Immobilization Of Lipase Research Articles

Improved lipase performance by covalent immobilization of Candida antarctica lipase B on amino acid modified microcrystalline cellulose as green renewable support

Development of immobilized lipase with excellent catalytic performance and low cost is the major challenge for large-scale industrial applications. In this study, green renewable microcrystalline cellulose (MCC) that was hydrophobically modified with D-alanine (Ala) or L-lysine (Lys) was used for immobilizing Candida antarctica lipase B (CALB). The improved catalytic properties were investigated by experimental and computational methods. CALB immobilized on MCC-Ala with higher hydrophobicity showed better catalytic activity than CALB@MCC-Lys because the increased flexibility of the lid region of CALB@MCC-Ala favored the formation of open conformation. Additionally, the low root mean square deviation and the high β-sheet and α-helix contents of CALB@MCC-Ala indicated that the structure became more stable, leading to a significantly enhanced stability (54.80% and 90.90% relative activity at 70 °C and pH 9.0, respectively) and good reusability (48.92% activity after 5 cycles). This study provides a promising avenue to develop immobilized lipase with high catalytic properties for industry applications.

Preparation of Ordered Macroporous ZIF-8-Derived Magnetic Carbon Materials and Its Application for Lipase Immobilization

Metal–organic framework materials (MOFs) and their derivatives are considered ideal immobilization carrier materials because of their large specific surface area, high porosity and excellent structural designability. Among them, ZIF-8 has great potential for immobilization of enzymes due to mild synthesis conditions, and good biocompatibility. However, conventional ZIF-8 crystals have poor separation and recovery efficiency due to their small pore size and poor acid stability, greatly limiting their application in enzyme immobilization and further application. Although the carbonization of ZIF-8 by pyrolysis has been shown to be one of the approaches that can enhance its chemical stability, this still does not effectively solve the problem of the difficulty of recycling. Herein, we developed a strategy of pre-carbonization immersion (immersion in aqueous FeSO4 solution before carbonization) to synthesize ordered macroporous ZIF-8-derived carbon materials with stable ferromagnetism (denoted as CZ-x-M-y, where x denotes the carbonization temperature and y denotes the concentration of the impregnated FeSO4 solution) and used them to immobilize lipases for biodiesel production. XRD analysis showed that the magnetic properties in the materials came from Fe3C species. We found that the magnetic carbon materials obtained by carbonization at 600 °C showed the best immobilization effect, where CZ-600-M-0.3 (using 0.3 mol·L−1 FeSO4 aqueous solution to soak ZIF-8 and carbonized at 600 °C) had the highest enzyme loading of 183.04 mg·g−1, which was 49.7% higher than that of the non-magnetic CZ-600. In addition, CZ-600-M-0.5 maintained the highest enzyme activity, which was 81.9% of the initial activity, after five batches of reuse. The stable magnetic support materials reported in this study have promising potential for the industrial application of immobilized lipase.

Activated carbon from sugarcane bagasse as support for lipase immobilization by physical adsorption technique

Lipases are recognized as the most important group of catalysts in biotechnology. However, utilization of free enzymes is often hampered by the need for more operational stability, high cost, and non-reusability. Most of these obstacles can be solved by lipase immobilization. This work's objective was to evaluate the performance of the activated carbon obtained from sugarcane straw (SAC) as a support for lipase immobilization. Two lipases were immobilized by physical adsorption on SAC: Aspergillus niger 11T53A14 lipase and CalB (lipase B from Candida antarctica, Novozymes). Results revealed that the lipase had been anchored on the activated carbon with the lipase binding efficiency of 89 % (A. niger lipase) and 100 % (CalB) at the optimum experimental conditions (initial protein concentration 0.1 mg mL-1, 0.15 g of SAC, 25 °C, and 120 min). Langmuir isotherm fitted the adsorption equilibrium data of the lipases on SAC. SAC presents a high surface area and protein adsorption capacity. These results show that activated carbon synthesized from the sugarcane straw is a promising support for enzyme immobilization.

Supramolecular immobilization of lipase inspired by octopus suckers for biodiesel production

It is still a challenge to design a supramolecular interaction system for immobilizing enzymes efficiently and stably. In this work, inspired by octopus suckers, we used cyclodextrin with a similar structure to the octopus sucker cavity as supramolecular interaction functional units of soft tentacles to immobilize lipase through physical adsorption. By modifying the surface of cyclodextrin with polar hydrophilic groups, the difference in hydrophilic and hydrophobic properties and stability within the molecule were effectively improved, thereby enhancing the cavity suction of cyclodextrin. The enzyme load of the soft tentacles reached 10 mg/g, and the lipase was adsorbed firmly under high strength mechanical action (the retention rate is 66.4%, other physical adsorption under the same conditions is 51.6%). The soft tentacle immobilized lipase showed good dispersibility in the organic phase and its water absorption characteristics could provide oil-water interface for lipase in organic phase reaction, promoting the forward transesterification reaction. Compared with free lipase, the soft tentacle immobilized lipase (L-CDST-Ms) showed the improved activity, which could reach 180% of that of free lipase. The L-CDST-Ms was applied to biodiesel production, and the conversion rate could reach 94%. The results showed that the immobilization method opened up a new way for the application of lipase in transesterification.

Different Substrate Selectivity and Product Patterns of Immobilized Thermophilic Lipases from Geobacillus stearothermophilus, Anoxybacillus flavithermus, and Thermomyces lanuginosus for Glyceryl Decanoate Synthesis

Lipases can catalyze synthesis reactions in a micro aqueous system, producing useful partial glycerides (mono- and diglycerides), and these compounds are commonly utilized in different products as surfactants. Depending on the microbial sources for lipases, immobilization conditions, and starting substrates for synthesis reaction, the composition and yields of the resulting partial glycerides could be variable. These differences could lead to the final efficacy of partial glycerides as surfactants in targeted products. Therefore, it is necessary to establish a group of immobilized lipases from different microbial sources with information about substrate specificity to produce effective partial glycerides for various product types. Here, lipases from thermophilic Geobacillus stearothermophilus and Anoxybacillus flavithermus were prepared with a simple partial purification method, and after immobilization, these lipases were tested to synthesize partial glycerides using different types of decanoic acids. The distinct product patterns were analyzed using HPLC. Both immobilized lipases showed the highest substrate selectivity to decanoic acids in common, producing mainly glyceryl monodecanoate. However, commercial immobilized lipases from Thermomyces lanuginosus produced the largest glyceryl monodecanoate from methyl decanoate. These results indicate the importance of immobilization conditions like different microbial sources and substrates and the need for their optimal combination.

Electrostatically Mediated In Situ Polymerization for Enzyme Immobilization and Activation.

Enzyme immobilization in nanoparticles is of interest for boosting their catalytic applications, yet rational approaches to designs achieving both high enzyme loading and activation remain a challenge. Herein, we report an electrostatically mediated in situ polymerization strategy that simultaneously realizes enzyme immobilization and activation. This was achieved by copolymerizing cationic monomers with a cross-linker in the presence of the enzyme lipase (anionic) as the template, which produces enzyme-loaded nanogels. The effects of different control factors such as pH, lipase dosage, and cross-linker fraction on nanogel formation are investigated systematically, and optimal conditions for enzyme loading and activation have been determined. A central finding is that the cationic polymer network of the nanogel creates a favorable environment that not only protects the enzyme but also boosts enzymatic activity nearly 2-fold as compared to free lipase. The nanogels improve the stability of the lipase to tolerate a broader working range of pH (5.5-8.5) and temperature (25-70 °C) and allow recycling such that after six cycles of reaction, 70% of the initial activity is conserved. The established fabrication strategy can be applied generally to different cationic monomers, and most of these nanogels exhibit adequate immobilization and activation of lipase. Our study confirms that in situ polymerization based on electrostatic interaction provides a facile and robust strategy for enzyme immobilization and activation. The wide variety of ionic monomers, therefore, features great potential for developing functional platforms toward satisfying enzyme immobilization and demanding applications.

Preparation and Application of Complexes Based on Biopolymers of Animal Origin

Immobilization of enzymes (IoE) from animal origin on natural carriers increases the system stability; facilitates the separation and accelerates the recovery of the enzyme; makes reuse possible; provides a significant reduction in operating costs. There are numerous IoE methods and systems, including immobilization of various lipases on major carbohydrate biopolymers (chitin, chitosan, cellulose, etc.), discussed in this review. The key points of the most encouraging methods “for increasing the activity and stability” of such biopolymer systems are the “chitosan particle activation” by “ultra-sonication” and multiplicative “addition of glutaraldehyde” to these abovementioned systems. The design of such complex biopolymer preparations (in their various forms) is an important area of modern agrosciences, biomedicine, veterinary, zootechnology and bionanotechnology.

Bio-Nanoparticles Mediated Transesterification of Algal Biomass for Biodiesel Production

Immense use of fossil fuels leads to various environmental issues, including greenhouse gas emissions, reduced oil reserves, increased energy costs, global climate changes, etc. These challenges can be tackled by using alternative renewable fuels such as biodiesel. Many studies reported that biodiesel production from microalgae biomass is an environment-friendly and energy-efficient approach, with significantly improved fuel quality in terms of density, calorific value and viscosity. Biodiesel is produced using the transesterification process and the most sustainable method is utilizing enzymes for transesterification. Lipase is an enzyme with excellent catalytic activity, specificity, enantio-selectivity, compatibility and stability and hence it is applied in microalgae biodiesel production. But, difficulty in enzymatic recovery, high enzyme cost and minimal reaction rate are some of its drawbacks that have to be addressed. In this aspect, the nanotechnological approach of lipase immobilization in producing microalgae biodiesel is a promising way to increase production yield and it is due to the adsorption efficiency, economic benefit, recyclability, crystallinity, durability, stability, environmental friendliness and catalytic performance of the bio-nanoparticles used. Through increasing post-harvest biomass yield, absorption of CO2 and photosynthesis in the photobioreactor, the use of nanoparticle immobilized lipase during the generation of biodiesel from microalgae has the potential to also remove feedstock availability constraints. This review article discusses the production of microalgae biodiesel, and effect of nanoparticles and immobilized lipase nanoparticles on biodiesel production. The advantages of using lipase nanoparticles and the challenges in introducing the immobilized lipase on nanoparticles in large-scale microalgae biodiesel production are also discussed. Reducing the water and land use, energy and nutrient footprints of integrated algae-based operations must be the main goal of larger-scale experiments as well as ongoing research and development in order to expedite the adoption of microalgae-based biodiesel production. Also, the cost-effectiveness and large-scale availability of nanoparticles and the impact of lipase nanoparticles on engine performance should be analyzed for commercialization of microalgae biodiesel.

Covalent immobilization of lipase on magnetic biochar for one-pot production of biodiesel from high acid value oil

Magnetic biochar was synthesized via chelation of Fe3+ with carboxymethyl cellulose and pyrolysis for covalently immobilizing Eversa® Transform lipase. The magnetic biochar had 75.8 mg/g lipase loading that was 54.1 % higher than that without magnetism. The immobilized lipase achieved 91.3 mg/g lipase loading with 19.2 U/mg lipase activity after optimization. It showed good thermal and acid stability with 82.5 % and 98.2 % relative activity at 45 °C and pH 4, respectively. Its relative activity was 90.8 % after stored for 30 d at 4 °C. After magnetically separated for 10 cycles, it still kept 70.1 % activity due to the strong covalent bonding. The lipase further catalyzed one-pot esterification and transesterification of high acid value oil (38 mg KOH/g) with 95.7 % biodiesel yield and cycled for 10 times at 85.7 % yield. Kinetic study gave the activation energy of 28.7 kJ/mol. The covalently immobilized lipase could find practical applications.

Immobilization of Candida rugosa lipase on optimized polyamidoamine dendrimer functionalized magnetic multiwalled carbon nanotubes for green manufacture of butyl butyrate ester

Mineral acid catalysis in Fisher-Speier esterification is hazardous and have other disadvantages. The objective of the study was immobilization of Candida rugosa lipase onto iron oxide (Fe3O4) impregnated magnetic mutiwalled carbon naotubes (MMWCNTs) using amine functionalized polyamidoamine (PAMAM) dendrimers for manufacture of butyl butyrate ester. Dendrimers were synthesized from reaction of ethyl methacrylate with varying chain length of diamines 1, 4 diaminobutane (DAB) / 1, 3 diaminopropane (DAP) / ethylene diamine (ED). The effect of aliphatic alkyl chain length (CH2) of functionalizing agents ED, DAP and DAB, and dendrimer generations on amount of terminal NH2 group, surface charge, protein loading and immobilized enzyme activities were analysed. Magnetic MWCNTs support surface modified with third generation PAMAM using ethylene diamine showed highest protein load 220.1 mg g−1 and enzymatic activity 8,276.21 U g−1 support. The immobilized enzyme particles were characterized by HRTEM, FTIR, XRD, TGA, VSM and Zeta potential analysis. The immobilized enzyme showed improvement in the dispersion, activity, stability and reusability characteristics. Maximum esterification yield 92.67 % and reuse upto 11 cycles with 65.2 % yield obtained. The immobilized preparation showed 2.3 folds esterification than free enzyme. Developed nanobiocatalyst expects to possess potential for various other applications of lipase catalysed reactions.

Enzymatic degradation pattern of polysorbate 20 impacts interfacial properties of monoclonal antibody formulations

Polysorbate 20 (PS20) is widely used to maintain protein stability in biopharmaceutical formulations. However, PS20 is susceptible to hydrolytic degradation catalyzed by trace amounts of residual host cell proteins present in monoclonal antibody (mAb) formulations. The resulting loss of intact surfactant and the presence of PS20 degradation products, such as free fatty acids (FFAs), may impair protein stability. In this study, two hydrolytically-active immobilized lipases, which primarily targeted either monoester or higher-order ester species in PS20, were used to generate partially-degraded PS20. The impact of PS20 degradation pattern on critical micelle concentration (CMC), surface tension, interfacial rheology parameters and agitation protection was assessed. CMC was slightly increased upon monoester degradation, but significantly increased upon higher-order ester degradation. The PS20 degradation pattern also significantly impacted the dynamic surface tension of a mAb formulation, whereas changes in the equilibrium surface tension were mainly caused by the adsorption of FFAs onto the air–water interface. In an agitation protection study, monoester degradation resulted in the formation of soluble mAb aggregates and proteinaceous particles, suggesting that preferential degradation of PS20 monoester species can significantly impair mAb stability. Additional mAbs should be tested in the future to assess the impact of the protein format.

Beef quality monitoring using immobilization lipase on modified polypropylene non-woven fabric based on time-temperature indicator

A lipase-based time-temperature indicator (TTI) was developed via the immobilization of lipase on polyethylene imine/glutaraldehyde/polypropylene non-woven fabric for the catalytic hydrolysis of glyceryl triacetate. pH of the reaction system gradually decreased and the color changed from green to orange or salmon pink. Four TTIs were selected by changing the concentration of glyceryl triacetate methanolic solution, the concentration of lipase-phosphate buffer solution, and the amount of phosphate buffer added, storing at 5–25 °C and measuring the coloration difference every 24 h. The Arrhenius activation energy (Ea) of obtained TTIs was calculated as 26.28, 47.98, 38.87, and 42.00 kJ/mol. For the application of TTIs to fresh beef monitoring, the kinetic properties of beef were studied and an Ea value of 37.60 kJ/mol was obtained. The similarity of these Ea shows accuracy in the prediction of beef quality with TTI color development.

Immobilization of Lipase on Agarose Beads for Enzymatic Hydrolysis/Transesterification of Castor Oil

Lipase was immobilized on agarose beads and used for enzyme-catalyzed hydrolysis/transesterification of castor oil at room temperature without surfactants and salts. The immobilization yield was evaluated by reacting p-nitrophenyl butyrate and immobilized lipase. The reaction was performed in a 25 mL phosphate buffer medium (pH 7.0) and incubated for 2 hours. The absorbance of the formed p-nitrophenol was measured at 346 nm. The highest immobilization rate was obtained using 5.0 mg lipase with 2.5 mL glyoxal agarose beads. The reaction was performed in 25.0 mL sodium bicarbonate buffer pH 10 for 12 h. The hydrolysis/transesterification of castor oil was performed in phosphate buffer (pH 7) – ethanol medium. The main products were ricinoleic acid and ethyl ricinoleate. The highest hydrolysis/transesterification yield was 87% using 1.0 g castor oil and 2.5 mL of lipase immobilized agarose beads in the phosphate buffer (pH 7): ethanol (1: 9, V: V) at 72 hours. The reaction products were analyzed using FTIR and RP-HPLC.

The preparation of Diacylglycerol-rich soybean oil by acetylated modification of arachin nanoparticles for W/O Pickering emulsion system.

Pickering emulsion catalytic system (PEC) stabilized by nanoparticles is an efficient catalytic platform. Herein, a high-performance PEC was constructed by acetylated modification of arachin nanoparticles (AAPs). The results showed the pI of arachin was decreased from pH 5.5 to pH 3.5. The surface hydrophobicity index was significantly increased (from 56.28±4.23 to 120.77±0.79) after acetylated modification. The three-phase contact angle of AAPs was 91.20±0.98°. AAPs were used as lipase immobilization carriers to increase the activity of free lipase fabricating lipase-AAPs. The immobilization efficiency and activity of lipase-AAPs were 12.95±0.03% and 1.74±0.07 U/mg, respectively. Enzymatic reaction kinetics showed that Vm of lipase-AAPs was twice of free lipase. Km was 1/5 of free lipase. The catalytic efficiency of PEC to prepare DAG was 2.36 times of biphasic catalytic system (BCS). This work provided a promising way to promote the efficiency of DAG preparation.

Fabrication of lipase immobilized-carboxylated cellulose nanocrystals as biocatalyst for enzymatic hydrolysis of palm oil and its kinetical study

This research focuses on the production of free fatty acids (FFA), one of the essential components in the oleochemical industry, through the enzymatic hydrolysis process of palm oil at low temperature and pressure using cellulose nanocrystals (CNCs) as lipase immobilization support. Furthermore, the kinetic behavior of this hydrolysis reaction with the presence of lipase immobilized-CNCs (lipase@CNCs) was studied to find the optimum condition for designing the reactor. According to FTIR and TEM analyses, CNCs were successful in immobilizing lipases, as ascertained by the appearance of amide peaks from the formation of bonds between CNCs and lipases and the thickening of the surface of the CNC morphology after immobilization. As a result, the optimum immobilization conditions were obtained at a lipase concentration of 1.5 mg/mL, an immobilization time of 2 h, and the CNCs/EDC ratio of 1:3. Moreover, the Michaelis-Menten model with the inhibition product fitted with the experiment and lipase@CNCs has the highest Vmax value at a temperature of 40 °C of 15.53 mM/h with a KM of 99.80 mM and an Ea of 41.07 kJ/mol. The simulation results show that the semi-batch reactor can achieve a higher conversion at the beginning of the reaction with a conversion of 28%. Therefore, using lipase@CNCs in hydrolysis processes is expected to be one of the strategies that can overcome the limitations associated with existing methods in terms of high energy consumption.

In-situ immobilization of lipase on α-alumina membrane for oil fouling control and cleaning

Enzyme immobilization on inorganic membranes is typically a difficult, time-consuming, multi-step process carried out ex-situ (outside the filtration system). To enable process scaling, an in-situ immobilization technique is essential. Therefore, an in-situ method to immobilize the lipase Eversa Transform 2.0 (ET2) on a ceramic membrane was developed using polydopamine as a bonding agent. The in-situ immobilization was successful, and optimum dopamine hydrochloride and ET2 concentrations (0.3 mg·mL−1 and 4 mg·mL−1, respectively) resulted in an enzyme loading of 10 g·m−2 that increased the membrane water affinity and hydrolytic activity towards soybean oil (38 mmol·min−1·m−2). The enzymatic membrane demonstrated great self-cleaning capability (pure water permeance recovery of 97 % after cleaning with deionized water at 40 °C for 6 h) and strong fouling resistance (pure water permeance decline limited to 43 % following oil emulsion filtration). To reuse the membrane after it lost its hydrolytic activity, calcination or in-situ chemical cleaning were tested. There were no morphological or chemical alterations on the membrane surface after five cycles of modification-regeneration. When compared to ex-situ immobilization techniques, in-situ enzyme immobilization and chemical regeneration can save costs, improve the sustainability of the process, and ease scale-up.

Carboxy-Functionalized Covalent Organic Framework as a Carrier for Lipase Immobilization and Its Application in Inhibitors Screening.

Covalent organic frameworks (COFs) with large specific surface areas, high porosity, good stability, and designable structure are promising carriers for immobilized enzymes. It is important to explore lipase inhibitors from natural foods as lipase inhibitors are closely related to the treatment of obesity. In this work, a carboxyl functionalized covalent organic framework (TpBD-3COOH) was prepared by solvothermal method for covalent immobilization of porcine pancreatic lipase (PPL) and obtained the enzyme-decorated COF (PPL@COF). The immobilized lipase showed wider pH and temperature tolerance with the same optimal pH and temperature of 7.5 and 50 ℃ compared to free lipase. After 6 successive reuses, the PPL@COF maintained 53.0% of its original activity. Immobilized lipase also displayed enhanced storage stability (55.4% after 14days at 4 ℃). When p-nitrophenyl acetate was applied as the substrate, the calculated Michaelis constant was 3.57mM and the half maximal inhibitory concentration of orlistat was 3.20μM. Finally, the PPL@COF was used for enzyme inhibitors screening from natural foods combined with UV spectrophotometry, and Hawthorn was screened for excellent lipase inhibitory activity.

Preparation and characterization of stable biopolymer-based nanogel for studying its application in immobilization of Candida rugosa lipase

Preparation and characterization of stable biopolymer-based nanogel for studying its application in immobilization of Candida rugosa lipase

Zinc hydroxide salts as new supports for the immobilization of Pseudomonas cepacia lipase

Layered double hydroxides (LDHs) are brucite-like nanomaterials that have been used to immobilize several enzymes. However, layered hydroxide salts (LHSs), another group of brucite-like nanomaterials, have not yet been used for enzyme immobilization. In this work, we prepared two types of layered hydroxide salts, zinc hydroxide nitrate (ZHN: Zn5(OH)8(NO3)2 .2H2O) and zinc hydroxide chloride (ZHC: Zn5(OH)8Cl2 .H2O) and used them to immobilize Pseudomonas cepacia lipase (LipPS). The best protein loading for both ZHN and ZHC was 162.5 mg g−1 of LHS, which gave high values of triolein-hydrolyzing activity in organic medium (103 U g−1 for LipPS-ZHN and 105 U g−1 for LipPS-ZHC), immobilization efficiencies above 90% and activity retentions above 170%. In the kinetic resolution of (R,S)-1-phenylethanol, LipPS-ZHN gave better results, with 50% conversion being obtained in 2 h and an ee s of 99%. With LipPS-ZHC, the conversion at 2 h was 40% and the ee s, was lower, only 73%. For both immobilized materials, the eep was higher than 99% and E was higher than 200. The immobilized materials were stable after 5 cycles of reuse in successive 2-h kinetic resolutions. These results demonstrate that the layered hydroxide salts ZHN and ZHC have good potential as supports for the immobilization of lipases.

Immobilization of Lipases on Chitosan Hydrogels Improves Their Stability in the Presence of the Products of Triglyceride Oxidation.

A significant bottleneck for the industrial application of lipases stems from their poor stability in the presence of commercial triglycerides. This is mainly due to the inactivating effect of the products of triglyceride oxidation (PTO), which are usually produced when oils and fats, being imported from far countries, are stored for long periods. In this study, the immobilization of a lipase from Candida rugosa on chitosan hydrogels has been carried out following two alternative approaches based on the enzyme adsorption and entrapment to increase the lipase stability under the operating conditions that are typical of oleochemical transformations. The effect of model compounds representing different classes of PTO on a lipase has been studied to optimize the enzyme immobilization method. Particular attention has been devoted to the characterization of the inactivating effect of PTO in nonaqueous media, which are adopted for most industrial applications of lipases.