Native Lipase Research Articles

Implementation of Spore Display in Paenibacillus polymyxa with Different Hydrolytic Enzymes.

Biotechnological processes are essential for producing climate-friendly high-value chemicals or pharmaceutical compounds, which can include steps catalyzed by enzymes. Therefore, establishing new, robust, and cheap enzyme production processes is desirable. One possible way to enhance processes is through the use of the spore display method. Spore display can present heterologous proteins on the surface of bacterial spores, offering numerous advantages in a range of biotechnological applications. This study demonstrates the implementation of the spore display method in Paenibacillus polymyxa, achieved by modifying the spore surface, incorporating an anchoring protein, and attaching green fluorescent protein to it, allowing the visualization of fluorescent spores. Following the initial experiment, a native lipase (Lip3), a heterologous lipase (LipA) from Bacillus subtilis, a native esterase (PnbA) from P. polymyxa, and a lipoyl synthase were expressed during sporulation and displayed on the spore surface. The activity profiles were determined in the temperature range from 4 °C to 70 °C. The PnbA reached its optimum at 4 °C, whereas the LipA from B. subtilis showed 4.4-fold higher activity at 42 °C compared to the control. Furthermore, we explored a possible new technique for the purification of enzymes with the TEV cleavage site between the anchor and the protein of interest. Finally, we showed a not-yet-described side activity of the lipoyl synthase over a wide temperature range.

A Synergetic Pore Compartmentalization and Hydrophobization Strategy for Synchronously Boosting the Stability and Activity of Enzyme.

Spatial immobilization of fragile enzymes using a nanocarrier is an efficient means to design heterogeneous biocatalysts, presenting superior stability and recyclability to pristine enzymes. An immobilized enzyme, however, usually compromises its catalytic activity because of inevasible mass transfer issues and the unfavorable conformation changes in a confined environment. Here, we describe a synergetic metal-organic framework pore-engineering strategy to trap lipase (an important hydrolase), which confers lipase-boosted stability and activity simultaneously. The hierarchically porous NU-1003, featuring interconnected mesopore and micropore channels, is precisely modified by chain-adjustable fatty acids on its mesopore channel, into which lipase is trapped. The interconnected pore structure ensures efficient communication between trapped lipase and exterior media, while the fatty acid-mediated hydrophobic pore can activate the opening conformation of lipase by interfacial interaction. Such dual pore compartmentalization and hydrophobization activation effects render the catalytic center of trapped lipase highly accessible, resulting in 1.57-fold and 2.46-fold activities as native lipase on ester hydrolysis and enantioselective catalysis. In addition, the feasibility of these heterogeneous biocatalysts for kinetic resolution of enantiomer is also validated, showing much higher efficiency than native lipase.

A silver cluster-assembled material as a matrix for enzyme immobilization towards a highly efficient biocatalyst.

Silver cluster-assembled materials (SCAMs) epitomize well-defined extended crystalline frameworks that combine the ingenious designability at the atomic/molecular level and high structural robustness. They have captivated the interest of the scientific fraternity because of their modular construction which enables to systematically tailor their functions, and their capacity to not only inherit the characteristics of component building units but also introduce their uniqueness in endowing the final material with extraordinary properties. Herein, we demonstrate the synthesis of a novel (3,6)-connected two-dimensional (2D) SCAM [Ag12(StBu)6(CF3COO)6(THIT)6]n (described as TUS 5, THIT = 2,4,6-tri(1H-imidazol-1-yl)-1,3,5-triazine) composed of Ag12 cluster nodes and tritopic imidazolyl linkers. We have leveraged, for the first time, this precisely architected extended SCAM structure as a support matrix for enzyme immobilization. The electrostatic attraction between the negatively charged amano lipase PS and positively charged TUS 5 as well as the surface hydrophobicity of TUS 5 catered to great binding of lipase onto the TUS 5 matrix, in addition to boosting the activity of lipase via interfacial activation. Capitalizing on the cooperative benefits of organic and inorganic support matrices wherein organic supports impart with cost-efficiency, biocompatibility, and improved enzyme stability and reusability and inorganic supports confer high thermal, mechanical and microbial resistance, we have utilized the immobilized lipase on TUS 5 SCAM (lipase@TUS 5) for the kinetic resolution of (R,S)-1-phenylethanol by transesterification reaction. Importantly, lipase@TUS 5 could attain appreciably higher conversion into (R)-1-phenylethyl acetate, besides featuring superior thermal stability, solvent tolerance and recyclability, over the native lipase.

Enhanced Esterification Activity and Thermostability of Imprinted Poly(Ethylene Glycol)-Lipase Complex

Although the range of applications for enzymatic reactions in organic solvents is <br /> rapidly expanding, this study focused on the enzymatic activity in the esterification of <br /> lauric acid with benzyl alcohol, and thermostability of lipase using poly(ethylene glycol) <br /> (PEG)-lipase complex and molecular imprinting techniques. The catalytic activity was <br /> enhanced through molecular imprinting and the PEG-lipase complex. The imprinting operation was particularly effective for catalytic activity after forming the PEG-lipase complex. The kinetic analysis of the lipase-catalyzed esterification revealed that the increase in esterification rate with imprinted lipases was mainly due to the higher maximum rate achieved by the system. The thermostability of the lipases was significantly improved by imprinting at all temperatures (50~70 °C). After forming a PEG-lipase complex, the imprinted lipase exhibited much higher reactivity and thermostability compared to the native lipase and the imprinted PEG-lipase complex.

In Silico and Experimental Studies on the Effect of α3 and α5 Deletion on the Biochemical Properties of Bacillus thermocatenulatus Lipase.

To investigate the effect of α3 and α5 helices on the biochemical characterization of Bacillus thermocatenulatus lipase (BTL2), both helices were deleted from native BTL2 lipase. After structural modeling and characterization, the truncated btl2 gene (Δbtl2) was cloned into E. coli BL21 under the control of the T7 promoter. After cultivation and induction of the recombinant bacteria, the Δα3α5 lipase was purified by Ni-NTA column chromatography. Next, the biochemical properties of the Δα3α5 lipase were compared with the previously expressed and purified native lipase. In the presence of the substrate tributyrin (C4), the maximum activity of native and Δα3α5 lipase was 9360 and 5000 U/mg, respectively. The deletion changed the substrate specificity from tributyrin (C4) to tricaprylin (C8) substrate. Native and Δα3α5 lipase showed similar activity patterns at all temperatures and pH values, with the activity of Δα3α5 lipase being approximately 20% lower than native lipase. Triton X100 increased the activity of native and Δα3α5 lipases by 2.1- and 2.5-fold, respectively.

Copper Phthalocyanine Improving Nonaqueous Catalysis of Pseudomonas cepacia Lipase for Ester Synthesis.

The nonaqueous catalysis of lipases is significant for synthesis of high pure esters, but they usually behave low catalytic activity due to denaturation and aggregation of enzyme protein in organic phases. To improve the nonaqueous catalysis, the inexpensive copper phthalocyanine was taken as a new carrier on which Pseudomonas cepacia lipase was immobilized by physical absorption, and used for synthesis of hexyl acetate, an important flavor, via transesterification of hexanol and vinyl acetate. Results showed that the desired loading was 10-mg lipase immobilized on 10-mg copper phthalocyanine powder. When the immobilized lipase was employed in the reaction system consisted of 1.5-mL hexanol and 1.5-mL vinyl acetate at 37°C and 160 rpm, the conversion was fivefolds of that catalyzed by native lipase after 1 h, and reached 99.0% after 8 h. In six times of 8-h reuses, the immobilized lipase behaved an activity attenuation rate 1.22% h-1, lower than 1.77% h-1 of native lipase, which meant that the immobilized lipase was more stable. Even at the room temperature and the static state without shaking or stirring, the immobilized lipase still brought conversion 42.8% after 10 h and the native lipase gave 20.1%. Obviously, the immobilized lipase is an available biocatalyst in organic phase and has great potential in food industry.

Unfolded Lipase at Interfaces Studied via Interfacial Dilational Rheology: The Impact of Urea

Unfolding can interrupt the activity of enzymes. Lipase, the enzyme responsible for triglyceride catalysis, can be deactivated by unfolding, which can significantly affect the yield of enzymatic processes in biochemical engineering. Different agents can induce lipase unfolding, among which we study the impact of urea as a strong denaturant. Unfolding weakens the rigidity and stability of globular proteins, thereby changing the viscoelastic properties of the protein adsorbed layers. These changes can be detected and quantified using interfacial dilational rheology. The urea-induced unfolding of lipase destructs its globular structure, making it more flexible. The interfacial tension and viscoelastic moduli of lipase adsorbed layers reduce upon the addition of urea in the range of studied concentrations. The results agree with the theory that, upon unfolding, a distal region of the loop and tail domain forms adjacent to the proximal region of the interface. The exchange of matter between these regions reduces the viscoelasticity of the unfolded lipase adsorbed layers. Additionally, unfolding reduces the rigidity and brittleness of the lipase adsorbed layers: the aged adsorbed layer of native lipase can break upon high-amplitude perturbations of the interfacial area, unlike the case for urea-induced unfolded lipase.

Copper Phthalocyanine Improving Nonaqueous Catalysis of Pseudomonas cepaciaLipase for Ester Synthesis.

The nonaqueous catalysis of lipases is significant for synthesis of high pure esters, but they usually behave low catalytic activity due to denaturation and aggregation of enzyme protein in organic phases. To improve the nonaqueous catalysis, the inexpensive copper phthalocyanine was taken as a new carrier on which Pseudomonas cepacia lipase was immobilized by physical absorption, and used for synthesis of hexyl acetate, an important flavor, via transesterification of hexanol and vinyl acetate. Results showed that the desired loading was 10mg lipase immobilized on 10mg copper phthalocyanine powder. When the immobilized lipase was employed in the reaction system consisted of 1.5 mL hexanol and 1.5 mL vinyl acetate at 37℃ and 160rpm, the conversion was five fold of that catalyzed by native lipase after 1h, and reached 99.0% after 8h. Undergoing six times of 8-h reuses, the immobilized lipase had an activity attenuation rate 1.22% h- 1, lower than 1.77% h- 1 of native lipase, which meant that the immobilized lipase was more stable. Even at the room temperature and the static state without shaking or stirring, the immobilized lipase could bring conversion 42.8% after 10h and the native lipase gave 20.1%. Obviously, the immobilized lipase is an available biocatalyst in organic phase and has great potential in food industry.

Improvement of enzymatic activity and stability of lipase A from Candida antartica onto halloysite nanotubes with Taguchi method for optimized immobilization

This research reports the immobilization by ion adsorption and interfacial activation of lipase A from Candida antarctica (CALA) in halloysite nanotubes (Hal) was optimized by the Taguchi method. Under optimized conditions (pH 5 to 5 mM, 5 °C and 4 h), it was possible to obtain an immobilization yield (IY) of 97.1 ± 0.10% with a mass activity (AtD) of 83.81 ± 0 0.50 U/g for the hydrolysis of p-nitrophenyl butyrate. pH and ionic strength were the parameters that most positively influenced the optimization of the enzymatic immobilization process, favoring the activity of the catalytic derivative. At pH 7, CALA-Hal exhibited a half-life 2–8 times longer than CALA at 50–90 °C. CALA showed maximum activity at pH 7.Especially at pH 9, CALA-Hal was more active than the native lipase at the pHs under study, expect at pH 7. CALA was partially desorbed from HNT after 2 h of incubation in 4% Triton X-100 and fully desorbed after 1 h of incubation in 1.0 M NaCl. Hal and CALA-Hal were characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy, Scanning Electron Microscope (SEM), elemental analysis (CHNS), Thermogravimetry (TG) and Differential Scanning Calorimetry (DSC), showing the immobilization of CALA on HNT and the maintenance of the nanotubes structure after immobilization. Therefore, the strategy employed in this study to immobilize CALA onto Hal allowed us to obtain promising results. Biocatalysts showed potential for use in diverse reactions of industrial interest.

PLANEJAMENTO FATORIAL NA SÍNTESE DO OLEATO DE ETILA CATALISADA PELA LIPASE NATIVA DE Aspergillus niger

FACTORIAL DESIGN FOR THE SYNTHESIS OF ETHYL OLEATE CATALYZED BY NATIVE LIPASE Aspergillus niger. This work reports the use of the Octave, a free computational software, to optimize the experimental design in the synthesis of ethyl oleate from oleic acid and ethanol catalyzed by a native lipase from Aspergillus niger. The central composite factorial design (CCD) was performed with 2 levels and 3 variables (temperature, mass of native A. niger lipase and oleic acid:ethanol molar ratio) in conjunction with response surface methodology (RSM). The results indicate that variable 1 (oleic acid:ethanol molar ratio; X1) and 2 (mass of A. niger lipase, X2), as well as the interaction between them, are statistically significant. The optimal reaction condition was estimated using ANOVA data and the response surface. The experimental results are consistent with those estimated, and thus, ethyl oleate was obtained with conversion (> 90%) using oleic acid and ethanol in a 1:2 molar ratio, A. niger lipase (8,00 mg) and temperature of 45 °C in 6 hours of reaction.

Partial purification, characterization and immobilization of a novel lipase from a native isolate of Lactobacillus fermentum.

Background and Objectives:Due to the widespread use of lipase enzymes in various industries, finding native lipase producing microorganisms is of great value and importance. In this study, screening of lipase-producing lactobacilli from native dairy products was performed.Materials and Methods:Qualitative evaluation of lipolytic activity of lipase-producing lactobacilli was performed in different media containing olive oil. A clear zone observation around the colonies indicated the lipolytic activity. The strain with the highest enzymatic activity was identified. Determination of optimal pH and temperature of lipase activity was measured by spectrophotometry using p-nitrophenyl acetate (ρ-NPA) substrate. Partial purification of lipase enzyme was performed using 20–90% saturation ammonium sulfate. Eventually, lipase was immobilized by physical adsorption on chitosan beads.Results:Among screened lipolytic bacterial strains, one sample (5c isolate) which showed the highest enzymatic activity (5329.18 U/ml) was close to Lactobacillus fermentum. During characterization, the enzyme showed maximum activity in Tris-HCl buffer with pH 7, while remaining active over a temperature range of 5°C to 40°C. The results of the quantitative assay demonstrated that the fraction precipitated in ammonium sulfate at 20% saturation has the highest amount of lipolytic activity, with a specific activity of 22.0425 ± 3.6 U/mg. Purification folds and yields were calculated as 8.73 and 44%, respectively. Eventually, the enzyme was immobilized by physical adsorption on chitosan beads with a yield of 56.21%.Conclusion:The high efficiency of enzyme immobilization on chitosan beads indicates the suitability of this method for long-term storage of new lipase from native 5c isolate.

Rice straw enhancing catalysis of Pseudomonas fluorescens lipase for synthesis of citronellyl acetate.

Citronellyl acetate as an important flavor, can be effectively synthesized by lipase catalysis in nonaqueous system. But lipases usually behave low catalytic activity due to aggregation and denaturation of them in organic phase. To enhance the nonaqueous catalysis, based on the mechanism of lipases activated at water/oil (organic phase) interface, the inexpensive race straw was processed into powder and filaments on which Pseudomonas fluorescens lipase was immobilized by physical adsorption, used for synthesis of citronellyl acetate via transesterification of citronellol and vinyl acetate. Results showed that the desired loading was 10mg lipase immobilized on 30mg rice straw filaments or 25mg rice straw powder. When the two immobilized lipases were employed in the reaction system consisted of 1-mL citronellol and 2-mL vinyl acetate at 37℃ and 160rpm, the conversions all reached 99.8% after 12h. Under the reaction condition, the conversion catalyzed by 10mg native lipase was 85.1%. Undergoing six times of 8-h reuses in the organic system, the filament and power immobilized lipases had weak activity attenuation rates 0.36 and 0.32% h-1, lower than 1.52% h-1 of native lipase. Even at the room temperature and the static state without shaking and stirring, the rice straw filaments immobilized lipase could brought conversion 62.9% after 10h but the native lipase only gave 37.0%. Obviously, the rice straw, especially its filaments, is an inexpensive and available natural material to prepare immobilized lipase with desired catalysis in organic phase, meant significant potential in flavor industry.

Melia azedarach leaf powder stabilizing Pseudomonas fluorescens lipase to catalyze synthesis of geranyl acetate

To improve the stability of lipases in organic phases and promote the enzymatic synthesis, the inexpensive Melia azedarach leaf powder was taken as carrier for lipase immobilization, which was considered as solid “water” that could stabilize enzyme proteins. On the leaf powder, Pseudomonas fluorescens lipase was immobilized by physic adsorption in a column glass bottle and used for synthesis geranyl acetate via transesterification of geraniol and vinyl acetate. It is found that the immobilized lipase prepared in a mass ratio of 10 : 25 (enzyme: leaf powder, mg: mg) not only has a fine catalytic activation but also has an excellent nonaqueous stability in solvent-free nonaqueous reaction system composed of 1 mL geraniol and 2 mL vinyl acetate. Namely, the best loading of this immobilized lipase was 11.7 mg mL −1 in the reaction system in which, at 37°C and 160 rpm, the reaction conversion reached 90.1% after 10 h and achieved at 99.3% after 16 h. And after undergoing six times of 10-h reuses, the immobilized lipase had no decrease in the catalytic ability of converting geraniol except a little fluctuation 0.09% h −1 in conversion during reuse. Differently, the native lipase showed an activity attenuation 0.61% h −1 in the organic system in the reuse. Obviously, this immobilized lipase has been significantly improved in the nonaqueous stability, meant great potential in practical application. • Melia azedarach leaf powder is a useful carrier for lipase immobilization. • Immobilized Pseudomonas fluorescens lipase is desired to prepare citronellyl acetate. • Reuses show that the immobilized lipase has little decrease in nonaqueous activity. • The bottle with immobilized lipase is a simple bioreactor with great potential.

Efficient biodegradation of aliphatic polyester by genetically engineered strains of the yeast Yarrowia lipolytica

The increased production and consumption of plastic materials in today's world has caused a huge number of environmental problems, the solution of which is now crucial to improving the state of nature of our planet. In this study, we expressed cutinases from Fusarium solani f. sp. pisi and from Trichoderma reesei, individually or in combination with overexpression of the native lipase Lip2 in the yeast Yarrowia lipolytica. The engineered strains showed biodegradation activity toward polyester in the pH range between 4.0 and 9.0. The highest esterase activity was observed at pH 9.0 for the strain overexpressing cutinase from F. solani and lipase from Y. lipolytica, it reached 63.5 U ml−1, moreover this strain was capable to degrade 0.5 g of polycaprolactone film within 144 h of shake flask culture. The obtained results showed that natural capability of Y. lipolytica combined with metabolic engineering results in a highly efficient biodegradation process.

Heterologous expression, kinetic characterization and molecular modeling of a new sn-1,3-regioselective triacylglycerol lipase from Serratia sp. W3

A recombinant Serratia sp. W3 lipase (rSmL) was expressed in E. coli and purified to homogeneity. The kinetic properties, regio-selectivity, and interfacial performances of rSmL were compared with those of the native lipase (nSmL). rSmL has a molecular mass of 67 kDa and a specific activity of 3530 U mg−1 which is around 12-fold higher than that of nSmL (300 U mg−1) when using olive oil as the substrate. Both rSmL and nSmL were able to hydrolyze the ester bond at the sn-1 and sn-3 positions but exhibited a clear regio-preference towards the sn-3 position of the surface-coated triglycerides (TG), which were esterified with α-eleostearic acid at the sn-1/3 position or dicaprin isomers spread as monomolecular films. Molecular modeling and docking of TG into the active site of rSmL indicated that this regio-preference may be due to steric hindrance, created by the residues Ile308 and Trp311, with distances between the sn-1 reactive carbon and the catalytic Serine residue ranging from 3.15 to 5.47 Å. In contrast, the sn-3 positioning within the enzyme catalytic pocket resulted in shorter distances, ranging from 2.79 to 4.15 Å, therefore facilitating the hydrolysis at the sn-3 position.

Understanding thermal and organic solvent stability of thermoalkalophilic lipases: insights from computational predictions and experiments.

Bacillus thermocatenulatus lipase (BTL2), a member of the isolated lipase family known as thermoalkalophilic lipases, carries potential for industrial applications owing to its ability to catalyze versatile reactions under extreme conditions. This study investigates the molecular effects of distinct solvents on the stability of BTL2 at different temperatures, aiming to contribute to lipase use in industrial applications. Initially, molecular dynamic (MD) simulations were carried out to address for the molecular impacts of distinct solvents on the structural stability of BTL2 at different temperatures. Two lipase conformations representing the active and inactive forms were simulated in 5 solvents including water, ethanol, methanol, cyclohexane, and toluene. Low temperature simulations showed that polar solvents led to enhanced lid fluctuations compared with non-polar solvents reflecting a more dynamic equilibrium between active and inactive lipase conformations in polar solvents including water, while the overall structure of the lipase in both forms became more rigid in non-polar solvents than they were in polar solvent. Notably, the native lipase fold was maintained in non-polar solvents even at high temperatures, indicating an enhancement of lipase's thermostability in non-polar organic solvents. Next, we conducted experiments for which BTL2 was expressed in a heterologous host and purified to homogeneity, and its thermostability in different solvents was assessed. Parallel to the computational findings, experimental results suggested that non-polar organic solvents contributed to BTL2's thermostability at concentrations as high as 70% (v/v). Altogether, this study provides beneficial insights to the lipase use under extreme conditions. Graphical Abstract.

A simple room temperature-static bioreactor for effective synthesis of hexyl acetate

AbstractFor green synthesis in organic phases, the catalysis of lipases needs to be further improved. Here a strategy is to simulate the activation of lipases at water/oil interface by immobilizing lipase on an available carrier material, which can replace the water at the interface to stabilize enzyme against denaturation from organic phase. The carrier is selected as cotton fiber on whichPseudomonas cepacialipase is immobilized by physical adsorption in a column glass bottle to form a simple bioreactor together with the bottle. In synthesis of fragrance ester hexyl acetate via transesterification of hexanol with vinyl acetate, the bioreactor exhibits a 3-fold increase in the ability to transform substrate, relative to native lipase in terms of the initial period of reaction at 37°C and 160 rpm. And also the bioreactor is very stable in catalysis for that it has an extra long half lifet1/2= 636 h, calculated from the decrease degree of molar conversions in six times of 6-hour transesterifications. More interestingly, the bioreactor behaves excellent activity at room temperature and in a static state, and can transform nearly 100% hexanol after 48 h. All these indicate that the bioreactor has great potential for industrial application.

Surface residues serine 69 and arginine 194 of metagenome-derived lipase influence catalytic activity

A lipase gene was identified and isolated from a fosmid metagenomics library constructed from forest topsoil samples. Error prone PCR and saturation mutagenesis were used to generate seven lipase variants: S69 P/R194Q, S69 G, S69 P, S69E, S69 P/T99S/N190S/R194Q, R194S, and R194A; by testing five different fatty acids of p-nitrophenyl (p-NP) ester, positions S69 and R194 were found to influence the catalytic activity. Specifically, variant R194S was identified that had 5.0, 4.5, 2.1, 2.2, and 1.4-fold higher catalytic efficiency (vmax/Km) towards p-NP acetate, p-NP butyrate, p-NP octanoate, p-NP dodecanoate, and p-NP palmitate, respectively, compared to the metagenome-derived native lipase. Among the S69 variants, variants S69 P/R194Q and S69 P hydrolyzed p-NP acetate 1.8- and 1.5-fold faster than wild-type in terms of apparent vmax/Km values, respectively. The activity is lowered by introducing mutations S69E, R194A, and S69 P/T99S/N190S/R194Q. Here, two additional residues, T99 and N190, were also identified that may have important roles in catalysis. Present work confirms the advantages of combining directed evolution and saturation mutagenesis approaches of protein engineering and expands the knowledge on the architecture of the industrially important lipase enzyme and its relation to its catalytic activity.

Two novel lipases purified from rice bran displaying lipolytic and esterification activities

In this study, two novel lipases, rice bran lipase 1 (RBL1) and rice bran lipase 2 (RBL2), were first identified in rice (Oryza sativa) bran. Through the purification by ammonium sulfate precipitation, ion-exchange chromatography and size-exclusion chromatography, RBL1 and RBL2 were purified to 36- and 339-fold with the final specific activity of 4.73 and 44.06 U/mg, respectively. The purified RBL1 and RBL2 had the molecular weight of 18.8 and 35.5 kDa, respectively. The Km values of RBL1 and RBL2 were 0.766 and 0.432 mM while catalytic efficiency (kcat/Km) values of RBL1 and RBL2 were 102.4 and 2559.3 s−1/mM, respectively. RBL1 and RBL2 both showed esterification activity, but had no transesterification activity. In a solvent-free system, RBL1 and RBL2 exhibited degree of esterification (ED) of 4.4% and 6.5%, respectively.These two novel lipases exerted great properties for their potentials in industrial applications. First, RBL1 and RBL2 showed both mild reaction pH of 7.0 and temperature of 35 °C and 50 °C, respectively. Secondly, they showed great tolerance to several organic solvents and detergents while RBL1 exhibited great pH stability across a very broad range of pH (pH 3–12). Lastly and most importantly, RBL1 and RBL2 both exhibited esterification activity better than a commercial lipase, Candida rugosa lipase (CRL), in a solvent-free system. In conclusion, two novel lipases, RBL1 and RBL2, are different from published native lipases in rice bran and may be alternative potential candidates of biocatalysts contributing to the development of diverse industrial application fields.

Burkholderia cepacia lipase immobilization for hydrolytic reactions and the kinetic resolution of the non-equimolar mixtures of isomeric alcohols

The major drawbacks of native lipase applications in processes occurring in water or in organic solvents include: difficulties in catalyst recycling, low activity and operational instability. The immobilization of Burkholderia cepacia lipase by adsorption or covalent binding onto 5 differently functionalized carriers (silica, acrylic, cellulose-based) was performed to overcome this problem. The optimization of the reaction preparation in water-rich media was based on the hydrolytic reactivity of the preparations, as well as the thermal, operational and storage stabilities. Aminated silica carrier, activated with glutaraldehyde, was determined to be the carrier of choice. Regarding processes in water-restricted media, carrier selection was based on reactivity after drying and five preparations were chosen for the resolution of a non-equimolar isomer mixture (85:15 ratio of R to S isomers), treating the kinetic resolution of ((+)-(S/R)-1-[(1S,5R)-6,6-dimethylbicyclo[3.1.0]hex-2-en-2-yl)]ethanol as a model. The resulting acetate of R configuration exhibits interesting sensory properties. The operational stability of the chosen catalysts was tested over 15 consecutive batch processes; the most beneficial results were obtained with lipase adsorbed on an acrylic carrier. Conversion increased gradually from 10 to 84% over the first five processes, which could be explained by the product sorption onto the carrier. Full kinetic resolution with maximal substrate conversion (approximately 84%) was achieved and remained stable during the next 10 runs, an excellent result, and thus, the proposed system might be regarded as an exceptionally attractive solution for the perfume and cosmetic industries.