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The lipase from Burkholderia cepacia (BCL) was immobilized through physical adsorption on pristine and functionalized multiwalled carbon nanotubes (MWCNTs) with carboxyl or amine groups and used in the stereoselective acylation of (R,S)-1-octen-3-ol (1) and (R,S)-(E)-4-phenyl-3-buten-2-ol (4) with vinyl acetate. All immobilized preparations produced better results than free BCL. For (R,S)-4, 50% conversion and E 200 were obtained in n-hexane or in solvent-free medium. For (R,S)-1, in solvent-free medium, the conversion was 38% with a slight increase in the E-value (E = 10).

This work aimed to produce porous poly-hydroxybutyrate (PHB) pellets in order to evaluate the pellets as a support for immobilization of the metagenomic lipase, LipG9. Four types of pelletized PHB particles with different morphological characteristics were obtained using the double emulsion and solvent evaporation technique (DESE). The micropores of these PHB pellets had similar average diameters (about 3 nm), but the pellets had different specific surface areas: 11.7 m

The present work deals with the preparation and characterization of Zn2Al layered double hydroxides (LDHs) intercalated with chloride and dodecyl sulfate ions and their use as supports for the immobilization of Pseudomonas cepacia lipase (LipPS). LipPS was immobilized on LDH by adsorption, in situ entrapment and delamination entrapment methods. The performance of free and immobilized LipPS in the transesterification of (R,S)-1-phenylethanol was compared. The immobilized derivatives prepared by adsorption gave the highest conversions (c = 50%), with an enantiomeric excess of the product (eep) greater than 99% and a high enantiomeric ratio (E > 200). For immobilization by in situ entrapment (coprecipitation), a conversion of 31.5% and E > 200 were obtained. For immobilization by delamination entrapment, a conversion of 36% and E= 155 were obtained. Although the best results were obtained by adsorption, these results show that the delamination entrapment method can also be used to prepare bioinorganic-enzyme materials based on LDH. Our work therefore increases the range of methods that can be used to immobilize lipases for use in the production of optically pure secondary alcohols.

The present work evaluates the immobilization of LipC12 on different supports in tandem with bioimprinting technique, in order to improve its activity and stability in organic medium. Oleic acid was selected as the bioimprinting molecule. The immobilized LipC12 was applied in the synthesis of pentyl oleate by esterification reaction and in the production of fatty acids, mono, and diglycerides via hydrolysis of triacylglycerols, in n-heptane reaction media. For all immobilized lipase preparations, an increase in the conversion of oleic acid to pentyl oleate was observed when immobilization in tandem with bioimprinting treatment was carried out versus immobilization without bioimprinting. The highest conversions were achieved using LipC12 immobilized on hydrophobic supports. The reuse potential of the immobilized preparations was evaluated. The preparations were used in eight successive cycles of esterification reactions and the best results were obtained for LipC12 immobilized on Immobead 150 and chitosan. The activity for the hydrolysis of soybean oil was improved by bioimprinting treatment only for LipC12 immobilized on commercial polypropylene and Accurel MP-1000. LipC12 immobilized on hydrophilic supports or on Immobead150 could be used to hydrolyze tricaprylin to obtain diglycerides with a high proportion of 1,2-diglycerides in reaction times as short as 30 min.

The production and direct employment in organic medium in the synthesis of ethyl oleate of a solid fermented (FS) containing lipases of Penicillium roqueforti ATCC 10110 (PR10110), was investigated. For the production of this FS, the solid state fermentation of different agro-industrial waste was used, testing: cocoa shell, sugarcane bagasse, sugarcane bagasse with cocoa shell and cocoa shell with soybean oil and nutrient solution. The response surface methodology was used to study the effect of independent variables of initial moisture content (BMI) and inductor concentration, as carbon source and inducer (CS&I) on lipase production. The characterization of the fermented solid in organic medium was also carried out. The highest lipase activity (53±5 U.g-1) was 16% higher than that obtained with the non-optimized conditions. The characterization studies, was observed high stability of the FS in organic solvents for 5 h at 30°C, as well as at different temperatures, and the residual activity was measured against triolein. The FS was also able to catalyze ethyl-oleate synthesis maintaining high relative conversion over 5 reaction cycles of 96 h at 40°C in n-heptane. These results are promising and highlight the use of the FS containing lipases for first time in biocatalytic processes. This article is protected by copyright. All rights reserved.

Packed-bed bioreactors are often used for aerobic solid-state fermentation, since the forced aeration supplies O2 and removes metabolic heat from the bed. Motivated by the potential for applications in biorefineries, we review studies conducted on packed-bed bioreactors over the last decade, evaluating the insights these studies provide into how large-scale packed beds should be designed and operated. Many studies have used low superficial air velocities and suffer from preferential airflow, such that parts of the bed are not properly aerated. Moreover, some studies have proposed ineffective strategies, such as reversing the direction of the airflow or introducing air through perforated pipes within the bed. Additionally, many studies have used narrow water-jacketed packed-bed bioreactors, but these bioreactors do not reflect heat removal in wide large-scale packed beds, in which heat removal through the side walls makes a minor contribution. Finally, we conclude that, although some attention has been given to characterizing the porosities, water sorption isotherms and volumetric heat and mass transfer coefficients of substrate beds, this work needs to be extended to cover a wider range of solid substrates, and work needs to be done to characterize how these bed properties change due to microbial growth.

Cetyl palmitate was produced by the esterification of palmitic acid with cetyl alcohol using n-hexane as solvent and a fermented solid of Burkholderia contaminans as catalyst. Effects of temperature (35–55 °C) and alcohol:acid molar ratio (0.5:1–2:1) on the reaction kinetics were evaluated for a fixed amount of catalyst (10 wt% related to the total mass of substrates) and a stirring speed (600 rpm) that was sufficiently high to avoid convective mass transfer limitations in the bulk phase. The temperature did not to influence the reaction rates and conversions above 90% were obtained for all temperatures after 12 h of reaction using a stoichiometric ratio (1:1) between the substrates. The prepared catalyst was recycled and reused in seven consecutive reactions of 8 h each and lost around 40% of its initial activity during the process. A Ping-Pong Bi–Bi kinetic model was proposed to represent the system reactions and was satisfactorily fitted to the experimental data.

Lipases are often used in immobilized form, but commercial immobilized lipases are costly. An alternative is to produce lipases in solid-state fermentation, dry the solids and then use the “dry fermented solids” (DFS) directly. We produced DFS by growing Burkholderia contaminans on a mixture of sugarcane bagasse and sunflower seed meal and used the DFS to esterify oleic acid with ethanol in subcritical and supercritical CO2 at 40 °C. Compared to a control without CO2 at atmospheric pressure, subcritical CO2 at 30 bar improved esterification activity 1.2-fold. Higher pressures, including supercritical pressures up to 150 bar, reduced activity to less than 80% of the control. At 30 bar, the esterification activity was improved a further 1.8-fold with the addition of 9% water (i.e. 9 g water per 100 g oleic acid) to the reaction medium. A subcritical CO2 atmosphere, with the addition of a small amount of water, improved the esterification activity of DFS containing lipases of Burkholderia contaminans.

In this work, ultrasound was used to assist the ethanolysis of castor oil in a solvent-free system, catalyzed by a dry fermented solid containing the lipase from Burkholderia contaminans (BCFS). Reactions were done at 45°C. The maximum conversion in Erlenmeyer flasks was 71% in 96 h, using a loading of 9% (mass of BCFS in relation to the mass of triacylglycerols in the castor oil) and a molar ratio of ethanol:oil of 6:1, with addition of ethanol in 12 steps. In a packed-bed reactor containing 12 g of BCFS, the conversions were 78% in 48 h, and 83% in 72 h with an ethanol to oil molar ratio of 3:1 and treatment with an ultrasound probe, with maximum power of 500 W, frequency of 20 kHz, and 75% of the maximum power. These results are promising given that, with an ultrasound assisted bioreactor, a higher conversion in a shorter time was achieved, with a lower ethanol to oil molar ratio than was the case in the Erlenmeyer flasks without ultrasound.

Metagenomics is a modern approach to discovery of new enzymes with novel properties. This article reports the structure of a new lipase, belonging to family I.1, obtained by means of metagenomics. Its structure presents a fold typical of α/β hydrolases, with the lid in closed conformation. The protein was previously shown to present high thermostability and to be stable in aqueous solutions of polar organic solvents at high concentrations [30% (V/V)]. Molecular dynamics studies showed that the protein maintains its structure well in organic solvents. They also suggested that its thermostability might be enhanced if it were mutated to present a disulfide bond similar to that typically found in lipase family I.2. These findings identify this lipase as a good candidate for further improvement through protein engineering.

LipMF3 is a new lipase isolated from a metagenomic library derived from a fat-contaminated soil. It belongs to the lipase subfamily I.1 and has identities of 68% and 67% with lipases of Chromobacterium violaceum and C. amazonense, respectively. Genes encoding LipMF3 and its cognate foldase, LifMF3, were cloned and co-expressed in Escherichia coli. The highest hydrolytic activity of purified Lip-LifMF3 was at 40 °C and pH 6.5. Under these conditions, the highest activity was against tributyrin (1650 U mg−1), but it also had high activity against olive oil (862 U mg−1). It was stable in hydrophilic organic solvents (25%, v/v in water) with residual activity around 100% after 24 h. It also showed stability over a wide pH range (5.5 to 11) with residual activity above 80% after 24 h. Lip-LifMF3 was immobilized by covalent bonding onto Immobead 150P and by adsorption onto Sepabeads FP-BU. The latter preparation gave the best results, producing 94% conversion after 5 h for the synthesis of ethyl oleate and a 90% enantiomeric excess of the product (R)‑1‑phenylethyl acetate for the kinetic resolution of (R,S)‑1‑phenyl‑1‑ethanol. The results obtained in this work provide a basis for the development of applications of Lip-LifMF3 in biocatalysis.

During solid-state fermentation, aerial hyphae occupy the interparticle spaces, binding adjacent particles and decreasing the efficiency of mass and heat transfer within the bioreactor bed. The present work explores two questions related to this phenomenon: (i) what determines the direction of growth of vegetative aerial hyphae and (ii) what makes them stop growing? We developed a discrete mathematical model to explore the factors affecting the distribution of aerial hyphae with height above the surface and adjusted the model parameters by fitting the model to literature data of Rhizopus oligosporus growing on potato dextrose agar. The adjusted rules were simple and indicate that vegetative aerial hyphae grow in any direction, as though their tips moved in a random-walk fashion. Vegetative aerial hyphae stop extending when they enter crowded spaces, resulting in a maximum local biomass concentration. With these rules, the layer model was used to study the influence of increasing inoculum concentration and to simulate, for the first time, growth between the surfaces of two adjacent particles.

Lipases are often used in immobilized form, but commercial immobilized lipases are costly. An alternative is to produce lipases in solid-state fermentation, dry the solids and then use the "dry fermented solids" (DFS) directly. We produced DFS by growing Burkholderia contaminans on a mixture of sugarcane bagasse and sunflower seed meal and used the DFS to esterify oleic acid with ethanol in subcritical and supercritical COCompared to a control without COA subcritical CO

Interest is increasing in the application of lipases to catalyze sequential hydrolysis or transesterification reactions with diacids. In such systems, there are two Ping Pong bi bi reaction loops, with the free enzyme being the only enzyme form that is common to both loops. The modeling of such systems has not received much attention to date. The current work derives an expression for a reaction loop selectivity factor, denominated L, which is given by the ratio of the specificity constant of the enzyme for the first substrate of the second loop to the specificity constant for the first substrate of the first loop. The value of L is determined for two case studies using literature data. For the hydrolysis of diethyl adipate by Rhizopus arrhizus lipase, L is equal to 0.062, indicating that the specificity of this lipase for hydrolyzing diethyl adipate is much higher than that for hydrolyzing monoethyl adipate. For the CALB-catalyzed transesterification of diethyl azelate with 2-ethylhexan-1-ol, producing ethyl 2-ethylhexyl azelate as an intermediate and bis (2-ethylhexyl) azelate as the final product, L is 0.37, meaning that CALB prefers to transesterify diethyl azelate, rather than the intermediate ethyl 2-ethylhexyl azelate.

Several biotechnologically important enzymes catalyze reactions by the Ping Pong bi bi mechanism. Traditional kinetic equations for these enzymes contain several terms with combinations of three or four lumped parameters, making it difficult to relate the terms directly to the reaction mechanism. Further, the Ping Pong bi bi reaction mechanism is symmetrical in the forward and reverse directions, but the traditional equations are not mathematically symmetrical. In the present work, we express the Ping Pong bi bi kinetic equation in terms of specificity constants. Our equation is consistent with the traditional equations, but has several conceptual advantages. First, the terms of our equation contain two parameters at most, making it easier to relate the terms to specific features of the reaction mechanism. Second, our equation has only eight lumped parameters, so it has no redundant parameters; traditional equations have nine lumped parameters, such that one is redundant. Third, our equation is mathematically symmetrical, explicitly reflecting the symmetry of the Ping Pong bi bi reaction mechanism. We demonstrate how the parameters of our new equation can be estimated in classic initial rate experiments.

This study reports on the synthesis and characterization of sericin–dimethylolurea films (SS–DMU) and the immobilization of Burkholderia cepacia LTEB11 lipases through the so-called “sandwich” adsorption method, wherein the enzyme is deposited on the film, forming an enzyme–film–enzyme surface. Measured film thicknesses were between 230 and 309 μm. Resistance to traction was approximately double in comparison to the film with no enzyme applied. Scanning electron microscopy images attest the formation, after the enzyme immobilization, of aggregate deposits, possibly proteins, over the film surface. Catalytic efficiency was ascertained in ethyl oleate esterification reactions. Ester yield was practically equal (96%, 24 h) for the free and immobilized enzymes. Film samples with different quantities of lyophilized enzyme deposits (56, 122 and 156 mg) were analyzed; during the first 5 h of observation the sample with the largest quantity showed faster reaction speeds. The immobilized lipase (56 mg) was reused, and catalytic efficiency decreased to 58% after seven reaction cycles. The results point to SS–DMU film as a promising biocatalysis material.

Chemical methods for the regioselective modification of carbohydrates require several steps that involve the addition and removal of protective groups. Enzymatic methods are promising since they can give high selectivity in a single step. We developed a new method for acetylation of methyl-α- d -glucopyranoside with vinyl acetate, the reaction being catalyzed by a fermented solid containing lipases from Burkholderia contaminans LTEB11. The main product of the synthesis, methyl 6-O-acetyl-α- d -glucopyranoside (6-OAc-α-MetGlc), was identified by NMR and quantified by HPLC. The conversion of methyl-α- d -glucopyranoside to 6-OAc-α-MetGlc was evaluated at laboratory scale (4 mL) in different solvent systems. The conversion increased with increasing hydrophobicity of the solvent, with the highest conversion, 65% in 72 h, being obtained in the solvent-free system. This system was scaled up to a 1-L reaction medium. A conversion of 76% in 72 h was obtained, with a calculated yield of 1.05 g (4.43 mmol) of 6-OAc-α-MetGlc. This is the largest amount of 6-OAc-α-MetGlc (in moles) produced by enzymatic catalysis that has been reported in the literature to date. These results suggest that the fermented solid produced by B. contaminans LTEB11, which is a low-cost biocatalyst, could potentially be used for the industrial production of esters of α- d -glucose.

Many polysaccharide hydrolases are capable of processive action, where they perform repeated attacks without dissociating from the substrate molecule. In this paper, we extend the “Fingerprinting method”, a method that can be used to determine the relative specificities that an enzyme has for the various reactions that it can catalyze in a multi-step reaction scheme, to reaction schemes involving processive action. We do this for three case studies for the hydrolysis of linear malto-oligosaccharides by β-amylase, an exoenzyme that successively removes maltose units from the non-reducing ends of the molecules. In these case studies, we demonstrate that it is possible to use reaction profiles to determine, simultaneously, the relative specificities that the enzyme has for the different species in the reaction mixture and the probabilities of processive action occurring. These parameters can be used in models for designing and optimizing hydrolysis processes and also to guide protein engineering programs.

We determined the effect of the His-tag on the structure, activity, stability and immobilization of LipC12, a highly active lipase from a metagenomic library. We purified LipC12 with a N-terminal His-tag and then removed the tag using tobacco etch virus (TEV) protease. Circular dichroism analysis showed that the overall structure of LipC12 was largely unaffected by His-tag removal. The specific hydrolytic activities against natural and artificial substrates were significantly increased by the removal of the His-tag. On the other hand, His-tagged LipC12 was significantly more active and stable in the presence of polar organic solvents than untagged LipC12. The immobilization efficiency on Immobead 150 was 100% for both forms of LipC12 and protein desorption studies confirmed that the His-tag does not participate in the covalent binding of the enzyme. In the case of immobilized LipC12, the His-tag negatively influenced the hydrolytic activity, as it had for the free lipase, however, it positively influenced the esterification activity. These results raise the possibility of tailoring recombinant lipases for different applications, where the His-tag may be retained or removed, as appropriate for the desired activity.

Free Candida antarctica lipase B (Lipozyme, CALB L®) was used to produce fatty acid ethyl esters (FAEE) from refined soybean oil in solvent-free media using the conventional (CHS) and microwave (MHS) heating systems. Statistical analyses (95% confidence level) for both reaction products, FAEE and free fatty acids (FFA), were performed. An increase in ethanol:oil molar ratio decreased the catalytic performance of CALB L (p

Lipases can resolve many chiral compounds of commercial interest. However, for some substrates, it has been difficult to find lipases with the necessary chemo-, regio- and enantioselectivities. Searching in nature for new lipases with the desired selectivities is a difficult task and is not always successful. An alternative strategy is to engineer existing lipases. However, until now, there has been no systematic evaluation to identify which positions in the protein molecule should be engineered to obtain variants with the desired selectivities. Such an evaluation is possible because, even though the catalytic clefts of lipases have differing shapes, there is sufficient structural similarity for one to identify the key regions involved. In this review, we analyze representative studies that have engineered bacterial and fungal lipases for increased enantioselectivity for application in racemic resolutions. Based on this analysis, we identify key sites of the catalytic cleft at which mutations should be targeted in attempts to improve the enantioselectivity of lipases. The locations of these key sites depend on whether the chiral substrate is a carboxylic acid or an alcohol.

Fermented solids containing lipases from Burkholderia contaminans LTEB (denominated BCFS) were obtained by solid-state fermentation using sugarcane bagasse impregnated with soybean oil. These solids were dried and used to catalyze the ethanolysis of palm oil in a solvent-free reaction medium. The reaction was optimized in 25-mL vials, containing 15 mmol of palm oil. Molar ratios (MR) of ethanol to palm oil of 3:1 and 4.5:1 were tested, with stepwise addition of ethanol. In these experiments, the maximum conversion of palm oil achieved in 48 h was 90%, with a MR of 4.5:1 and ethanol addition in 3 steps. We then used BCFS in a packed-bed reactor with recirculation. Ethanol was added in four steps, with an overall molar ratio of ethanol added to initial oil of 5.5:1, giving a conversion of 89% in 30 h. When the same fermented solids were used under the same conditions in successive 30-h batches in this packed-bed bioreactor, with sequential washings with n -hexane and tert -butanol between cycles, the conversion in the 5th cycle was 66%. These results are comparable to those that have been obtained for transesterification of palm oil in solvent-free reaction media with commercial lipases, with our catalyst having the advantage that it is much cheaper to prepare.

A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse (50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from 10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from 265 U g−1 at 18 h in the laboratory bioreactor to 113 U g−1 at 20 h in the pilot bioreactor. However, the esterification activity decreased by only 14%: from 12.1 U g−1 to 10.4 U g−1. When the FS produced in the laboratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h.

There has been a surge of interest in the establishment of citrus waste biorefineries. One strategy in such biorefineries would be to extract pectin and saccharify it enzymatically to produce d -galacturonic acid, which has potential as a platform chemical for the production of compounds of commercial interest, such as meso-galactaric acid and l -galactonic acid. A mathematical model of the hydrolysis of pectic substrates by the pool of enzymes present in pectinase preparations would be a useful tool for guiding the design and optimization of the saccharification process. Current deterministic models of this process are highly simplified and, as a result, are not useful tools. In the current manuscript, we develop stochastic models, based on the Monte Carlo method, to describe the action of two important enzymes found within pectinase preparations, namely endopolygalacturonases and exopolygalacturonases. Our work represents a first step towards the development of a complete model that integrates the action of the various enzymes present in pectinase preparations.

The enzymatic route for biodiesel production is environmentally attractive, but costs need to be reduced and reaction times shortened. Costs can be reduced by producing lipases by solid-state fermentation and using the fermented solids directly to catalyze the ethyl-esterification of fatty acids in solvent-free media. However, the esterification studies done to date with fermented solids have involved 12 g or less of fermented solids and reaction times greater than 30 h. The aim of the current work was therefore to increase the scale of the process by an order of magnitude, while also decreasing the reaction time, using bi-distilled olein as the source of fatty acids. The best result was obtained with a molar ratio of ethanol to fatty acids of 1.5:1, in a process in which 245 g of ethanol and 1000 g of olein were circulated in a closed-loop system through a column containing 120 g of dry fermented solids. A two-compartment reservoir was used, with the water in the outflow of the column being decanted in the first compartment. The organic phase that passed to the second compartment was agitated and fed back to the column. This system gave 88% conversion in 24 h. This system was used in 6 successive 48-h batches with the same dry fermented solids, in which a total of 4.6 kg of ester was produced. These are promising results that lay the basis for further scale-up studies.

Solid-state fermentation could be used to produce low-cost pectinases that could then be used to saccharify pectin in citrus waste biorefineries. Recently, we produced pectinases in a pilot-scale packed-bed bioreactor, growing Aspergillus niger on a substrate mixture consisting of 27 kg of wheat bran and 3 kg of sugarcane bagasse (dry mass). However, the agglomeration of particles and shrinkage of the bed created preferential flow paths, leading to overheating within the bed and poor uniformity of pectinase levels at the end of the fermentation. In the current work, we used intermittent agitation as a strategy to minimize agglomeration, comparing one agitation (10 h), three agitations (at 8, 10 and 12 h) and five agitations (every 2 h from 8 to 16 h). The pectinase activity in the bed was uniform after agitation, but poor uniformity occurred when the bed was left unmixed for more than 10 h. The best regime was that with three agitations: For 15 samples removed from different vertical and horizontal positions of the bed at 20 h, the average pectinase activity was 22 U g−1, with a sample standard deviation of 2 U g−1. We conclude that the use of intermittently-mixed packed-bed bioreactors is a promising strategy for producing pectinases in solid-state fermentation.

A low-cost enzymatic catalyst for biodiesel production can be produced by growing a lipase-producing organism in solid-state fermentation and then drying the solids after the fermentation. Recently, we cultivated Burkholderia cepacia on a mixture of sugar cane bagasse and sunflower seed meal and used the dried fermented solid to catalyze the esterification of fatty acids with ethanol in a solvent-free system (Soares et al., Fuel (2015) 159, 364–372). During the reaction, up to 30% of the reaction medium sorbed onto the dried fermented solid, with the sorbed medium having a composition different from that of the bulk phase. In the current work, we develop a combined sorption-kinetic model to describe the reaction kinetics in this system. The sorption of the medium components onto the fermented solid follows a multicomponent Langmuir isotherm, while the kinetic equation is expressed in terms of the composition of the sorbed phase. With a single set of parameters, the model adjusted well to the experimental results obtained by Soares et al. for three different ethanol to fatty acid molar ratios (1:1, 1.5:1 and 3:1), not only for the overall conversion profile, but also for the compositions of the bulk phase and the sorbed phase. The model has potential to be used to guide the scale-up of our system.