Immobilization Of Lipase Research Articles

Optimizing Thermal Stability: Evaluating the Impact of Heterofunctional Hydrophobic Supports on Immobilized Flaxseed Lipase.

Lipases have catalytic capacity in various processes such as hydrolysis. Those derived from plant sources, such as linseed, offer an economical alternative. The immobilization process facilitates the recovery and reuse of lipase, providing advantages such as resistance to high temperatures and difficulties in recovering and reusing free lipases, which makes product separation difficult. This study presents the immobilization of lipases extracted from flax seeds on octylfunctional hydrophobic supports. Additionally, the thermal stability of the derived products was evaluated in comparison with freely soluble lipase. The lipase exhibited a strong affinity for the evaluated heterofunctional hydrophobic supports, with DVS-activated octylagarose emerging as the most efficient method for immobilization, thus increasing catalytic activity upon resuspension. Furthermore, the octylagarose derivative demonstrated a notable increase in thermal stability. The main results of the study include that the soluble enzyme showed greater activity after 24 h, regardless of the temperature evaluated. The benzamide extract showed greater thermal stability, and all supports evaluated showed greater activity than the soluble enzyme after immobilization. Notably, lipase immobilized on octyl glyoxyl agarose showed the highest activity, demonstrated stability for 840 h at 60 °C, and had a half-life of 1242 h. Furthermore, the lipase immobilized in octyl glyoxyl agarose showed a stabilization factor approximately nine times greater than the free enzyme. These results suggest that immobilization, probably achieved through interfacial activation and multipoint covalent bonds, prevented the release of the enzyme into the medium with increasing temperature. This study thus highlights the significant potential of immobilizing flaxseed-derived lipase.

In-situ aqueous phase encapsulation immobilized lipase assisted by acidic ionic liquids for the synthesis of isoamyl acetate

In this study, two acidic ionic liquids were innovatively selected to replace traditional acetic acid as catalysts and templates for the preparation of covalent organic frameworks (COFs) materials in aqueous phase at room temperature. At the same time, the synthesized COFs carrier achieved in-situ encapsulation and immobilization of Candida rugosa lipase (CRL) in aqueous phase at room temperature. The optimum immobilized lipases COF-COOH-M and COF-SO3H-M were obtained by adjusting the reaction conditions. Their enzyme loadings were 210.17 mg/g and 258.47 mg/g, respectively, and the specific activity retention compared to free lipase was 67.11% and 78.42%, respectively. The carrier and immobilized lipase were characterized by FT-IR, XRD, BET and TG. The catalytic performance and operational stability of the immobilized lipase were investigated. The results showed that both immobilized lipases showed better stability than free lipases, and COF-SO3H-M had the best performance. The relative activity retention of COF-SO3H-M after incubation at 60 °C for 60 minutes was 4.35 times that of free lipase. After 28 days of storage at 4 °C, the relative activity of COF-SO3H-M remained 75.10%, while that of free lipase was only 24.61%. The COF-SO3H-M was applied as biocatalyst for isoamyl acetate preparation at 50 °C for 7 hours, and the yield of isoamyl acetate was as high as 86.94%, which was 22.12% higher than that of free lipase.

Enzymatic synthesis and antioxidation characterization of various acylated chlorogenic acids via regioselective and recyclable immobilized lipases

Enzymatic synthesis and antioxidation characterization of various acylated chlorogenic acids via regioselective and recyclable immobilized lipases

Optimization of Candida antarctica lipase immobilization in xerogels using an ionic liquid additive: enhanced esterification activity and thermal stability

Optimization of Candida antarctica lipase immobilization in xerogels using an ionic liquid additive: enhanced esterification activity and thermal stability

Interfacial modulation of hierarchically porous UIO-66 for the immobilization of Rhizomucor miehei lipase towards the efficient synthesis of 1,3-dioleic acid glycerol.

Herein, UiO-66 was selected as the immobilization carrier of Rhizomucor miehei lipase (RML). After etching and hydrophobic modification, the functionalized UIO-66 (H-UIO-66-OPA) was utilized for RML immobilization and the obtained RML@H-UIO-66-OPA showed about 70 % relative activity after incubation at 60 °C, which was much better than RML (20 %). RML@H-UIO-66-OPA was used in the synthesis of 1,3-dioleic acid glycerol (1,3-DAG) and the effects of reaction conditions (temperature, enzyme addition, substrate molar ratio, and time) on 1,3-DAG yield were investigated. Under the optimal conditions (temperature of 65 °C, enzyme addition of 2 wt%, substrate molar ratio of 2:1, and reaction time of 4 h), 1,3-DAG yield reached 77 %. Finally, Box-Behnken experimental design method was utilized to optimize the process parameters for the preparation of 1,3-DAG, and the final 1,3-DAG content was 79.80 %, which demonstrated that the model could predict the relationship between the factors and be suitable for the process optimization in 1,3-DAG preparation.

Sustainable Lipase Immobilization: Chokeberry and Apple Waste as Carriers.

In the modern world, the principles of the bioeconomy are becoming increasingly important. Recycling and reusability play a crucial role in sustainable development. Green chemistry is based on enzymes, but immobilized biocatalysts are still often designed with synthetic polymers. Insoluble carriers for immobilized biocatalysts, particularly those derived from agro-industrial waste such as mesoporous lignocellulosic materials, offer a promising alternative. By using waste materials as support for enzymes, we can reduce the environmental impact of waste disposal and contribute to the development of efficient bioprocessing technologies. The current study aimed to assess the possibility of using apple and chokeberry pomace as carriers for the immobilization of Palatase 20000L (lipase from Rhizomucor miehei). The analysis of lignocellulosic materials revealed that chokeberry pomace has a higher neutral detergent fiber (NDF) and lignin contents than apple pomace. Moreover, Scanning Electron Microscopy (SEM) observations indicated similar compact structures in both pomaces. The lipase activity assays demonstrated that immobilization of lipase from R. miehei onto apple and chokeberry pomace improves their properties, especially the synthetic activity. The findings highlight the potential of utilizing fruit pomaces not only as a source of bioactive compounds but also in enhancing enzyme stability for industrial applications.

In-situ immobilization of lipase within hydrogen-bonded organic framework for rapid screening inhibitors from traditional Chinese medicines

In-situ immobilization of lipase within hydrogen-bonded organic framework for rapid screening inhibitors from traditional Chinese medicines

Agri-Food and Food Waste Lignocellulosic Materials for Lipase Immobilization as a Sustainable Source of Enzyme Support-A Comparative Study.

Enzyme immobilization is a crucial method in biotechnology and organic chemistry that significantly improves the stability, reusability, and overall effectiveness of enzymes across various applications. Lipases are one of the most frequently applied enzymes in food. The current study investigated the potential of utilizing selected agri-food and waste materials-buckwheat husks, pea hulls, loofah sponges, and yerba mate waste-as carriers for the immobilization of Sustine® 121 lipase and Yarrowia lipolytica yeast biomass as whole-cell biocatalyst and lipase sources. Various lignocellulosic materials were pretreated through extraction processes, including Soxhlet extraction with hexane and ethanol, as well as alkaline and acid treatments for loofah sponges. The immobilization process involved adsorbing lipases or yeast cells onto the carriers and then evaluating their hydrolytic and synthetic activities. Preparations' activities evaluation revealed that alkaline-pretreated loofah sponge yielded the highest hydrolytic activity (0.022 U/mg), while yerba mate leaves under brewing conditions demonstrated superior synthetic activity (0.51 U/mg). The findings underscore the potential of lignocellulosic materials from the agri-food industry as effective supports for enzyme immobilization, emphasizing the importance of material selection and pretreatment methods in optimizing enzymatic performance through giving an example of circular economy application in food processing and waste management.

Hierarchically ordered truncated single crystal mesoporous ZIF-8 as a solid platform for lipase immobilization

Most pristine metal organic frameworks (MOFs) are inherently microporous. To introduce desired porosity for easy enzyme infiltration and immobilization, polystyrene spheres was used as sacrificial template to design hierarchical ordered micro-mesoporous truncated single-crystalline ZIF-8 (SOM-ZIF-8). The surface area and pore diameter of the SOM-ZIF-8 were 821 m2/g and 7.09 nm which changed to 669 m2/g and 4.98 nm respectively, after lipase immobilization, suggesting the pores and surface of the SOM-ZIF-8 served as binding sites for the enzyme, and could reach a loading capacity of 134 mg/g after 24 h. The optimal conditions for achieving maximum lipase activity for free LipaseELP, ZIF-8@LipaseELP and SOM-ZIF-8@LipaseELP were pH 7.8 at 45 °C, and maintained 33.67 %, 45.6 % and 57.78 % residual activity after incubation at 80 °C for 2 h. The specific activity towards tributyrin and p-nitrophenyl acetate were observed to be SOM-ZIF-8@LipaseELP (0.25 and 45.85 U/mg) compared to ZIF-8@LipaseELP (0.211 and 43.62 U/mg) and LipaseELP (0.179 and 41.09 U/mg) respectively. The SOM-ZIF-8@LipaseELP could further be recovered and reused for 9 rounds while maintaining 73.8 % of its original activity. This study demonstrates that introducing mesoporous structures into ZIF-8 could improve its binding enzyme property for enhanced hydrolytic function.

Immobilization of lipase on zeolite, silica, and silica-aluminas and its use in hydrolysis, esterification, and transesterification reactions

Lipases have been immobilized on various supports to catalyze hydrolysis, esterification, and transesterification reactions efficiently. Among a broad range of materials, mesoporous silica has attracted attention thanks to its distinct characteristics and advantages, being widely used for biocatalysis applications. In this work, the lipase from Thermomyces lanuginosus (TLL) was immobilized on six different carriers: two zeolites HZSM-5 (SAR 25 and 280), mesoporous Si-MCM-41, and silica-aluminas Siral 10, 20, and 40. TLL was efficiently immobilized in Siral 20 (99.9 %) and Siral 40 (99.9 %) using 26 mg g−1 of enzyme loading. Due to its more hydrophobic nature, Siral 40 was selected as the most suitable support for TLL immobilization using 5 mmol L−1 of sodium phosphate buffer solution, pH 7, and rotational stirring as the optimum condition. The effect of protein concentration on the TLL immobilization was investigated, and the results adjusted well (R2 > 0.99) on the Langmuir isotherm model. The Siral 40 presented a maximum adsorption capacity equal to 169 mgprotein gsupport−1. The heterogeneous biocatalyst (TLL-S40) was applied in biodiesel synthesis, olive oil hydrolysis, p-nitrophenyl-laurate hydrolysis, and ethyl oleate synthesis. The esterification reaction was successfully catalyzed by TLL-S40, leading to a conversion 2.6-fold higher than free TLL at 30 °C. The biocatalyst was reused for three operational cycles with a retention of 34 % of its initial conversion. The results show that Siral 40, a silica-alumina material, can potentially be employed in lipase immobilization for esterification reactions.

Revealing the potential of a novel approach for assessing the effect of pyrolysis conditions on sisal waste-based activated carbon for lipase immobilization and ethyl lactate biosynthesis

Revealing the potential of a novel approach for assessing the effect of pyrolysis conditions on sisal waste-based activated carbon for lipase immobilization and ethyl lactate biosynthesis

PVP-assisted in situ immobilizing lipase on covalent organic framework for enhanced catalytic activity and stability in bioconversions

Covalent organic frameworks (COFs) are crystalline, porous organic materials that have significant potential as supports for enzyme immobilization. Nevertheless, the in situ preparation of biocatalysts during the COF formation process remains a considerable challenge. Herein, we developed a one-pot in situ preparation strategy. The immobilized lipase PS@TPB-TFPB COF-I was fabricated by mixing the polyvinylpyrrolidone (PVP)-lipase PS complex with precursors 1,3,5-tris(4-aminophenyl)benzene (TPB) and 1,3,5-tris(4-formylphenyl)benzene (TFPB) in acetonitrile catalyzed by acetic acid at room temperature for 48 h. The formation mechanism was systematically investigated using time-dependent microscopy techniques. PVP acts as a guiding reagent, controlling the morphological changes that occur during this process. Furthermore, the biocatalyst was employed in the kinetic resolution of racemic 1-phenylethanol, resulting in a significant enhancement in the conversion rate, with a range of 2.1 to 10.6 times higher compared to free PS at the same reaction time. The robust biocatalyst maintained high catalytic activity and enantioselectivity even after 10 cycles. The strategy described here is promising for lipase immobilization and expands the range of applications for COFs in biomanufacturing.

Enhancing Lipase Immobilization via Physical Adsorption: Advancements in Stability, Reusability, and Industrial Applications for Sustainable Biotechnological Processes.

Immobilization of lipases by physical adsorption improves their stability, recovery, and reusability in biotechnological processes. The present review provides an advanced bibliometric analysis and a comprehensive overview of research progress in this field. By searching Web of Science, 39,575 publications were analyzed, and 325 relevant articles were selected. Key journals, countries, institutions, and authors were identified. The most cited articles focus on biofuel production and industrial applications. The analysis revealed four research themes with a focus on the production of biofuel. The physical adsorption method is effective when the appropriate support is used. Despite a decrease in patent applications, industrial interest remains high. Future studies should focus on optimizing support materials and exploring new applications of this technique. The present review provides a detailed understanding of the immobilization of lipases by physical adsorption.

Preparation of two new chiral metal-organic frameworks for lipase immobilization and their use as biocatalysis in the enantioselective hydrolysis of racemic naproxen methyl ester

Considering the selective pharmacological activity of chiral drugs, it is important to develop new chiral materials to synthesize them. In this work, two new chiral MOFs (UiO-66@Np and UiO-66@Ib) were prepared by the covalent attachment of the chiral compounds (S-naproxen and S-ibuprofen) to the amine-functionalized Zr-MOF (UiO-66-NH2). Then, Candida rugosa lipase (CRL) was immobilized on these chiral MOFs to fabricate two new biocomposites (UiO-66@Np@CRL and UiO-66@Ib@CRL) as effective biocatalysts, which enable significant enhancement in the catalytic activity and enantioselectivity of lipase. The FTIR, SEM, EDX, TGA, and PXRD analyses were carried out to confirm the formation of the biocomposites. The catalytic performances of the biocomposites (UiO-66@Np@CRL and UiO-66@Ib@CRL) were evaluated in the typical hydrolysis of p-nitro-phenyl palmitate (p-NPP) and the catalytic enantioselective hydrolysis of (R,S)-naproxen methyl ester. Under optimal conditions, UiO-66@Np@CRL showed a higher enantiomeric excess of the substrate (ees) value and a 98 % and 50 % conversion rate, respectively, than that of UiO-66@Ib@CRL. Besides, an excellent enantioselectivity (E) value of 458 was obtained in the presence of the biocomposite (UiO-66@Np@CRL). In addition, it was observed that the catalytic activity, conversion rate and ees value of this composite (UiO-66@Np@CRL) remained almost unchanged in reuse after 6 months. The results showed that in enantioselective reactions, the highest conversion and enantioselectivity could be achieved when the chiral compound bound to the MOF and the model compound used are the same.

Application of Spectrogel clay as a support for immobilization of lipase from Burkholderia cepacia

AbstractThe interest in maximizing the production of ethyl esters in a sustainable way and with lower energy costs has increased the use of immobilized enzymes as catalysts. This study aimed to apply the commercial clay Spectrogel® as a support for the immobilization of lipase from Burkholderia cepacia by adsorption and covalent bonding methods. The immobilizations were carried out using a 23 factorial design to study the effects of the activity offered (U g−1), pH, and the molar concentration of the enzyme solution buffer (mol L−1) on the enzyme activity obtained (U g−1). From the statistical analysis of the results, the best conditions for immobilization were pH 7.0 and 0.1 mol L−1 for both tested immobilization methods, with the best offered activity being 5709 and 7600 U g−1. The activities obtained were 1219.81 ± 7.51 and 1274.89 ± 14.99 U g−1 for adsorption and covalent bonding, respectively. The biocatalysts exhibited protein leaching of 33.55 ± 1.08% and 19.44 ± 2.43% when immobilized by adsorption and covalent bonding, respectively. The optimal activity temperature and thermal stability were obtained at 40°C. Additionally, the immobilization of lipase in Spectrogel® by both methods was efficient, showing higher thermal stability than the free enzyme. Thus, this work contributed scientifically to the development of a new and economical biocatalyst for ethyl ester production.

Improved catalytic stability of immobilized Candida antarctica lipase B on macroporous resin with organic polymer coating for biodiesel production.

Lipase is one of the most widely studied and applied biocatalysts. Due to the high enzyme leakage rate of the immobilization method of physical adsorption, we propose a new lipase immobilization method, based on the combination of macroporous resin adsorption and organic polymer coating. The immobilized Candida antarctica lipase B (CALB@resin-CAB) was prepared by combining the macroporous resin adsorption with cellulose acetate butyrate coating, and its structure was characterized by various analytic methods. Immobilized lipase was applied for biodiesel production using acidified palm oil as the starting material, the conversion rate achieved as high as 98.5% in two steps. Furthermore, the immobilized lipase displayed satisfactory stability and reusability in biodiesel production. When the aforementioned reaction was carried out in a continuous flow packed bed system, the yield of biodiesel was 94.8% and space-time yield was 2.88g/(mL∙h). The immobilized lipase CALB@resin-CAB showed high catalytic activity and stability, which has good potential for industrial application in the field of oil processing.

One‐Pot Synthesis of (S)‐Flavanones by a Double‐Face Promiscuous Chemo‐Enzymatic Cascade of Lipases

AbstractThe one‐pot stereoselective synthesis of (S)‐flavanones from 2′‐hydroxyacetophenone and substituted aromatic aldehydes was obtained by a double‐face promiscuous chemo‐enzymatic cascade of porcine pancreas and Mucor javanicus lipases. The reaction pathway comprises: A) cross‐aldol condensation catalysed by porcine pancreas lipase to yield chalcone intermediates; B) unprecedented intramolecular oxa‐Michael addition of chalcone intermediates to (S)‐flavanones. Mucor javanicus lipase was the most effective enzyme in step B. Imidazole and 2‐methylimidazole were studied as additive in order to improve the efficacy of the overall transformation. The sustainability of the chemo‐enzymatic cascade was increased by immobilization of lipases on cross‐linked hydroxy‐methylated kraft lignin nanoparticles, by use of concanavalin A. Immobilization conferred considerable stability and reusability at the system for 4 runs. Noteworthy, the reaction mixture was significantly enriched in (S)‐flavanones under both homogeneous and heterogeneous conditions. Computational studies encompassing docking and molecular dynamic analyses showed the role played by evolutionary conserved oxyanion holes and catalytic triad of Mucor javanicus lipase in the stereocontrol of the intra‐molecular oxa‐Michael addition.

Improvement of Thermo-Stability and Solvent Tolerant Property of Streptomyces sp. A3301 Lipase by Immobilization Techniques with Application in Poly (lactic acid) Polymerization by Using Biological Process

The thermo-solvent-tolerant lipase-producing actinomycete, Streptomyces sp. A3301, was utilized as a biocatalyst for poly (lactic acid) or PLA polymerization. The study aimed to optimize lipase immobilization conditions, characterize the immobilized lipase and apply it for PLA polymerization. The results showed using a sponge as the immobilizing matrix was the most effective method, achieving a maximum activity of 277 U/g of sponge. The optimal sponge size was determined to be 0.125 cm³ and pre-soaking the sponge in 0.1 M phosphate buffer at pH 7.0 for 24 h before use proved advantageous. Immobilization significantly enhanced the thermo-stability of the enzyme, with a relative activity ranging from 140 to 190 % within the temperature range of 30 to 60 °C. In contrast, the crude lipase exhibited thermo-stability only within the 30 - 50 °C range. The immobilized lipase demonstrated stability under PLA polymerization conditions, which involved a reaction mixture containing toluene and lactic acid and performed at 60 °C for 8 h. The immobilized lipase maintained its activity under this condition for 5 h, retaining a relative activity of 230 %, which was 1.2 times higher than the activity of the crude lipase. When the immobilized lipase was used in PLA polymerization, the resulting PLA product exhibited a molecular weight of 5,333 ± 0.02 Da, and the degree of polymerization was approximately 72. These findings underscore the potential of the immobilization technique to enhance lipase activity for PLA polymerization. HIGHLIGHTS PLA polymerization via a biological process is demonstrated. PLA can be polymerized by enzyme lipase produced by Streptomyces sp. A3301. Improving PLA polymerization with enzyme immobilization techniques. Sponge is the best immobilizer for PLA polymerization. PLA can be synthesized under mild conditions. GRAPHICAL ABSTRACT

Characteristic study of Candida rugosa lipase immobilized on lignocellulosic wastes: effect of support material.

For the first time is reported the comparison of solid biocatalysts derived from Candida rugosa lipase (CRL) immobilized on different lignocellulosic wastes (rice husk, brewer's spent grain, hemp tea waste, green tea waste, vine bark, and spent coffee grounds) focusing on the characterization of these materials and their impact on the lipase-support interaction. The wastes were subjected to meticulous characterization by ATR-FTIR, BET, and SEM analysis, besides lignin content and hydrophobicity determination. Investigating parameters influencing immobilization performance revealed the importance of morphology, textural properties, and hydrophobic interactions revealed the importance of morphology, textural properties and especially hydrophobic interactions which resulted in positive correlations between surface hydrophobicity and lipase immobilization efficiency. Hemp tea waste and spent coffee grounds demonstrated superior immobilization performances (7.20 U/g and 8.74 U/g immobilized activity, 102.3% and 33.5% efficiency, 13.4% and 15.4% recovery, respectively). Moreover, they demonstrated good temporal stability (100% and 92% residual activity after 120days, respectively) and retained 100% of their immobilized activity after five reuses in the hydrolysis of p-nitrophenyl palmitate in hexane. In addition, the study of enzymatic desorption caused by ionic strength and detergent treatments indicated mixed hydrophobic and electrostatic interactions in rice husk, vine bark, and spent coffee grounds supports, while hemp tea waste and green tea waste were dominated by hydrophobic interactions.

Zr-based MOF as a support for lipase immobilization to enhance enzymatic transesterification for biodiesel production

The development of novel biocatalysts is essential to promote the commercialization of biodiesel production by transesterification reaction. In this paper, Rhizopus oryzae lipase (ROL) was immobilized on an amino-functionalized zirconium-based metal organoskeleton by interfacial adsorption. The immobilization conditions were optimized and the enzymatic properties were tested, and the resulting novel biocatalysts exhibited higher stability and heat resistance. SEM, XRD and BET analyses were used to characterize the biocatalysts and carrier materials. The catalytic performance of ROL@UiO-66-NH2 in the production of biodiesel by transesterification reaction was explored, and the production process was optimized by response surface method. The results showed that the conversion rate of FAEE reached 82.05% at molar ratio of ethanol/oil of 15.43:1, reaction temperature of 50.28°C, reaction time of 120.9 min, DES addition of 48.08 wt%, biocatalyst addition of 3 wt%, and ultrasonic power of 90 W. In addition, ROL@UiO-66-NH2 demonstrated good recyclability, with the catalytic efficiency remaining at 71.87% after five cycles.