Immobilization of the metagenomic lipase, LipG9, on porous pellets of poly-hydroxybutyrate produced by the double emulsion solvent evaporation technique.

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 m2 g-1 for the PHB powder, 8.4 m2  g-1 for the control pellets (Ø < 0.5 mm, produced without the pore forming agent), 10.0 m2  g-1 for the small pellets (Ø < 0.5 mm), 9.5 m2  g-1 for the medium pellets (0.5 < Ø < 0.8 mm) and 8.4 m2  g-1 for the large pellets (Ø > 1.4 mm). Purified LipG9 was immobilized by adsorption on these pellets, and the results were compared with those obtained with PHB powder. The highest immobilization yield (83%) was obtained for the medium PHB pellets, followed by large (76%) and small (55%) PHB pellets. The activity of LipG9 immobilized on the pellets, for the synthesis of ethyl oleate in n-hexane, was highest for the medium pellets (22 U g-1 ). The immobilization yield was high for PHB powder (99%) but the esterification activity was slightly lower (20 U g-1 ). These results show that pelletized PHB beads can be used for the immobilization of lipases, with the advantage that pelletized PHB will perform better than PHB powder in large-scale enzyme bioreactors.

Transesterification of castor oil catalyzed by a fermented solid produced by Burkholderia contaminans using a bioreactor coupled with ultrasound irradiation.

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.

Potential of time-stepping stochastic models as tools for guiding the design and operation of processes for the enzymatic hydrolysis of polysaccharides - A review.

Processes for the enzymatic hydrolysis of polysaccharides in biorefineries are becoming increasingly important. The complex network of reactions involved in polysaccharide hydrolysis can be described by stochastic models that advance in steps of time. Such models have the potential to be important tools for guiding process design and operation, and several have been developed over the last two decades. We evaluate these models. Many of the current stochastic models for the hydrolysis of colloidal polysaccharides use empirical parameters that have no recognized biological meaning. Only one model uses classical parameters of enzyme kinetics, namely specificity constants and saturation constants. Recent stochastic models for the hydrolysis of insoluble cellulose give valuable insights into the molecular-level phenomenon that limit hydrolysis rates. We conclude that, if stochastic models of enzymatic polysaccharide hydrolysis are to become widely used tools for guiding process development, then further improvements are required.

Performing under pressure: esterification activity of dry fermented solids in subcritical and supercritical CO2.

OBJECTIVE: 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. RESULTS: 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. CONCLUSION: A subcritical CO2 atmosphere, with the addition of a small amount of water, improved the esterification activity of DFS containing lipases of Burkholderia contaminans.

Metagenomics: Is it a powerful tool to obtain lipases for application in biocatalysis?

In recent years, metagenomic strategies have been widely used to isolate and identify new enzymes from uncultivable components of microbial communities. Among these enzymes, various lipases have been obtained from metagenomic libraries from different environments and characterized. Although many of these lipases have characteristics that could make them interesting for application in biocatalysis, relatively little work has been done to evaluate their potential to catalyze industrially important reactions. In the present article, we highlight the latest research on lipases obtained through metagenomic tools, focusing on studies of activity and stability and investigations of application in biocatalysis. We also discuss the challenges of metagenomic approaches for the bioprospecting of new lipases.

Immobilization of Pseudomonas cepacia lipase on layered double hydroxide of Zn/Al-Cl for kinetic resolution of rac-1-phenylethanol.

Layered double hydroxides (LDHs) are cheap materials suitable for immobilization of enzymes. In this study, we prepared Zn/Al-Cl LDHs with different Zn:Al molar ratios for immobilization of the lipase from Pseudomonas cepacia. The best values for activity retention (188%), immobilization efficiency (96%) and hydrolytic activity in organic medium (279 U g-1) were obtained with a molar ratio of Zn:Al of 4:1, a protein loading of 162 mg g-1 and Tris-HCl buffer (10 mmol L-1, pH 7.5) as the solvent for preparing the lipase solution. The immobilized lipase keeps its activity when stored at 4 °C during 30 days. The immobilized lipase gave a conversion of 50% in 1 h for the kinetic resolution of the alcohol rac-1-phenylethanol, with both ees and eep higher than 99% and E higher than 200. In the reutilization study, 30 successive 1-h kinetic resolutions were done with the same batch of immobilized enzyme. For all 30 resolutions, 50% conversion was maintained, with ees and eep higher than 99% and E higher than 200. These are promising results that lay the basis for further studies of immobilization of lipases onto LDHs for applications in organic media.

Biochemical characterization and application of a new lipase and its cognate foldase obtained from a metagenomic library derived from fat-contaminated soil.

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)­1­phenylethyl acetate for the kinetic resolution of (R,S)­1­phenyl­1­ethanol. The results obtained in this work provide a basis for the development of applications of Lip-LifMF3 in biocatalysis.

Genome sequencing of Burkholderia contaminans LTEB11 reveals a lipolytic arsenal of biotechnological interest.

Burkholderia contaminans LTEB11 is a Gram-negative betaproteobacterium isolated as a contaminant of a culture in mineral medium supplemented with vegetable oil. Here, we report the genome sequence of B. contaminans LTEB11, identifying and analyzing the genes involved in its lipolytic machinery and in the production of other biotechnological products.

Design and Operation of a Pilot-Scale Packed-Bed Bioreactor for the Production of Enzymes by Solid-State Fermentation.

In this review, we describe our experience in building a pilot-scale packed-bed solid-state fermentation (SSF) bioreactor, with provision for intermittent mixing, and the use of this bioreactor to produce pectinases and lipases by filamentous fungi. We show that, at pilot scale, special attention must be given to several aspects that are not usually problematic when one works with laboratory-scale SSF bioreactors. For example, it can be a challenge to produce large amounts of inoculum if the fungus does not sporulate well. Likewise, at larger scales, the air preparation system needs as much attention as the bioreactor itself. Sampling can also be problematic if one wishes to avoid disrupting the bed structure. In the fermentations carried out in the pilot bioreactor, when the substrate bed contained predominantly wheat bran, the bed shrank away from the walls, providing preferential flow paths for the air and necessitating agitation of the bed. These problems were avoided by using beds with approximately 50% of sugarcane bagasse. We also show how a mathematical model that describes heat and water transfer in the bed can be a useful tool in developing appropriate control schemes. Graphical Abstract.

Fermented Solids and Their Application in the Production of Organic Compounds of Biotechnological Interest.

We review the application of dry fermented solids (DFS) containing naturally immobilized enzymes as catalysts in synthesis and in hydrolysis reactions. The most studied application is the use of DFS containing lipases in the synthesis of biodiesel esters, by transesterification of oils or by esterification of fatty acids with short-chain alcohols in solvent-free reaction media. Other applications of DFS that have been studied include the use of DFS containing pectinases to liberate D-galacturonic acid from pectin and the production of high-value compounds by DFS containing lipases, such as the synthesis of sugar esters and the production of pure enantiomers by resolution of racemic mixtures. To date, studies are limited to proof of concept, and there are still many challenges to be faced in the development of industrial-scale processes using DFS as catalysts. A key challenge is the relatively low activity of DFS compared to commercial enzyme preparations. Attention needs to be given to scale up, not only of the bioreactor for the application of the DFS but also for the production of the fermented solids. There is also a need for economic feasibility studies to determine whether the production of DFS and their use as catalysts can be competitive at industrial scale. Graphical Abstract.

Tailoring recombinant lipases: keeping the His-tag favors esterification reactions, removing it favors hydrolysis reactions.

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.

Co-expression, purification and characterization of the lipase and foldase of Burkholderia contaminans LTEB11.

Genes encoding lipase LipBC (lipA) and foldase LifBC (lipB) were identified in the genome of Burkholderia contaminans LTEB11. Analysis of the predicted amino acid sequence of lipA showed its high identity with lipases from Pseudomonas luteola (91%), Burkholderia cepacia (96%) and Burkholderia lata (97%), and classified LipBC lipase in the lipase subfamily I.2. The genes lipA and lipB were amplified and cloned into expression vectors pET28a(+) and pT7-7, respectively. His-tagged LipBC and native LifBC were co-expressed in Escherichia coli and purified. LipBC and LifBC have molecular weights of 35.9 kDa and 37 kDa, respectively, and remain complexed after purification. The Lip-LifBC complex was active and stable over a wide range of pH values (6.5-10) and temperatures (25-45 °C), with the highest specific activity (1426 U mg-1) being against tributyrin. The Lip-LifBC complex immobilized on Sepabeads was able to catalyze the synthesis of ethyl-oleate in n­hexane with an activity of 4 U g-1, maintaining high conversion (>80%) over 5 reaction cycles of 6 h at 45 °C. The results obtained in this work provide a basis for the development of applications of recombinant LipBC in biocatalysis.

Optimization of biodiesel synthesis by esterification using a fermented solid produced by Rhizopus microsporus on sugarcane bagasse.

A fermented solid containing lipases was produced by solid-state fermentation of Rhizopus microsporus on sugarcane bagasse enriched with urea, soybean oil, and a mineral solution. The dry fermented solid produced using R. microsporus (RMFS) was used to catalyze the synthesis of alkyl-esters by esterification in a solvent-free system containing ethanol and oleic acid (as a model system) or a mixture of fatty acids obtained from the physical hydrolysis of soybean soapstock acid oil (FA-SSAO) in subcritical water. The conversions were 93.5 and 84.1%, for oleic acid and FA-SSAO, respectively, at 48 h and 40 °C, at a molar ratio (MR) of ethanol to fatty acid of 5:1. A further increase in the MR to 10:1 improved the production of ethylic-esters, giving conversions at 48 h of 98 and 86% for oleic acid and FA-SSAO, respectively. The results obtained in this work foster further studies on scaling-up of an environmentally friendly process to produce biofuels.

The introduction of the fungal D-galacturonate pathway enables the consumption of D-galacturonic acid by Saccharomyces cerevisiae.

BACKGROUND: Pectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. One possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. However, the ethanol-producing organism of choice, Saccharomyces cerevisiae, is not able to catabolize D-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, namely, 18 % (w/w) from citrus pulp and 16 % (w/w) sugar beet pulp. RESULTS: In the current work, we describe the construction of a strain of S. cerevisiae in which the five genes of the fungal reductive pathway for D-galacturonic acid catabolism were integrated into the yeast chromosomes: gaaA, gaaC and gaaD from Aspergillus niger and lgd1 from Trichoderma reesei, and the recently described D-galacturonic acid transporter protein, gat1, from Neurospora crassa. This strain metabolized D-galacturonic acid in a medium containing D-fructose as co-substrate. CONCLUSION: This work is the first demonstration of the expression of a functional heterologous pathway for D-galacturonic acid catabolism in Saccharomyces cerevisiae. It is a preliminary step for engineering a yeast strain for the fermentation of pectin-rich substrates to ethanol.

A model for growth of a single fungal hypha based on well-mixed tanks in series: simulation of nutrient and vesicle transport in aerial reproductive hyphae.

Current models that describe the extension of fungal hyphae and development of a mycelium either do not describe the role of vesicles in hyphal extension or do not correctly describe the experimentally observed profile for distribution of vesicles along the hypha. The present work uses the n-tanks-in-series approach to develop a model for hyphal extension that describes the intracellular transport of nutrient to a sub-apical zone where vesicles are formed and then transported to the tip, where tip extension occurs. The model was calibrated using experimental data from the literature for the extension of reproductive aerial hyphae of three different fungi, and was able to describe different profiles involving acceleration and deceleration of the extension rate. A sensitivity analysis showed that the supply of nutrient to the sub-apical vesicle-producing zone is a key factor influencing the rate of extension of the hypha. Although this model was used to describe the extension of a single reproductive aerial hypha, the use of the n-tanks-in-series approach to representing the hypha means that the model has the flexibility to be extended to describe the growth of other types of hyphae and the branching of hyphae to form a complete mycelium.

Immobilization and characterization of a new regioselective and enantioselective lipase obtained from a metagenomic library.

In previous work, a new lipase and its cognate foldase were identified and isolated from a metagenomic library constructed from soil samples contaminated with fat. This new lipase, called LipG9, is a true lipase that shows specific activities that are comparable to those of well-known industrially-used lipases with high activity against long-chain triglycerides. In the present work, LipG9 was co-expressed and co-immobilized with its foldase, on an inert hydrophobic support (Accurel MP1000). We studied the performance of this immobilized LipG9 (Im-LipG9) in organic media, in order to evaluate its potential for use in biocatalysis. Im-LipG9 showed good stability, maintaining a residual activity of more than 70% at 50 °C after incubation in n-heptane (log P 4.0) for 8 h. It was also stable in polar organic solvents such as ethanol (log P -0.23) and acetone (log P -0.31), maintaining more than 80% of its original activity after 8 h incubation at 30 °C. The synthesis of ethyl esters was tested with fatty acids of different chain lengths in n-heptane at 30 °C. The best conversions (90% in 3 h) were obtained for medium and long chain saturated fatty acids (C8, C14 and C16), with the maximum specific activity, 29 U per gram of immobilized preparation, being obtained with palmitic acid (C16). Im-LipG9 was sn-1,3-specific. In the transesterification of the alcohol (R,S)-1-phenylethanol with vinyl acetate and the hydrolysis of the analogous ester, (R,S)-1-phenylethyl acetate, Im-LipG9 showed excellent enantioselectivity for the R-isomer of both substrates (E> 200), giving an enantiomeric excess (ee) of higher than 95% for the products at 49% conversion. The results obtained in this work provide the basis for the development of applications of LipG9 in biocatalysis.

Modeling the Growth of Filamentous Fungi at the Particle Scale in Solid-State Fermentation Systems.

Solid-state fermentation (SSF) with filamentous fungi is a promising technique for the production of a range of biotechnological products and has the potential to play an important role in future biorefineries. The performance of such processes is intimately linked with the mycelial mode of growth of these fungi: Not only is the production of extracellular enzymes related to morphological characteristics, but also the mycelium can affect bed properties and, consequently, the efficiency of heat and mass transfer within the bed. A mathematical model that describes the development of the fungal mycelium in SSF systems at the particle scale would be a useful tool for investigating these phenomena, but, as yet, a sufficiently complete model has not been proposed. This review presents the biological and mass transfer phenomena that should be included in such a model and then evaluates how these phenomena have been modeled previously in the SSF and related literature. We conclude that a discrete lattice-based model that uses differential equations to describe the mass balances of the components within the system would be most appropriate and that mathematical expressions for describing the individual phenomena are available in the literature. It remains for these phenomena to be integrated into a complete model describing the development of fungal mycelia in SSF systems.

Evaluation of the structural composition and surface properties of rhamnolipid mixtures produced by Pseudomonas aeruginosa UFPEDA 614 in different cultivation periods.

We studied the production of rhamnolipids by Pseudomonas aeruginosa UFPEDA 614 in submerged culture, using glycerol as the carbon source. A rhamnolipid yield of 15.9 g/L was obtained with 40 g/L glycerol and 5 g/L sodium nitrate as nitrogen source after 7 days of cultivation. Structural analysis carried out at different cultivation periods showed that the four major mono-rhamnolipid homologues are present in higher proportion in the first 48 h. Over time, the corresponding four major di-rhamnolipid homologues predominated, representing about 75 % of the total rhamnolipids after 96 h. Physicochemical analysis of the rhamnolipid mixtures obtained at different cultivation periods showed that the sample obtained from the first day of cultivation had the lower critical micelle concentration (15.6 mg/L), which is probably related to the higher proportion of mono-rhamnolipids. The results presented here show that the composition of the mixture of rhamnolipid homologues produced by P. aeruginosa UFPEDA 614 varies over time and that this variation influences the physicochemical properties of the mixture. These findings can be used in order to produce rhamnolipid mixtures that have suitable properties for different applications.

First co-expression of a lipase and its specific foldase obtained by metagenomics.

BACKGROUND: Metagenomics is a useful tool in the search for new lipases that might have characteristics that make them suitable for application in biocatalysis. This paper reports the cloning, co-expression, purification and characterization of a new lipase, denominated LipG9, and its specific foldase, LifG9, from a metagenomic library derived from a fat-contaminated soil. RESULTS: Within the metagenomic library, the gene lipg9 was cloned jointly with the gene of the foldase, lifg9. LipG9 and LifG9 have 96% and 84% identity, respectively, with the corresponding proteins of Aeromonas veronii B565. LipG9 and LifG9 were co-expressed, both in N-truncated form, in Escherichia coli BL21(DE3), using the vectors pET28a(+) and pT7-7, respectively, and then purified by affinity chromatography using a Ni(2+) column (HiTrap Chelating HP). The purified enzyme eluted from the column complexed with its foldase. The molecular masses of the N-truncated proteins were 32 kDa for LipG9, including the N-terminal His-tag with 6 residues, and 23 kDa for LifG9, which did not have a His-tag. The biochemical and kinetic characteristics of the purified lipase-foldase preparation were investigated. This preparation was active and stable over a wide range of pH values (6.5-9.5) and temperatures (10-40°C), with the highest specific activity, of 1500 U mg(-1), being obtained at pH 7.5 at 30°C. It also had high specific activities against tributyrin, tricaprylin and triolein, with values of 1852, 1566 and 817 U mg(-1), respectively. A phylogenetic analysis placed LipG9 in the lipase subfamily I.1. A comparison of the sequence of LipG9 with those of other bacterial lipases in the Protein Data Bank showed that LipG9 contains not only the classic catalytic triad (Ser(103), Asp(250), His(272)), with the catalytic Ser occurring within a conserved pentapeptide, Gly-His-Ser-His-Gly, but also a conserved disulfide bridge and a conserved calcium binding site. The homology-modeled structure presents a canonical α/ß hydrolase folding type I. CONCLUSIONS: This paper is the first to report the successful co-expression of a lipase and its associated foldase from a metagenomic library. The high activity and stability of Lip-LifG9 suggest that it has a good potential for use in biocatalysis.

Pectinase activity determination: an early deceleration in the release of reducing sugars throws a spanner in the works!

Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.