Lipase Biocatalyst Research Articles

English

The use of fungal biomass as a lipase biocatalyst represents an attractive approach for the treatments of oil wastewater as well as for the production of biodiesel from oil and residual grease, due to its greater stability, possibility of reuse, and lower cost. In this work, 20 filamentous fungi were isolated from the grease trap scum of a restaurant at the Federal University of Espírito Santo, Brazil. The fungi included those belonging to the genera Aspergillus, Beauveria, Botrytis, Cladosporium, Colletotrichum, Fusarium, Geotrichum, Penicillium, Rhizomucor, and Verticillium. Fungal lipase activity and biomass production were quantified. Lipase activity ranged from 0.13 U mg-1 protein of Rhizomucor sp. ECGF18 to 18.06 U mg-1 protein of Penicillium sp. ECGF02, and the biomass production ranged from 7.61 mg mL-1 for Cladosporium sp. ECGF19 to 12.68 mg mL-1 for Rhizomucor sp. ECGF18. In the sequence, Penicillium sp. ECGF02 and Rhizomucor sp. ECGF18, were previously select and, further evaluated in solid-state fermentation. Results confirmed the high extracellular lipase-activity of Penicillium sp. ECG02 and the high intracellular lipase activity of Rhizomucor sp. ECG18. Rhizomucor sp. ECG18 showed potential for use in future research, in the form of whole-cell lipases, wastewater treatment, and as a biocatalyst in the production of biodiesel from oil residues.   Key words: Lipase-producing fungi, wastewater treatment, bioenergy.

Probing the roles of two tryptophans surrounding the unique zinc coordination site in lipase family I.5.

A unique zinc domain found in all of the identified members of the lipase family I.5 is surrounded by two conserved tryptophans (W61 and W212). In this study, we investigated the role of these hydrophobic residues in thermostability and thermoactivity of the lipase from Bacillus thermocatenulatus (BTL2) taken as the representative of the family. Circular dichroism spectroscopy revealed that the secondary structure of BTL2 is conserved by the tryptophan mutations (W61A, W212A, and W61A/W212A), and that W61 is located in a more rigid and less solvent exposed region than is W212. Thermal denaturation and optimal activity analyses pointed out that zinc induces thermostability and thermoactivity of BTL2, in which both tryptophans W61 and W212 play contributing roles. Molecular explanations describing the roles of these tryptophans were pursued by X-ray crystallography of the open form of the W61A mutant and molecular dynamics simulations which highlighted a critical function for W212 in zinc binding to the coordination site. This study reflects the potential use of hydrophobic amino acids in vicinity of metal coordination sites in lipase biocatalysts design.

Immobilization of Alcaligenes sp. lipase as catalyst for the transesterification of vegetable oils to produce biodiesel

The work refers to the immobilization of the Alcaligenes sp. lipase for its use in the synthesis of biodiesel from canola oil, which has never been reported before. The enzyme was immobilized on different supports: polyethyleneimine, agarose, glutaraldehyde agarose, octyl agarose, glyoxyl agarose, Sepabeads® and also by aggregation and crosslinking to produced enzyme aggregates (CLEAs), so to consider different catalyst options for the reaction under study. The lipase biocatalysts were evaluated and compared based on their specific hydrolytic activities, immobilization yields and thermal stabilities under non-reactive and reactive (in the presence of oil and methanol) conditions. The lipase immobilized on Sepabeads® Ec-BU and CLEAs were selected to catalyze the methanolysis of canola oil. The synthesis of methyl esters from vegetable oil was performed at 40°C, using oil:methanol molar ratios of 1/3 and 1/4, and also using a six-step addition of methanol to the reaction mixture (oil/methanol molar ratio of 1/6) in order to decrease its inhibitory effect. At oil:methanol molar ratios of 1/3 and 1/4 a maximum conversion yield of 70% was obtained after 30h of reaction; on the other hand, using the six-step addition of methanol the maximum conversion yield obtained was of 80% in only 10h of reaction, demonstrating that the inhibitory effect of methanol was significantly reduced. The operational stability of the catalysts was assessed in three sequential batches of 10h each, obtaining a yield decay of 13% and 3,2% for lipase immobilized on Sepabeads® Ec-BU and CLEAs respectively, so the latter had a higher operational stability; however, recovery is simpler in the former so both catalyst are competitive for biodiesel production.

Alkylative Amination of Biogenic Furans through Imine-to-Azaallyl Anion Umpolung

Starting from biogenic furfurals, an operationally simple and scalable condensation-umpolung-alkylation protocol was employed in the synthesis of racemic furfurylamines. Subsequent enzymatic kinetic resolution by ω-transaminase or lipase biocatalysts allows for the preparation of functionalized heterocyclic building blocks from biogenic base chemicals in optically pure form.

Enhanced Biocatalytic Activity of Lipase Immobilized on Biodegradable Copolymer of Chitosan and Polyvinyl Alcohol Support for Synthesis of Propionate Ester: Kinetic Approach

The objective of this work was to investigate a kinetic and mechanistic analysis for the synthesis of benzyl propionate which has diverse applications. Furthermore this study illustrates the synthesis and beneficial uses of an ecofriendly support that is made up of chitosan (CHI) and polyvinyl alcohol (PVA) to immobilize lipase Pseudomonas cepacia (PCL), and it showed remarkable enhancement of biocatalytic activity over free lipase. Among various biocatalysts prepared, CHI:PVA:PCL (5:5:2) was found as a robust biocatalyst composition. The lower activation energy (Ea) of immobilized lipase (12.8 ± 0.11 kcal/mol) than that of free lipase (16.6 ± 0.65 kcal/mol) showed better catalytic activity of immobilized lipase. Kinetic parameters were determined which showed that benzyl propionate synthesis catalyzed by immobilized PCL followed the order bi–bi mechanism with benzyl alcohol inhibition. CHI/PVA immobilized lipase biocatalyst showed enhanced (3.5 fold) catalytic activity and efficient recyclability over th...

The chemo-enzymatic Baeyer–Villiger oxidation of cyclic ketones with an efficient silica-supported lipase as a biocatalyst

The synthesis and characterisation of new, silica-supported lipase biocatalysts and studies of their performance in Baeyer–Villiger oxidation (BVO) of cyclic ketones to lactones were described. Biocatalysts were obtained by immobilisation of Candida Antarctica lipase B onto siliceous materials with multimodal pore structure (MH) and also on typical SBA-15, chemically modified with organosilanes terminated with methyl, octyl and hexadecyl group. Biocatalysts structure was characterised by nitrogen adsorption and TEM/SEM imaging. The lipase presence was confirmed by FTIR spectroscopy, whereas protein loads were obtained from thermogravimetric data. They appeared to be notably larger for MH catalysts than SBA-15s and correlated with surface hydrophobicity of supports which decreased in the order Hd>Oc>Me, from 198 to 80mg/g, for MH-Hd-L and MH-Me-L biocatalysts, respectively.Investigation of the biocatalysts performance (activity, stability, reusability) in BVO of cyclic ketones, using urea hydrogen peroxide as oxidant and ethyl acetate as both the peracid precursor and solvent, clearly demonstrated very large activity of MH biocatalysts. For the same lipase load activity decreased in the order MH-Me-L>MH-Oc-L>MH-Hd-L, with that of least active (MH-Hd-L) catalysts being even trice larger than of Novozyme-435 under equal mass basis. The biocatalysts showed good stability, even in the presence of 60% aq. hydrogen peroxide, and facile reuse. On this basis we proposed a general chemo-enzymatic BVO method for oxidation of cyclohexanones and cyclobutanone to obtain adequate lactones with high yields (80–98%).

ChemInform Abstract: Synthesis of Some Novel Pyrimidinedione and Pyrimidinetrione Derivatives by a Greener Method: Study of Their Antimicrobial Activity and Photophysical Properties.

AbstractThe title compounds such as (III), (V), and (VII) are obtained by Knoevenagel condensation under classical, conventional condition, in the presence of a lipase biocatalyst or with a deep eutectic solvent (DES).

Environmental-friendly strategy for biocatalytic conversion of waste glycerol to glycerol carbonate

In this work, the conversion of waste glycerol from biodiesel production to glycerol carbonate (GlyC) as added-value product has been investigated. The transformation of waste glycerol to GlyC has been achieved taking the advantage of dimethyl carbonate (DMC) reaction with glycerol catalyzed by heterogeneous lipase biocatalyst under solvent-free conditions. The biocatalyst design was the lipase enzyme anchored on magnetic nano-particle surface via covalent binding. Waste glycerol with different matrixes according to the feedstock patterns (e.g. soybean, sunflower, rape, corn, olive, palm, and residual oil) has been tested for GlyC bio-synthesis. Content of MeOH, H2O, metals and salts from sample matrix (e.g. waste glycerol) was characterized using gravimetric and energy dispersive X-ray fluorescence (EDXRF) spectrometry techniques. Impurities effects of the waste glycerol on the performance of the biocatalytic process and also different strategies to avoid them were investigated.

Synthesis of β-amino alcohol derivatives from phenols in presence of phase transfer catalyst and lipase biocatalyst

Article history: Received June 25, 2012 Received in Revised form November 6, 2012 Accepted 6 November 2012 Available online 6 November 2012 A simple and environmentally friendly reaction has been developed for the first time for onepot synthesis of β-amino alcohol derivatives from aromatic phenols, epichlorohydrin and amines by using phase transfer catalysts (PTC) and Aspergillus Oryzae lipase biocatalyst. This method provides access to pharmaceutically relevant products in excellent yields with high regioselectivity. The remarkable catalytic activity and reusability of lipase was possible up to four consecutive cycles. © 2013 Growing Science Ltd. All rights reserved.

Synthesis of Some Novel Pyrimidinedione and Pyrimidinetrione Derivatives by a Greener Method: Study of Their Antimicrobial Activity and Photophysical Properties

A series of novel pyrimidinedione- and pyrimidinetriones-based compounds were synthesized by different techniques such as with conventional Knoevenagel condensation alone, with a Rhizopus oryzae lipase biocatalyst, and with a deep eutectic solvent (DES). The yield was found to be maximum by using lipase and DES. Reuse of the lipase and DES was possible up to four consecutive cycles. These methods are mild, highly efficient, and amenable to scaleup. The products were found to exhibit appreciable in vitro antibacterial activity against Echerichia coli, Pseudomonas neumoniae, and Micrococcus and in vitro antifungal activity against Aspergillus niger and Candida albicans. All the compounds exhibited appreciable in vitro activity against the tested strains. The photophysical properties and thermal stability of the products were also investigated.

Lipase and Deep Eutectic Mixture Catalyzed Efficient Synthesis of Thiazoles in Water at Room Temperature

Lipase bio-catalyst or ammonium based deep eutectic mixture efficiently catalyzed aqueous phase synthesis of methyl-thiazole and amino-thiazole derivatives. The simple ammonium deep eutectic catalyst, easily synthesized from choline chloride and urea, is inexpensive, recyclable and bio-degradable, making it suitable for industrial applications. An efficient and eco-friendly process for methyl and amino-thiazole synthesis in aqueous media involving bio-catalyst or ammonium deep eutectic mixtures as catalysts is described. Both the catalysts are bio-degradable, recyclable and non-toxic.

Synthesis of novel dihydropyrimidin-2(1h)-ones derivatives using lipase and their antimicrobial activity

Article history: Received January 11, 2012 Received in Revised form Feb 19, 2011 Accepted 2 March 2011 Available online 2 March 2012 A series of novel dihydropyrimidin-2(1H)-ones derivatives was synthesized by using Rhizopus Oryzae lipase biocatalyst in deep eutectic solvent. The reaction is characterized by high efficiency and selectivity, short reaction time, mild and environmentally friendly reaction conditions. The yields were found to be significantly higher and reuse of both the lipase and deep eutectic solvent was possible up to four consecutive cycles. The products are found to exhibit appreciable in vitro antibacterial activity against Echerichia coli, Pseudomonas neumoniae and in vitro antifungal activity against Aspergillus niger and Candida albicans. © 2012 Growing Science Ltd. All rights reserved.

Immobilization of lipase B from Candida antarctica on porous styrene–divinylbenzene beads improves butyl acetate synthesis

A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.

Enzyme Surface Glycosylation in the Solid Phase: Improved Activity and Selectivity of Candida Antarctica Lipase B

AbstractTailor‐made oligosaccharides and polymers were investigated for a specific surface glycosylation of Candida antarctica lipase (fraction B) (CAL‐B) already immobilized on octyl‐Sepharose by interfacial activation. The chemical modification was performed in the N‐terminal amino acid enzyme residue by using low oxidized aldehyde–dextran polymers through a reductive amination. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) indicated that polymer/enzyme conjugates were obtained in all cases. Circular dichroism experiments revealed interesting conformational changes in secondary and tertiary structures of the protein after modification. The formed immobilized glycosylated lipase biocatalysts were more stable, active, and selective toward different substrates than unmodified CAL‐B. These immobilized conjugates were compared with a genetically glycosylated version of CAL‐B expressed in Pichia pastoris immobilized in the same way. Enzyme thermostability was improved after chemical modification with Dextran‐1500 and also by the genetic glycosylation, retaining 90–96 % activity after 24 h at 55 °C. The catalytic activity of CAL‐B was improved by the incorporation of dextran polymers (Mw=1500 or 6000) more than twofold in the hydrolysis of p‐nitrophenylbutyrate and more than threefold in the hydrolysis of methyl mandelate at pH 7. However, the activity of the genetically glycosylated CAL‐B was threefold lower in the hydrolysis of both substrates. The enantioselectivity of CAL‐B increased for all formed bioconjugates, with the Dextran‐1500–CAL‐B conjugate being the most selective in the hydrolysis of racemic methyl mandelate (up to 88.1 % ee at pH 5). This glycosylated CAL‐B also demonstrated the highest synthetic activity in the transesterification of methyl butyrate with glycerol, with 80 % yield of monoglyceryl ester at 100 % conversion compared to 57 % yield obtained with unmodified CAL‐B or other polymer–lipase conjugates.

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

Synthesis of Diphenylamine-Based Novel Fluorescent Styryl Colorants by Knoevenagel Condensation Using a Conventional Method, Biocatalyst, and Deep Eutectic Solvent

Novel Y-shaped acceptor-pi-donor-pi-acceptor-type compounds, synthesized from 4,4'-hexyliminobisbenzaldehyde as electron donors and different active methylene compounds as electron acceptors, were produced by conventional Knoevenagel condensation alone, with a deep eutectic solvent, or with a lipase biocatalyst to compare the yield and recyclability among the three methods. Yield, reaction time, reaction temperature, and recyclability were compared among the three methods. The photophysical properties and thermal stability of the products were also investigated.

Combined strategy for preparation of a bioimprinted Geotrichum sp. lipase biocatalyst effective in non-aqueous media

Geotrichum sp. lipase with enhanced activity and operational stability was prepared for use in non-aqueous media. A combined strategy comprising bioimprinting with dual imprint molecules and a co-solvent coupled to pH tuning, KCl salt activation, lecithin coating and immobilization on macroporous resin effectively enhanced the activity and operational stability of Geotrichum sp. lipase. The modified lipase exhibited 18.4-fold enhanced esterification activity towards methyl oleate synthesis, and retained 90% activity following repeated use in 10 cycles. The combined strategy exhibited a significant synergistic effect and was suitable for lipase modification, dramatically enhancing the enzyme activity and operational stability. This approach is applicable to the preparation of other enzyme biocatalysts, since the methods are effective for upgrading crude enzyme to a refined product with high activity and stability for use in non-aqueous media.

Biodiesel fuel production via transesterification of oils using lipase biocatalyst

AbstractBiodiesel has gained widespread importance in recent years as an alternative, renewable liquid transportation fuel. It is derived from natural triglycerides in the presence of an alcohol and an alkali catalyst via a transesterification reaction. To date, transesterification based on the use of chemical catalysts has been predominant for biodiesel production at the industrial scale due to its high conversion efficiency at reasonable cost. Recently, biocatalytic transesterification has received considerable attention due to its favorable conversion rate and relatively simple downstream processing demands for the recovery of by‐products and purification of biodiesel. Biocatalysis of the transesterification reaction using commercially purified lipase represents a major cost constraint. However, more cost‐effective techniques based on the immobilization of both extracellular and intracellular lipases on support materials facilitate the reusability of the catalyst. Other variables, including the presence of alcohol, glycerol and the activity of water can profoundly affect lipase activity and stability during the reaction. This review evaluates the current status for lipase biocatalyst‐mediated production of biodiesel, and identifies the key parameters affecting lipase activity and stability. Pioneer studies on reactor‐based lipase conversion of triglycerides are presented.

Solid-phase lipase biocatalysts for kinetic resolutions

Solid-phase lipase biocatalysts for kinetic resolutions

Lipases Enantioselectivity Alteration by Immobilization Techniques

Lipases are the most used enzymes as biocatalyst in the resolution of chiral compounds. However many times the selectivity towards different intermediates is very low. A simple strategy has recently been reported to permit greatly enhancing the lipase selectivity. The strategy based on the great conformational changes of these enzymes during catalysis consists in the preparation of a library of lipase biocatalysts by using different immobilization protocols that may permit to immobilize them via different orientations, with different rigidity or generating different environments. This review examines how this solid phase strategy has permitted greatly modulated the enantioselectivity of lipases in kinetic resolutions of racemic mixtures. Keywords: Conformational changes, lipases, immobilization, modulation, kinetic resolution, racemic mixtures, enantioselectivity