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, Krzywoustego 4, Gliwice 44-100, Poland.
Department of Chemical and Process Engineering, Silesian University of Technology, Strzody 7, 44-100 Gliwice, Poland.
Enzyme Microb Technol. 2016 Jun;87-88:61-9. doi: 10.1016/j.enzmictec.2016.02.015. Epub 2016 Mar 2.
Aiming at the preparation of efficient, stable on storage and recyclable nanobiocatalysts for enantioselective transesterification, alkaline lipase from Pseudomonas fluorescens was covalently immobilized (up to 8.5wt.%) on functionalized multi-wall carbon nanotubes (f-MWCNTs). f-MWCNTs were synthesized via: (a) (2+1)-cycloaddition of a nitrene to the C-sp(2) nanotube walls (3.2mmolg(-1), a novel synthetic approach) and, (b) oxidative treatments, i.e. Fenton reagent (3.5mmolg(-1)) and nitrating mixture (2.5mmolg(-1)), yielding aminoalkyl-, hydroxyl- and carboxyl-MWCNTs, respectively. Amino- and epoxy- functionalized mesoporous silica (f-SBA-15) were used as the reference supports. Transesterification of vinyl n-butyrate by racemic Solketal with a chromatographically (GC) traced kinetics was selected as the model reaction. The studies revealed that different chemical functionalization of morphologically identical nanotube supports led to various enzyme loadings, catalytic activities and enantioselectivities. MWCNT-NH2-based nanobiocatalyst was found to be the most active composite among all of the tested systems (yield 20%, t=0.5h, 1321Ug(-1)), i.e. 12 times more active than the native enzyme. In turn, lipase immobilized on MWCNT-COOH emerged as the most enantioselective system (ex aequo with SBA-NH2) (eeR=74%, t=0.5h at yield of 3-5%). The activity of the MWCNT-NH2-based nanobiocatalyst after 8 cycles of transesterification dropped to 60% of its initial value, whereas for SBA-NH2-based composite remained unchanged. Importantly, stability on storage was fully maintained for all MWCNT-based nanobiocatalysts or even 'extra-enhanced' for MWCNT-OH.
为了制备用于对映选择性酯交换反应的高效、储存稳定且可循环使用的纳米生物催化剂，将荧光假单胞菌碱性脂肪酶共价固定（高达8.5wt.%）在功能化多壁碳纳米管（f-MWCNTs）上。f-MWCNTs通过以下方法合成：（a）氮烯与C-sp(2)纳米管壁的(2+1)-环加成反应（3.2mmol g-1，一种新型合成方法），以及（b）氧化处理，即芬顿试剂（3.5mmol g-1）和硝化混合物（2.5mmol g-1），分别得到氨基烷基、羟基和羧基功能化的MWCNTs。氨基和环氧功能化的介孔二氧化硅（f-SBA-15）用作参考载体。以外消旋索尔克他尔与丁酸乙烯酯的酯交换反应为模型反应，采用气相色谱（GC）追踪动力学。研究表明，形态相同的纳米管载体的不同化学功能化导致了不同的酶负载量、催化活性和对映选择性。在所有测试体系中，基于MWCNT-NH2的纳米生物催化剂是活性最高的复合物（产率20%，t = 0.5h，1321U g-1），即比天然酶活性高12倍。反过来，固定在MWCNT-COOH上的脂肪酶成为对映选择性最高的体系（与SBA-NH2相当）（eeR = 74%，产率为3 - 5%时t = 0.5h）。基于MWCNT-NH2的纳米生物催化剂在8次酯交换反应循环后的活性降至其初始值的60%，而基于SBA-NH2的复合物则保持不变。重要的是，所有基于MWCNT的纳米生物催化剂在储存时的稳定性都能完全保持，对于MWCNT-OH甚至“额外增强”。
