Immobilization Of Lipase Research Articles

Functionalized corn stalk carboxymethyl cellulose and mesoporous silica SBA-15 composite hydrogel-immobilized lipase for the resolution of racemic ibuprofen ethyl ester

ABSTRACT The immobilization of enzymes is an important strategy to improve their stability and reusability. However, it often comes with a trade off of reduced biological activity and catalytic efficiency. In this study, a composite hydrogel based on corn stalk carboxymethyl cellulose, acrylic acid, and mesoporous silica SBA-15 was developed as a support for the immobilization of lipase from Pseudomonas S.P. (PSL). The surface morphology and functional properties of the composite hydrogels were investigated by scanning electron microscopy, Fourier transform infrared, X-ray diffraction and simultaneous thermal analyzer, respectively. The composite hydrogel materials had a large number of mesopores and macropores were present in the wall of the hydrogel sheet. The hydrogel had a network structure that reduced the diffusion resistance of the substrate and improved the accessibility of the enzyme. The immobilized PSL exhibited high enantioselectivity (E value: 88.2) and enzyme activity (316.8 µmol/g/min) in the preparation of (S)-ibuprofen by enzymatic hydrolytic resolution of racemic ibuprofen ethyl ester. This prepared novel composite hydrogel is a promising supporting matrix for the immobilization of enzymes.

Alloyed Trimetallic Nanocomposite as an Efficient and Recyclable Solid Matrix for Ideonella sakaiensis Lipase Immobilization.

In this work, a trimetallic (Ni/Co/Zn) organic framework (tMOF), synthesized by a solvothermal method, was calcinated at 400 and 600 °C and the final products were used as a support for lipase immobilization. The material annealed at 400 °C (Ni-Co-Zn@400) had an improved surface area (66.01 m2/g) and pore volume (0.194 cm3/g), which showed the highest enzyme loading capacity (301 mg/g) with a specific activity of 0.196 U/mg, and could protect the enzyme against thermal denaturation at 65 °C. The optimal pH and temperature for the lipase were 8.0 and 45 °C but could tolerate pH levels 7.0-8.0 and temperatures 40-60 °C. Moreover, the immobilized enzyme (Ni-Co-Zn@Lipase, Ni-Co-Zn@400@Lipase, or Ni-Co-Zn@600@Lipase) could be recovered and reused for over seven cycles maintaining 80, 90, and 11% of its original activity and maintained a residual activity >90% after 40 storage days. The remarkable thermostability and storage stability of the immobilized lipase suggest that the rigid structure of the support acted as a protective shield against denaturation, while the improved pH tolerance toward the alkaline range indicates a shift in the ionization state attributed to unequal partitioning of hydroxyl and hydrogen ions within the microenvironment of the active site, suggesting that acidic residues may have been involved in forming an enzyme-support bond. The high enzyme loading capacity, specific activity, encouraging stability, and high recoverability of the tMOF@Lipase indicate that a multimetallic MOF could be a better platform for efficient enzyme immobilization.

Evaluation of mesoporous silica particles as a support for lipase immobilization in biodiesel production: Enhanced ethyl ester synthesis from algal oil

The utilization of novel materials in the biofuel production domain has been regarded as a significant advancement in catalyst development. Incorporating lipases in biodiesel manufacturing is often viewed as a cost-restrictive phase since the expenses associated with recovering these catalysts can render market expectations unfeasible. Within this investigation, mesoporous silica (MPS) particles were assessed as a supporting medium for immobilizing commercial lipase, subsequent to their utilization in ethyl ester synthesis. Initially, the production of mesoporous silica was achieved through the co-precipitation technique at a consistent pH and was subsequently chosen as an immobilization support for the enzyme. The characterization of the biocatalyst support materials produced was performed using various analytical methods including X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and BET analysis. The Novozym 435 lipase was then immobilized within the matrices, followed by the transesterification process of algal oil derived from Nannochloropsis. The resulting biodiesel samples demonstrated compliance with commercial standards, exhibiting an ester conversion rate exceeding 94.8 %. Additionally, properties such as density (0.93 g cm−3) and viscosity (5.01 mm2 s−1) satisfied the specifications mandated by ASTM for biofuel application. Experimental planning techniques showed that increasing lipase loading and using temperatures between 38 and 48 °C resulted in higher conversion rates of ethyl esters. This confirms the effectiveness of the enzymatic catalyst in generating primary fatty acid esters, even with residual lipid feedstocks.

Lipolytic production from solid-state fermentation of the filamentous fungus Penicillium polonicum and its applicability as biocatalyst in the synthesis of ethyl oleate.

Lipases represent versatile biocatalysts extensively employed in transesterification reactions for ester production. Ethyl oleate holds significance in biodiesel production, serving as a sustainable alternative to petroleum-derived diesel. In this study, our goal was to prospect lipase and assess its efficacy as a biocatalyst for ethyl oleate synthesis. For quantitative analysis, a base medium supplemented with Rhodamine B, olive oil, and Tween 80 was used. Solid-state fermentation utilized crambe seeds of varying particle sizes and humidity levels as substrates. In the synthesis of ethyl oleate, molar ratios of 1:3, 1:6, and 1:9, along with a total enzymatic activity of 60 U in n-heptane, were utilized at temperatures of 30°C, 37°C, and 44°C. Reactions were conducted in a shaker at 200rpm for 60min. As a result, we first identified Penicillium polonicum and employed the method of solid-state fermentation using crambe seeds as a substrate to produce lipase. Our findings revealed heightened lipolytic activity (22.5 Ug-1) after 96h of fermentation using crambe cake as the substrate. Optimal results were achieved with crambe seeds at a granulometry of 0.6mm and a fermentation medium humidity of 60%. Additionally, electron microscopy suggested the immobilization of lipase in the substrate, enabling enzyme reuse for up to 4 cycles with 100% enzymatic activity. Subsequently, we conducted applicability tests of biocatalysts for ethyl oleate synthesis, optimizing parameters such as the acid/alcohol molar ratio, temperature, and reaction time. We attained 100% conversion within 30min at 37°C, and our results indicated that the molar ratio proportion did not significantly influence the outcome. These findings provide a methodological alternative for the utilization of biocatalysts in ethyl oleate synthesis.

Lipase-Catalyzed Preparation and Optimization of Structured Phosphatidylcholine Containing Nervonic Acid.

This study investigated the incorporation of nervonic acid into the chemical structure of phosphatidylcholine via a lipase-catalyzed acidolysis reaction to obtain a functional phospholipid. Lipase immobilization was conducted, and Amberlite XAD7-HP was selected as a carrier to immobilize phospholipase A1 (PLA1) for subsequent experiments. The main acidolysis reaction parameters, including enzyme load, substrate ratio, temperature, and water content, were studied against the reaction time. The optimum reaction conditions obtained were enzyme load, 20%; reaction temperature, 55 °C; water content, 1%; and reaction time, 9 h. The maximum incorporation of nervonic acid into phosphatidylcholine was 48 mol%, with PC recovery at 61.6 mol%. The positional distribution of structured phosphatidylcholine shows that nervonic acid was found in the sn-1 position due to enzyme specificity and in the sn-2 position, possibly due to acyl migration.

Reaction kinetics involved in esterification between the fatty acids in castor oil and furfuryl alcohol

Kinetics of esterification reaction between furfuryl alcohol (FA) and castor oil fatty acid (COFA) using immobilized Candida antartica lipase B in a solvent free system is investigated in present study. The well-known ping-pong bi bi mechanism with two-substrate inhibition has been confirmed to correctly describe the governing reaction pathway. Subsequently, the rate equation was used to evaluate pertinent kinetic parameters. Parametric optimization study was also carried out with respect to temperature, molar ratio of reactants and enzyme loading to maximize the equilibrium yield of COFA ester. Furthermore, functional variations of the kinetic parameters, which include Michaelis-Menten constants and inhibition coefficients, were evaluated with respect to three process parameters. Corresponding inferences are of high utility, specifically, in cases where the global optimum is beyond the scope of the existing production facility in any industrial platform.

Deciphering the immobilization of lipases on hydrophobic wrinkled silica nanoparticles

In this work, the adsorption of Candida antarctica B (CALB) and Rhizomucor miehei (RML) lipases into hydrophobic wrinkled silica nanoparticles (WSNs) is investigated. WSNs are hydrophobized by chemical vapor deposition. Both proteins are homogeneously distributed inside the pores of the nanoparticles, as confirmed by Transmission Electron Microscopy and Energy Dispersive X-ray measurements. The maximum enzyme load of CALB is twice that obtained for RML. Fourier Transform Infrared Spectroscopy confirms the preservation of the enzyme secondary structure after immobilization for both enzymes. Adsorption isotherms fit to a Langmuir model, resulting in a binding constant (KL) for RML 4.5-fold higher than that for CALB, indicating stronger binding for the former. Kinetic analysis reveals a positive correlation between enzyme load and RML activity unlike CALB where activity decreases along the enzyme load increases. Immobilization allows for enhancing the thermal stability of both lipases. Finally, CALB outperforms RML in the hydrolysis of ethyl-3-hydroxybutyrate. However, immobilized CALB yielded 20 % less 3-HBA than free lipase, while immobilized RML increases 3-fold the 3-HBA yield when compared with the free enzyme. The improved performance of immobilized RML can be explained due to the interfacial hyperactivation undergone by this lipase when immobilized on the superhydrophobic surface of WSNs.

Iron oxide immobilized lipase bioconjugate platform for sensing of triglycerides in biological samples

As a major biomarker in several health conditions, an efficient detection strategy to monitor triglyceride (TGs) level is imperative. In this study, enzyme-based triglyceride detection platform is reported by employing surface functionalized iron oxide nanoparticles (IONPs). The green synthesis method (using green tea extract) was adopted for the synthesis of IONPs exhibiting magnetic properties. The synthesized nanoparticle was characterized using SEM, FT-IR and UV–Vis spectroscopy. The SEM data confirmed the synthesis of spherical nanoparticles with an average size of 200 nm. Moreover, the additional coating of aspartate made the immobilization of biomolecule lipase on the IONPs more stable and was confirmed via the acquired data from SEM. The surface functionalization of IONPs using aspartic acid was confirmed by FT-IR spectra. Enzyme immobilization was performed by adding glutaraldehyde as the crosslinker, leading to formation of a nanoparticle-enzyme bioconjugate. The resultant bioconjugate was used for colorimetric estimation of triglyceride in biological samples using palmitic acid as the standard and methyl red as the indicator. The designed bioconjugate exhibited accuracy of 7.5 folds. The nanoparticle-enzyme bioconjugate platform renders a simple, economical, and easy-to-use biosensing approach with higher stability and activity when compared to its soluble counterpart.

Recent insight into the advances and prospects of microbial lipases and their potential applications in industry.

Enzymes play a crucial role in various industrial sectors. These biocatalysts not only ensure sustainability and safety but also enhance process efficiency through their unique specificity. Lipases possess versatility as biocatalysts and find utilization in diverse bioconversion reactions. Presently, microbial lipases are gaining significant focus owing to the rapid progress in enzyme technology and their widespread implementation in multiple industrial procedures. This updated review presents new knowledge about various origins of microbial lipases, such as fungi, bacteria, and yeast. It highlights both the traditional and modern purification methods, including precipitation and chromatographic separation, the immunopurification technique, the reversed micellar system, the aqueous two-phase system (ATPS), and aqueous two-phase flotation (ATPF), moreover, delves into the diverse applications of microbial lipases across several industries, such as food, vitamin esters, textile, detergent, biodiesel, and bioremediation. Furthermore, the present research unveils the obstacles encountered in employing lipase, the patterns observed in lipase engineering, and the application of CRISPR/Cas genome editing technology for altering the genes responsible for lipase production. Additionally, the immobilization of microorganisms' lipases onto various carriers also contributes to enhancing the effectiveness and efficiencies of lipases in terms of their catalytic activities. This is achieved by boosting their resilience to heat and ionic conditions (such as inorganic solvents, high-level pH, and temperature). The process also facilitates the ease of recycling them and enables a more concentrated deposition of the enzyme onto the supporting material. Consequently, these characteristics have demonstrated their suitability for application as biocatalysts in diverse industries.

Innovative approach for semi‐continuous production of puerarin palmitate via transesterification with novel immobilized lipase

BackgroundPuerarin, a flavonoid abundantly found in the roots of Pueraria lobata, exhibits antioxidant, anti-cancer, anti-inflammatory, and anti-aging properties. Despite its numerous benefits, the low bioavailability of puerarin hinders its widespread industrial application. To improve this, flavonoid acylation is required. Lipase is a biocatalyst primarily used for flavonoid acylation; immobilized lipase is preferred given the limited stability of free lipase. Although research on flavonoid acylation for structural modification is actively being conducted, there is a need to explore continuous production for enhancing the industrial application of flavonoid esters. Therefore, in this study, we aimed to produce puerarin palmitate innovatively by selecting an effective lipase, developing an immobilized lipase, characterizing the enzymatic reaction, and designing a semi-continuous reactor system.ResultsLipase from Thermomyces lanuginosus (TL) was found to be the most suitable for the synthesis of puerarin palmitate; it was successfully immobilized on glutaraldehyde-activated silica gel (GASG). TL GASG showed greater solvent, thermal, and operational stability than those of commercial immobilized lipases. Furthermore, TL GASG use resulted in a puerarin palmitate conversion rate of 97.17% within 3 h. Additionally, the reusability of TL GASG for this reaction was higher than that of commercially available immobilized lipases. Upon using a semi-continuous reactor, the cumulative conversion rate of puerarin palmitate remained above 85% after more than ten substrate injections.ConclusionsThe results support the feasibility of the continuous production of flavonoid esters, underscoring their diverse industrial applications. A biorefinery strategy is anticipated to be proposed to utilize P. lobata extract biomass for flavonoid ester production. Moreover, optimizing continuous reactors through statistical experimental design is expected to enhance flavonoid ester production efficiency; these aspects will be explored in future research.Graphical

Enhancing biocatalyst performance through immobilization of lipase (Eversa® Transform 2.0) on hybrid amine-epoxy core-shell magnetic nanoparticles

Magnetic nanoparticles were functionalized with polyethylenimine (PEI) and activated with epoxy. This support was used to immobilize Lipase (Eversa® Transform 2.0) (EVS), optimization using the Taguchi method. XRF, SEM, TEM, XRD, FTIR, TGA, and VSM performed the characterizations. The optimal conditions were immobilization yield (I.Y.) of 95.04 ± 0.79 %, time of 15 h, ionic load of 95 mM, protein load of 5 mg/g, and temperature of 25 °C. The maximum loading capacity was 25 mg/g, and its stability in 60 days of storage showed a negligible loss of only 9.53 % of its activity. The biocatalyst demonstrated better stability at varying temperatures than free EVS, maintaining 28 % of its activity at 70 °C. It was feasible to esterify free fatty acids (FFA) from babassu oil with the best reaction of 97.91 % and ten cycles having an efficiency above 50 %. The esterification of produced biolubricant was confirmed by NMR, and it displayed kinematic viscosity and density of 6.052 mm2/s and 0.832 g/cm3, respectively, at 40 °C. The in-silico study showed a binding affinity of −5.8 kcal/mol between EVS and oleic acid, suggesting a stable substrate-lipase combination suitable for esterification.

Calcium-based MOFs as scaffolds for shielding immobilized lipase and enhancing its stability

The enzyme immobilization technology has become a key tool in the field of enzyme applications; however, improving the activity recovery and stability of the immobilized enzymes is still challenging. Herein, we employed a magnetic carboxymethyl cellulose (MCMC) nanocomposite modified with ionic liquids (ILs) for covalent immobilization of lipase, and used Ca-based metal–organic frameworks (MOFs) as the support skeleton and protective layer for immobilized enzymes. The ILs contained long side chains (eight CH2 units), which not only enhanced the hydrophobicity of the carrier and its hydrophobic interaction with the enzymes, but also provided a certain buffering effect when the enzyme molecules were subjected to compression. Compared to free lipase, the obtained CaBPDC@PPL–IL–MCMC exhibited higher specific activity and enhanced stability. In addition, the biocatalyst could be easily separated using a magnetic field, which is beneficial for its reusability. After 10 cycles, the residual activity of CaBPDC@PPL–IL–MCMC could reach up to 86.9%. These features highlight the good application prospects of the present immobilization method.

Isocyanate-mediated immobilization of Candida antarctica lipase B onto resin for efficient 1,3-diacylglycerol bio-synthesis

In this work, isocyanate-based strategy was developed for efficient immobilization of Candida antarctica lipase B (CALB). Of 41 commercial resins, EP109 was chosen and hydrophobized with hydroxyl-terminated polydimethylsiloxane (HTPDMS) for tert-butyl isocyanate (tBIC)-based CALB immobilization due to high loading amount and specific activity. The tBIC-based strategy exhibited shorter immobilization time, higher loading and activity than adsorption strategy by hydrophobic interaction. When compared with free CALB, EP109-HTPDMS-tBIC@CALB owned improved stability (against acid, alkali, and heat) and storability. Subsequently, EP109-HTPDMS-tBIC@CALB was employed in the bio-synthesis of 1,3-diacylglycerol in a solvent-free system with a yield of 76.31%, which was higher than commercial Novozym 435 (53.93%). EP109-HTPDMS-tBIC@CALB exhibited 76.34% of residual activity after 20 repeat uses.

Preparation of Proline-Modified UIO−66 Nanomaterials and Investigation of Their Potential in Lipase Immobilization

Metal–organic frameworks (MOFs) are regarded as excellent carriers for immobilized enzymes due to their substantial specific surface area, high porosity, and easily tunable pore size. Nevertheless, the use of UIO−66 material is significantly limited in immobilized enzymes due to the absence of active functional groups on its surface. This study comprised the synthesis of UIO−66 and subsequent modification of the proline (Pro) on UIO−66 through post-synthetic modification. UIO−66 and UIO−66/Pro crystals were employed as matrices to immobilize Rhizopus oryzae lipase (ROL). The contact angle demonstrated that the introduction of Pro onto UIO−66 resulted in favorable conformational changes in the structure of ROL. The immobilized enzyme ROL@UIO−66/Pro, produced via the covalent-bonding method, exhibited greater activity (0.064715 U/mg (about 1.73 times that of the free enzyme)) and stability in the ester hydrolysis reaction. The immobilized enzymes ROL@UIO−66 (131.193 mM) and ROL@UIO−66/Pro (121.367 mM), which were synthesized using the covalent-bonding approach, exhibited a lower Km and higher substrate affinity compared to the immobilized enzyme ROL@UIO−66/Pro (24.033 mM) produced via the adsorption method. This lays a solid foundation for the industrialization of immobilized enzymes.

Octadecyl and sulfonyl modification of diatomite synergistically improved the immobilization efficiency of lipase and its application in the synthesis of pine sterol esters.

Phytosterols usually have to be esterified to various phytosterol esters to avoid their disadvantages of unsatisfactory solubility and low bioavailability. The enzymatic synthesis of phytosterol esters in a solvent-free system has advantages in terms of environmental friendliness, sustainability, and selectivity. However, the limitation of the low stability and recyclability of the lipase in the solvent-free system, which often requires a relatively high temperature to induce the viscosity, also increased the industrial production cost. In this context, a low-cost material, namely diatomite, was employed as the support in the immobilization of Candida rugosa lipase (CRL) due to its multiple modification sites. The Fe3 O4 was also then introduced to this system for quick and simple separation via the magnetic field. Moreover, to further enhance the immobilization efficiency of diatomite, a modification strategy which involved the octadecyl and sulfonyl group for regulating the hydrophobicity and interaction between the support and lipase was successfully developed. The optimization of the ratio of the modifiers suggested that the -SO3 H/C18 (1:1.5) performed best with an enzyme loading and enzyme activity of 84.8mg·g-1 and 54 U·g-1 , respectively. Compared with free CRL, the thermal and storage stability of CRL@OSMD was significantly improved, which lays the foundation for the catalytic synthesis of phytosterol esters in solvent-free systems. Fortunately, a yield of 95.0% was achieved after optimizing the reaction conditions, and a yield of 70.0% can still be maintained after six cycles.

Adsorption study of isotherms, kinetics modelling, and thermodynamics of immobilized Candida rugosa lipase on metal oxide for esterification of waste cooking oil

The problem regarding improper disposal of waste cooking oil (WCO) can be solved via biotransformation of WCO to value added product using lipases as a biocatalyst. Thus, immobilization method was proposed for the recyclability of the biocatalyst. However, a comprehensive discussion on adsorption study is limited for lipase immobilization. In this study, Candida rugosa lipase (CRL) was immobilized on modified metal oxide (Mg2+/Al2O3) and the support was improved by addition of activating agent (L-lysine/Mg2+/Al2O3). Subsequently, the biocatalysts were used for esterification of WCO in the presence of methanol. Maximum adsorption capacity for the support was improved after addition of L-lysine resulting in 0.262 mg protein/g of support. The equilibrium adsorption data for both systems were fitted to the Redlich Peterson isotherm model while the kinetics study was most fitted with Elovich model, and the adsorption process were influenced by intraparticle and film diffusion mechanisms. Thermodynamic analysis explained that the adsorption process of CRL/L-lysine/Mg2+/Al2O3 was more spontaneous and stronger as compared to CRL/Mg2+/Al2O3. Furthermore, CRL/L-Lysine/Mg2+/Al2O3 exhibited its potential in esterification of WCO by converting 62.95% of free fatty acid (FFA), while CRL/Mg2+/Al2O3 converted lower FFA (53.62%). This study offers a fundamental understanding on designing an effective immobilization system by addition of activating agent to develop a good biocatalyst for biotransformation of WCO.

Green synthesis of iron-alginate nanoparticle for Taguchi-assisted immobilization of Candida rugosa lipase and its application in the synthesis of butyl butyrate ester

The synthesis of butyl butyrate, medium chain flavored esters by green chemistry is of immense importance to the food industry. In the present work, green synthesis of iron-alginate nanogels is done using a water-in-oil microemulsion method using food-grade oil for immobilization of Candida rugosa lipase (CrL). The morphological characterization of Fe-Alg nanogels was accomplished using dynamic light scattering (DLS) and scanning electron microscopy (SEM). Elongated tube-shaped Fe-Alg showed a hydrodynamic diameter of 100 ± 2 nm and a zeta potential of − 28 ± 4 mV. The nanogels exhibit an immobilization efficiency of 98%. Taguchi design-assisted immobilization of CrL onto Fe-Alg was effective in predicting the maximum response against immobilization parameters (0.7 µgm/ml of substrate, 20 min immobilization, 53⁰ C and pH 8.5). The esterification of butyl butyrate (∼90%) was completed in 6 h, which is confirmed by gas chromatography. The application of food-grade oil in the microemulsion method (water-in-oil) of nanogel synthesis is an attractive improvement for the development of support matrix in enzyme immobilization and ester synthesis.

Potential Application of Bacterial Lipase from Bacillus subtilis in the Production of Biodisel

This work aims to evaluate the potential of bacterial lipase of Bacillus subtilis in biodiesel production. Assays were performed to obtain, purify, and immobilize the enzyme and compare chemical and enzymatic transesterification tests. The Tryptic Soy Broth (TSB) culture medium without supplementation showed the highest enzymatic activity and purification yields when using 60% of (NH4)2SO4. Citral was the most efficient agent for lipase immobilization. Even though the free enzymes showed a higher activity rate, the immobilized enzymes are associated with a better-quality product. In conclusion, the lipase enzyme showed positive potential in biodiesel production.

Covalent Immobilization of Aspergillus niger Lipase on Epoxy-Activated Silver Oxide Nanoparticles-Impregnated Chitosan Surfaces: Characterization, Stability & Kinetic Studies

Covalent Immobilization of Aspergillus niger Lipase on Epoxy-Activated Silver Oxide Nanoparticles-Impregnated Chitosan Surfaces: Characterization, Stability & Kinetic Studies

Experimental and Computational Analysis of Synthesis Conditions of Hybrid Nanoflowers for Lipase Immobilization.

This work presents a framework for evaluating hybrid nanoflowers using Burkholderia cepacia lipase. It was expanded on previous findings by testing lipase hybrid nanoflowers (hNF-lipase) formation over a wide range of pH values (5-9) and buffer concentrations (10-100 mM). The free enzyme activity was compared with that of hNF-lipase. The analysis, performed by molecular docking, described the effect of lipase interaction with copper ions. The morphological characterization of hNF-lipase was performed using scanning electron microscopy. Fourier Transform Infrared Spectroscopy performed the physical-chemical characterization. The results show that all hNF-lipase activity presented values higher than that of the free enzyme. Activity is higher at pH 7.4 and has the highest buffer concentration of 100 mM. Molecular docking analysis has been used to understand the effect of enzyme protonation on hNF-lipase formation and identify the main the main binding sites of the enzyme with copper ions. The hNF-lipase nanostructures show the shape of flowers in their micrographs from pH 6 to 8. The spectra of the nanoflowers present peaks typical of the amide regions I and II, current in lipase, and areas with P-O vibrations, confirming the presence of the phosphate group. Therefore, hNF-lipase is an efficient biocatalyst with increased catalytic activity, good nanostructure formation, and improved stability.