Microbial Lipases Immobilized On High-Loaded Magnetic Carbon Nanotubes Composites And Its Application In Biodiesel

Recently,magnetic carbon nanotubes have been extensively studied for the immobilization of enzymes due to the advantages like nanometer-grade size,high specific surface area,abundant polar functional groups,rich pore structure,effective thermal conductivity,biocompatibility,lower mass transfer resistance and easy recovery of the supported-enzyme conjugates via a simple magnet,etc.Nevertheless,magnetic carbon nanotubes do not provide enough active sites to immobilize enzymes.It has been demonstrated,however,the magnetic carbon nanotubes functionalized with dendrimer molecules PAMAM can provide massive active sites to increase the loading of enzymes.Because PAMAM have the characteristics of controllable molecular weight,cavity structure,nano size and rich in functional groups at molecular end.So as to select the suitable amino acid residues on enzyme as covalent binding sites,the bioinformatics was used to analyze the structure of enzymes.The enzymes were oriented immobilized on PAMAM funcationalized magnetic carbon nanotube composite material,which can prevent the channel that substrate access to the enzyme active center from blocking.In this article,we proposed to obtain immobilized enzyme with excellent catalytic performance by oriented-immobilization based on the structural information of enzyme molecules.Typical representative lipases of the three sub-group with different substrates specificity,Burkholderia cepacia(BCL),Rhizomucor miehei lipase(RML)and Candida rugose lipase(CRL),were oriented immobilized on two kinds of PAMAM funcationalized magnetic carbon nanotube composites(surface coated and tube filling)by various strategies.The derivatives were used to prepare biodiesel.The main work and innovations were summarized bellow:1.Fe3O4 coated carbon nanotubes magnetic nanocomposites(m-MWCNTs)were synthesized by coprecipitation,following modified with(3-aminopropl)triethoxysilane(APTES),named as amino functionalized m-MWCNTs(m-MWCNTs-NH2),then dendritic PAMAM was grafted onto the surface of m-MWCNTs by diffusion method(m-MWCNTs-G3).The nanocomposites were characterized by SEM,TEM,XRD,SQUID,FT-IR and elemental analyzer.The TEM shows that the surface of MWCNTs is coated with a lot of nanoparticles.The XRD spectra confirmes that the magnetic composite is Fe3O4/MWCNTs and the Fe3O4 phase shows inverse spinel;SQUID analysis proves that the magnetic composite has good magnetic response;FT-IR spectrum suggests that PAMAM dendrimer has been successfully grafted onto the surface of m-MWCNTs.2.The distribution of amino acid residues on the surface of three kinds of lipase molecules(BCL,RML and CRL)were analyzed.The results showed that the distribution of ?-NH2 residues on BCL and RML was regional,far away from the active center;For CRL,most of ?-NH2 away from the catalytic sites except for a very few residues.Based on the result and carrier properties,the-COOH(as control)and ?-NH2 on the enzyme molecules were selected as covalent binding sites,respectively.The results showed that the activity recovery of immobilized BCL,RML and CRL covalently bound via carboxyl and amino groups were 120.4%,1,607%,38.4% and 192.3%,2,780%,49.3%,respectively,the latter were 1.6,1.73,and 1.29 times more than the former.So BCL,RML and CRL were covalently orientedly immobilized on m-MWCNTs-G3 in aqueous phase via amino groups,and the immobilization conditions were optimized through single factorial experiment and response surface methodology.The results indicated that the immobilization efficiencies of the three lipases were all higher than 85%,and the activity recoveries were 194.1%(BCL),2,808%(RML)and 50.4%(CRL)under the optimal conditions.After being induced by imprinted molecules,the activity recoveries were up to 1,244%(BCL),3,437%(RML)and 64.8%(CRL),respectively.The results of enzymatic properties detection displayed that the pH and temperature stability of the immobilized enzyme were markedly improved compared with the free enzyme.3.Fe3O4 filling carbon nanotubes magnetic nanocomposites(mMWCNTs)were synthesized by siphonage,then dendritic PAMAM was grafted onto the surface of mMWCNTs by diffusion method(mMWCNTs-G3).A series of instruments were used to characterize the nanocomposites.The XRD and XPS patterns show that the MWCNTs/Fe3O4 nanocomposites were successfully prepared.The TEM analysis suggests that Fe3O4 nanoparticles were filled into MWCNTs;FT-IR spectrum and elemental analysis manifest that the PAMAM functionalized mMWCNTs composites with a large number of active groups were successfully prepared.4.BCL were covalently orientedly immobilized on mMWCNTs-G3 in aqueous phase via amino groups(BCL-mMWCNTs-G3),and the immobilization conditions were optimized through single factorial experiment after being induced by imprinted molecules.The optimal immobilization parameters for BCL were glutaraldehyde addition amount 8.5 wt%,pH value 8,enzyme dosage 300 mg/0.1 g,cross-linking time 2.5 h and immobilization temperature 30°C,the activity recovery was up to 1,716%.Compared with the free enzyme,the pH and temperature stability of the immobilized BCL were significantly improved.5.The immobilized lipases were then employed as catalysts for biodiesel production from soybean oil.The achieved optimum conditions were: for BCL&m-MWCNTs-G3,water content 5 wt%,reaction temperature 35°C,and with tert-butanol as reaction medium,methanol:oil molar ratio 4:1,its highest biodiesel yield attained 96.4% at 24 h;for RML&m-MWCNTs-G3,water content 10 wt%,reaction temperature 50°C,n-octane as the reaction medium,methanol:oil molar ratio 5:1,the utmost biodiesel conversion rate was up to 96.2% at 36 h;for CRL&m-MWCNTs-G3,water content 7.5 wt%,reaction temperature 40°C,isooctane as the reaction medium,methanol:oil molar ratio 4:1,the best yield reached 85.1% at 40 h.After 10 cycles of repeated use,the yields catalyzed by three immobilized lipases were 89.3%(BCL),81.3%(RML),and 58.4%(CRL),respectively.Moreover,RML&m-MWCNTs-G3 exhibited the best catalytic activity on the transesterification of waste vegetable oil in its corresponding solvent systems(92.1%).6.BCL-mMWCNTs-G3 as catalysts and soybean oil as substrate,the maximum yield of biodiesel was 93.1% reacting 16 h at the optimum conditions(water content 4 wt%,methanol:oil molar ratio 5:1,reaction temperature 35°C and enzyme amount 3.5 wt%).After 20 cycles of repeated use,it still retained ca.90% of its original activity,almost no loss of enzyme activity.