Immobilized Porcine Pancreas Lipase Research Articles

Fabrication of an organic–inorganic nanocomposite carrier for enzyme immobilization based on metal–organic coordination

An organic–inorganic nanocomposite which combined mesoporous silica SBA-15 and chitosan using a carboxyl functionalized ionic liquid as the bridging agent (SBA@CS) was successfully fabricated, and was used to immobilize porcine pancreas lipase (PPL) by physical adsorption, cross-linking and metal–organic coordination, respectively. The as-prepared carriers were characterized by scanning electron microscopy, Fourier transform infrared and energy-dispersive X-ray spectroscopy. Compared with immobilization onto the pure mesoporous silicon material SBA-15, all the batches of PPL immobilized onto organic–inorganic nanocomposites showed higher activity, improved stability and reusability as well as better resistance to pH and temperature changes. Among the immobilized PPLs, immobilization based on Co2+ coordination (SBA@CS-Co-PPL) produced the best enzymatic properties. The maximum immobilization efficiency and specific activity of 79.6% and 1975.8 U g−1 were obtained with SBA@CS-Co, separately. More importantly, the activity of immobilized enzyme can still maintain 84.0% after 10 times of reuse. These results demonstrated that thus prepared organic–inorganic nanocomposite could be an ideal carrier for enzyme immobilization by metal–organic coordination.

Surfactant assisted production of ricinoleic acid using cross-linked and entrapped porcine pancreas lipase

This work studied hydrolysis of castor oil to ricinoleic acid, catalyzed by immobilized porcine pancreas lipase (PPL) and impact of surfactant on its augmentation. In immobilization of lipase through entrapment in calcium alginate beads, concentration of calcium chloride (CaCl2) was the most significant factor. Optimum conditions for immobilization were, 1.3 M CaCl2 and 12% (w/v) sodium alginate. On cross-linking with glutaraldehyde after entrapment in calcium alginate bead, PPL led to 2.7-fold higher hydrolysis compared to only entrapped PPL. Most suitable surfactant and speed of agitation were Span 80 and 300 rpm, respectively, in enhancing ricinoleic acid production involving cross-linked and entrapped PPL. Central composite design and subsequent optimization with response surface methodology (RSM) found optimum production (recovery) as 21.2% (in 3 h and 25 °C) under the optimum set of process conditions like 9.96 mg lipase/ml, pH of 8.8, 0.014 M Span 80, 2.98% (v/v) glutaraldehyde, 1.3 M CaCl2 and 12% (w/v) sodium alginate. Each of concentration of lipase, pH, surfactant (Span 80) concentration and glutaraldehyde concentration was found to be highly significant. Interactions between any two of these four variables remained insignificant. On varying temperature keeping other variables at their optima, maximum recovery for free lipase (31.29% in 3 h) was obtained at 35 °C; whereas, for immobilized lipase, recovery was maximum (26.27% in 3 h) at 45 °C. Immobilized lipase acted much better than free lipase at higher temperatures.

Biodiesel from soybean oil using Porcine pancreas lipase immobilized on a new support: p-nitrobenzyl cellulose xanthate

The objective of the present work was to obtain methyl biodiesel from soybean oil (BMS) using immobilized Porcine pancreas lipase (PPL) on the support of p-nitrobenzyl cellulose xanthate (NBXCel). The influence of the parameters immobilized enzyme quantity, reaction time and buffered reaction medium were studied. As the reaction time increased, the conversion rate increased because using 5.16% mPPL/mNBXCel with 24 h reaction, the conversion rate achieved was 50.35%, and after 48 h the conversion increased to 71.19% conversion using the same amount. The pH 7.0 buffered reaction medium also contributed to decrease the required amount of immobilized lipase to achieve satisfactory conversion, because in the experiment using 4.27% mPPL/mNBXCel over 36 h of reaction, there was an increase in the OSjC conversion rate. for BMS ranging from 50.54% in bufferless reaction medium to 89.94% in buffered reaction medium. After the 40 experiments performed, the conditions of six experiments allowed a conversion higher than 96.5%, as required by the regulators, and under these conditions the reuse of the support was performed by evaluating the conversion obtained in 10 reuse cycles.

A novel mesoporous nanocarrier: Integrating hollow magnetic fibrous silica with PAMAM into a single nanocomposite for enzyme immobilization

Abstract A well-designed mesoporous nanocarrier, integrating hollow magnetic fibrous silica with polyamidoamine (PAMAM) (H-MFS/PAMAM), was obtained for the first time to immobilize porcine pancreas lipase (PPL). The H-MFS, based on the light-weight and hollow Fe3O4 core and the fibrous silica shell, was first synthesized as a matrix. Following, PAMAM dendrimers were grafted onto H-MFS by using reiterative Michael addition and amidation reactions, and subsequently PPL was effectively immobilized. We discussed the effect of silica surface micro-morphologies and the length of flexible chains on the performance of immobilized lipase. As a result, the G-3 generation of PAMAM-grafted H-MFS still maintained intact fibrous morphology and proper BET surface area (89 m2/g), which exhibited the highest activity among the different generations (730 ± 2.57 U/g protein). Moreover, after recycling 8 times, the immobilized PPL still retained about 90% of its initial activity. Therefore, this fabrication strategy provided a useful reference platform to design magnetic nanobiocatalyst with high accessible surface, excellent catalytic ability and operational stability.

Stability of immobilized porcine pancreas lipase on mesoporous chitosan beads: A comparative study

Porcine pancreas lipase was immobilized on mesoporous chitosan beads. Glutaraldehyde as coupling agent was used through several immobilization techniques. With the aid of FESEM, BET and BJH analysis, the effect of glutaraldehyde on porosity of chitosan was evaluated. It was observed that the total surface area and pore volume of the carrier were significantly improved by addition of glutaraldehyde as cross-linking agent. The surface area exposure and pore volume were substantially increased (both by 4.4 folds). In addition, distribution of enzyme on the carrier was illustrated by fluorescence image. The characteristics of the immobilized lipases such as immobilization efficiency, enzyme activity, pH stability, thermal stability, reusability, storage stability and enzyme leakage were evaluated. In kinetic studies of enzyme, Michaelis–Menten kinetic coefficients of the hydrolytic activity for the immobilized lipase were defined using Lineweaver–Burk plot. The low value of ionization constant, Km (∼0.008mM) and high value of specific rate, Vmax(∼200μM/ml.min) indicate strong affinity and high activity of enzyme. The obtained results demonstrate that use of glutaraldehyde has an excellent impact on expansion of the porosity of chitosan and enzyme distribution. The immobilization efficiency increased by1.6 folds and enzyme leakage was minimized (17% reduced to zero); while, glutaraldehyde has improved pH stability (in acidic range), thermal stability, reusability and storage stability of immobilized lipase.

Enzymatic hydrolysis of coconut oil using free and immobilized porcine pancreas lipases

The aim of this study is to evaluate the effect of some factors on the hydrolysis of coconut oil (CO) in the present of two kind of enzymes, the free lipases and immobilized lipases porcine pancreas. The activities of these two lipases under the optimal hydrolysis conditions was determined. The effects of factors on hydrolysis degree of coconut oil was investigated: the ratio of enzyme to substrate, the pH condition, and the temperature. The best conditions for the high hydrolysis degree in case of using lipase from porcine pancreas ascatalyst included: the ratio of the enzyme to substrate of 90(U/mL), and the pH condition of 8.5 at the temperature of 40oC. The best reaction condition the case of using immobilized porcine pancreas lipase as the catalyst was determined, including: the ratio enzyme to substrate of 393U/g, the pH condition of 7.5 and the temperature of 35oC. The hydrolysis degree of CO by immobilized porcine pancreas lipase was increased slower than free lipase at the first time. The highest hydrolysis degree achieved with immobilized porcine pancreas and free porcine pancreas lipase was 72.26% and 68.61%, respectively.

Thymine-functionalized amphiphilic biodegradable copolymers for high-efficiency loading and controlled release of methotrexate

In this study, a novel thymine-functionalized six-membered cyclic carbonate monomer (TAC) was synthesized by the Michael-addition reaction between thymine and acryloyl carbonate (AC). The corresponding functional amphiphilic block copolymer mPEG-b-PTAC was further successfully synthesized by ring-opening polymerization using immobilized porcine pancreas lipase (IPPL) as the catalyst and mPEG as the macroinitiator. Meanwhile, mPEG-b-P(TAC-co-DTC) and mPEG-b-PDTC were also synthesized by the same enzymatic methods for comparison on different TAC contents. The structures of monomer and copolymers were characterized by 1H-NMR, 13C-NMR and FTIR. All the amphiphilic block copolymers could self-assemble to form nano-sized micelles in aqueous solution. Transmission electron microscopy (TEM) observation showed that the micelles dispersed in spherical shape with nano-size before and after MTX loading. 1H-NMR and FTIR results confirmed the successful formation of multiple hydrogen-bonding interactions between exposed thymine groups of hydrophobic PTAC segments and 2,6-diaminopyridine (DAP) groups of MTX molecules, which resulting in the higher drug loading capacity and the pH-sensitive drug release behavior. MTT assays also indicated lower toxicity of copolymer but higher potent cytotoxic activity of MTX-loaded copolymer against HeLa cells.

Amphiphilic copolymers with pendent carboxyl groups for high-efficiency loading and controlled release of doxorubicin

In this paper, biodegradable amphiphilic block copolymer based on methoxy poly(ethylene glycol)-b-poly(5-allyloxy-1,3-dioxan-2-one) (mPEG-b-PATMC) was successfully synthesized in bulk using immobilized porcine pancreas lipase (IPPL) as the catalyst. After thiol-ene “click” reactions occur between thiol group of thioglycolic acid and carbon–carbon double bonds of PATMC segments, the pendent carboxyl-modified copolymer mPEG-b-PATMC-g-SCH2COOH was obtained for high-efficiency loading and controlled release of doxorubicin (DOX) to cancer cells. Both the carboxyl-modified and unmodified copolymers could self-assemble to form nano-sized micelles in aqueous solution, while transmission electron microscopy (TEM) observation showed that the micelles dispersed in spherical shape with nano-size before and after DOX loading. Compared with the unmodified copolymer, the pendent carboxyl-modified structure in mPEG-b-PATMC-g-SCH2COOH could markedly enhance the drug-loading capacity and entrapment efficiency via the electrostatic interaction. The in vitro release studies showed more sustained drug release behavior of mPEG-b-PATMC-g-SCH2COOH without an initial burst, which could be further adjusted by the conditions of ionic strength and pH. Confocal laser scanning microscopy (CLSM) indicated efficient cellular uptake of DOX delivered by mPEG-b-PATMC-g-SCH2COOH, while MTT assays also demonstrated potent cytotoxic activity against HeLa cells.

Folate-conjugated amphiphilic block copolymers for targeted and efficient delivery of doxorubicin

In this paper, novel biodegradable amphiphilic block copolymers based on folate-conjugated poly(ethylene glycol)-b-copolycarbonates (FA-PEG-b-P(MAC-co-DTC)) and methoxy poly(ethylene glycol)-b-copolycarbonates (mPEG-b-P(MAC-co-DTC)) were successfully synthesized for targeted and efficient delivery of doxorubicin (DOX) to cancer cells. Immobilized porcine pancreas lipase (IPPL) was employed as the catalyst to perform the ring-opening copolymerization in bulk, while the folate-conjugated poly(ethylene glycol) (FA-PEG) or methoxy poly(ethylene glycol) (mPEG) was used as the initiator. The resulting copolymers, characterized by 1H NMR and GPC, could self-assemble to form nano-sized micelles in aqueous solution by dialysis method. P(MAC-co-DTC) acted as the hydrophobic core, thereby aggregating hydrophilic PEG chains as the outer shell with FA as targeting ligand located at the surface of the polymeric micelles. Transmission electron microscopy (TEM) observation showed that the micelles dispersed in spherical shape with nano-size before and after DOX loading. Both the FA-conjugated and non-conjugated block copolymers showed low cellular cytotoxicity. Furthermore, as compared to the non-conjugated copolymers, much more efficient cellular uptake of the FA-conjugated copolymers via FA-receptor-mediated endocytosis could be observed by confocal laser scanning microscopy (CLSM), while MTT assays also demonstrated highly potent cytotoxic activity against HeLa cells.

Biodegradable amphiphilic block-graft copolymers based on methoxy poly(ethylene glycol)-b-(polycarbonates-g-polycarbonates) for controlled release of doxorubicin

In this paper, novel biodegradable amphiphilic block-graft copolymers based on methoxy poly(ethylene glycol)-b-(polycarbonates-g-polycarbonates) (mPEG-b-(PATMC-g-PATMC)) were synthesized successfully for controlled release of doxorubicin (DOX). Backbone block copolymer, methoxy poly(ethylene glycol)-b-poly(5-allyloxy-1,3-dioxan-2-one) (mPEG-b-PATMC) was synthesized in bulk catalyzed by immobilized porcine pancreas lipase (IPPL). Then, mPEG-b-PATMC-O, the allyl epoxidation product of mPEG-b-PATMC, was further grafted by PATMC itself also using IPPL as the catalyst. The copolymers were characterized by (1)N HMR and gel permeation chromatography results showed narrow molecular weight distributions. Stable micelle solutions could be prepared by dialysis method, while a monomodal and narrow size distribution could be obtained. Transmission electron microscopy (TEM) observation showed the micelles dispersed in spherical shape with nano-size before and after DOX loading. Compared with the block copolymers, the grafted structure could enhance the interaction of polymer chains with drug molecules and improve the drug-loading capacity and entrapment efficiency. Furthermore, the amphiphilic block-graft copolymers mPEG-b-(PATMC-g-PATMC) had low cytotoxicity and more sustained drug release behavior.

Amphiphilic polycarbonate conjugates of doxorubicin with pH-sensitive hydrazone linker for controlled release

Novel amphiphilic polycarbonates-graft-doxorubicin (mPEG-b-P(ATMC-co-DTC)-g-DOX) were successfully designed and synthesized for pH-triggered intercellular drug release in cancer cells. The amphiphilic block copolymer, mPEG-b-P(ATMC-co-DTC), was synthesized in bulk using immobilized porcine pancreas lipase (IPPL) as the catalyst. After allyl epoxidation of ATMC units, DOX was covalently conjugated to the hydrophobic polycarbonates block through a hydrazone linkage. The resulting mPEG-b-P(ATMC-co-DTC)-g-DOX prodrugs could self-assemble to form nano-sized micelles in aqueous solution, while DOX contents in the hydrophobic core were 9.9 and 12.5wt.%. DOX release rate from the prodrug micelles increased in acidic medium due to the acid-cleavable hydrazone linkage between the DOX and polycarbonates. MTT assays demonstrated that DOX prodrug micelles in this study showed effective cytotoxic effects to HeLa cells. Furthermore, confocal laser scanning microscopy (CLSM) observations also revealed that mPEG-b-P(ATMC-co-DTC)-g-DOX prodrugs could efficiently deliver and release DOX into the nuclei of HeLa cells.

Poly[(5-methyl-5-allyloxycarbonyl-trimethylene carbonate)-co-(5,5-dimethyl-trimethylene carbonate)] with Grafted Polyethylenimine as Biodegradable Polycations for Efficient Gene Delivery

In this paper, biodegradable polycations based on polycarbonates with grafted polyethylenimine (PEI) were synthesized as a nonviral vector for gene delivery. Immobilized porcine pancreas lipase (IPPL) was employed to perform the copolymerization of 5-methyl-5-allyloxy carbonyl-trimethylenecarbonate (MAC) with 5,5-dimethyl-trimethylene carbonate (DTC). The DTC molar percent X was equal to 6.7, 12.5, and 45.4, respectively. The resulting copolymers with different compositions (P(MAC-co-DTCx) underwent additional allyl epoxidation and thereby grafted by low molecular weight PEI1800. The MWs of P(MAC-co-DTCx)-g-PEI, measured by GPC-MALLS, were 219800, 179100, and 51700 g/mol with polydispersities of 1.5, 1.4, and 1.2, respectively. Physicochemical properties of these vectors were characterized and the DNA loading was evaluated. P(MAC-co-DTCx)-g-PEI could form nanosized particles (less than 100 nm) with pDNA. The three P(MAC-co-DTCx)-g-PEI/DNA polyplexes had similar buffer capabilities that were better than that of PEI25K and PMAC-g-PEI. Despite a slightly lower DNA binding ability, the PEI-grafted polycarbonates, especially P(MAC-co-DTC45.4)-g-PEI, presented apparently low cytotoxicity and much higher gene transfection efficiency in comparison with PEI25K in 293T cells. Moreover, preincubation of P(MAC-co-DTC6.7)-g-PEI showed a quickly weakening DNA binding capacity, while a suitable degradation rate of vectors would facilitate the efficient release of pDNA from polyplexes after cellular uptake and also reduce cell cytotoxicity. The results of this study demonstrated the promise of P(MAC-co-DTCx)-g-PEI copolymers for efficient gene delivery.

Polyethylenimine-grafted polycarbonates as biodegradable polycations for gene delivery

Polycations as one of non-viral vectors have gained increasing attentions. In this paper, polyethylenimine(PEI)-grafted polycarbonates (PMAC-g-PEIx) were synthesized as a kind of biodegradable polycations for gene delivery. Backbone polymer, poly(5-methyl-5-allyloxycarbonyl-trimethylene carbonate) (PMAC), was synthesized in bulk catalyzed by immobilized porcine pancreas lipase (IPPL). Then, PMAC–O, the allyl epoxidation product of PMAC, was further modified by PEIx with low molecular weight (x = 423, 800 and 1800). The MWs of PMAC-g-PEIx, measured by GPC–MALLS, were 81,900, 17,9900 and 200,600 g/mol with polydispersities of 1.2, 1.4 and 1.7, respectively. PMAC-g-PEIx could form positively charged nano-sized particles (30–90 nm) with pDNA, and all the three PAMC-g-PEIx/DNA polyplexes had similar buffer capabilities. In vitro experiments demonstrated that the PAMC-g-PEIx showed much low cytotoxicity and enhanced transfection efficiency could be found in comparison with PEI25K in 293T cells. Furthermore, pre-incubation of PMAC-g-PEI1800 showed a weakening binding capacity with DNA. The biodegradability of PMAC-g-PEIx can facilitate the efficient release of pDNA from polyplexes and reduce cell cytotoxicity. These results suggested that PMAC-g-PEIx would be a promising non-viral biodegradable vector for gene delivery system.

Synthesis, characterization and ring-opening polymerization of a novel six-membered cyclic carbonate bearing pendent allyl ether group

A novel six-membered cyclic carbonate with pendent allyl ether group, 5-allyloxy-1,3-dioxan-2-one (ATMC), was synthesized from glycerol, and the corresponding polycarbonate, poly(5-allyloxy-1,3-dioxan-2-one) (PATMC) was further synthesized by ring-opening polymerization in bulk at 120°C. Two kinds of catalyst, tin(II) 2-ethylhexanoate (Sn(Oct)2) and immobilized porcine pancreas lipase on silica particles (IPPL), were employed to perform the polymerization. The structures of the novel monomer and the resulting functional polymers were confirmed by FTIR, 1H NMR, 13C NMR, GPC and DSC. The molecular weight (Mn) of PATMC decreased rapidly with the increase of IPPL or Sn(Oct)2 concentration. The highest molecular weight (Mn=48,700g/mol) of PATMC with the polydispersity of 1.31 was obtained at 0.1wt% concentration of IPPL for 48h. Postpolymerization oxidation reactions to epoxidize the unsaturated bonds of the PATMC were also achieved. The epoxide-containing polymers could afford facilities for further modification.

Metal-free synthesis of amphiphilic functional polycarbonates

Amphiphilic block copolymers of poly(5-benzyloxy trimethylene carbonate) (PBTMC) and poly(ethylene glycol) (PEG) were synthesized through enzymatic polymerization using immobilized porcine pancreas lipase (IPPL). The obtained copolymers with different compositions were characterized by GPC and 1H NMR. The copolymer composition was in agreement with the feed ratio. The molecular weight of the copolymers showed an increasing trend with the decrease of PEG contents. Micelles of the copolymers were formed by dialysis procedure, and characterized by transmission electron microscopy (TEM).

Enzymatic Synthesis and Characterization of Novel Biodegradable Copolymers of 5-Benzyloxy-trimethylene Carbonate with 1,4-Dioxan-2-one

Enzymatic ring-opening copolymerization of 5-benzyloxy-trimethylene carbonate (BTMC) and 1,4-dioxan-2-one (DON) was investigated for the first time. Immobilized porcine pancreas lipase (IPPL) on silica particles was selected to perform the copolymerization. A series of novel biodegradable copolymers with different compositions were characterized by (1)H NMR, (13)C NMR, and GPC. The influences of reaction conditions such as polymerization time and catalyst concentration on the yield and molecular weight of the copolymers were also studied. The copolymerizations of different monomer feed ratios were carried out in bulk at 150 degrees C with 4.5 wt per thousand IPPL as a catalyst for 24 h. With the increase of the BTMC molar feed ratio from 20% to 79%, the M(n) of the resulting copolymers increased from 5600 to 63400. Water uptake and static contact angle experiments showed that the hydrophilicity of copolymers could be improved with increasing DON content in the copolymers. Moreover, the in vitro drug release rate (ibuprofen as the model drug) of the resulting copolymers also increased along with the DON content in the copolymers.

Enantioselective enzymatic hydrolysis of racemic glycidyl esters by using immobilized porcine pancreas lipase with improved catalytic properties

The crude porcine pancreas lipase (PPL) extract is a mixture of several proteins (mainly lipases and esterases). In order to develop enzymatic catalysts with good catalytic properties for hydrolytic enantioselective reactions in aqueous homogeneous medium, we studied the immobilization of the different enzymes contained in the crude PPL extracts by selective sequential adsorption on hydrophobic supports bearing octyl, octadecyl and phenyl groups. Some minor proteins were selectively adsorbed on octyl and octadecyl supports while the most abundant lipase was adsorbed on the support bearing phenyl groups. The enantioselectivity of the different lipase derivatives were tested considering the hydrolysis of esters of 1,2-epoxi-1-propanol (glycidol). The most abundant lipase contained in the commercial crude PPL extract resulted almost inactive while some lipases contained in low concentrations displayed high activities and enantioselectivities. The most interesting results were obtained with a 28-kDa protein selectively adsorbed on octyl-agarose. With this enzyme derivative, the residual butyric ester of glycidol was recovered with 96% enantiomeric excess at 55% conversion.