Magnetic biocatalysts and their uses to obtain biodiesel and biosurfactants.

Nanobiocatalysis, as the synergistic combination of nanotechnology and biocatalysis, is rapidly emerging as a new frontier of biotechnology. The use of immobilized enzymes in industrial applications often presents advantages over their soluble counterparts, mainly in view of stability, reusability and simpler operational processing. Because of their singular properties, such as biocompatibility, large and modifiable surface and easy recovery, iron oxide magnetic nanoparticles (MNPs) are attractive super-paramagnetic materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field. Cross-linked enzyme aggregates (CLEAs) have several benefits in the context of industrial applications since they can be cheaply and easily prepared from unpurified enzyme extracts and show improved storage and operational stability against denaturation by heat and organic solvents. In this work, by using the aforementioned advantages of MNPs of magnetite and CLEAs, we prepared two robust magnetically-separable types of nanobiocatalysts by binding either soluble enzyme onto the surface of MNPs functionalized with amino groups or by cross-linking aggregates of enzyme among them and to MNPs to obtain magnetic CLEAs. For this purpose the lipase B of Candida antarctica (CALB) was used. The hydrolytic and biosynthetic activities of the resulting magnetic nanobiocatalysts were assessed in aqueous and organic media. Thus, the hydrolysis of triglycerides and the transesterification reactions to synthesize biodiesel and biosurfactants were studied using magnetic CLEAs of CALB. The efficiency and easy performance of this magnetic biocatalysis validates this proof of concept and sets the basis for the application of magnetic CLEAs at industrial scale.

Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica lipase: an efficient and stable biocatalyst for biodiesel synthesis.

Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with -NH2 groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available.

Polyester hydrolytic and synthetic activity catalyzed by the medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Streptomyces venezuelae SO1.

The extracellular medium-chain-length polyhydroxyalkanote (MCL-PHA) depolymerase from an isolate identified as Streptomyces venezuelae SO1 was purified to electrophoretic homogeneity and characterized. The molecular mass and pI of the purified enzyme were approximately 27 kDa and 5.9, respectively. The depolymerase showed its maximum activity in the alkaline pH range and 50 °C and retained more than 70 % of its initial activity after 8 h at 40 °C. The MCL-PHA depolymerase hydrolyzes various p-nitrophenyl-alkanoates and polycaprolactone but not polylactide, poly-3-hydroxybutyrate, and polyethylene succinate. The enzymatic activity was markedly enhanced by the presence of low concentrations of detergents and organic solvents, being inhibited by dithiothreitol and EDTA. The potential of using the enzyme to produce (R)-3-hydroxyoctanoate in aqueous media or to catalyze ester-forming reactions in anhydrous media was investigated. In this sense, the MCL-PHA depolymerase catalyzes the hydrolysis of poly-3-hydroxyoctanoate to monomeric units and the ring-opening polymerization of ß-butyrolactone and lactides, while ε-caprolactone and pentadecalactone were hardly polymerized.

Characterization of a novel subgroup of extracellular medium-chain-length polyhydroxyalkanoate depolymerases from actinobacteria.

Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from natural sources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganisms to hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(ε-caprolactone) (PCL), poly(ethylene succinate) (PES), and poly(l-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Streptomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted of one polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depolymerase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximum activity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) was used as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of several Actinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomyces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity to mcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new subgroup of mcl PHA depolymerases.

Production of chiral (R)-3-hydroxyoctanoic acid monomers, catalyzed by Pseudomonas fluorescens GK13 poly(3-hydroxyoctanoic acid) depolymerase.

The extracellular medium-chain-length polyhydroxyalkanoate (MCL-PHA) depolymerase of Pseudomonas fluorescens GK13 catalyzes the hydrolysis of poly(3-hydroxyoctanoic acid) [P(3HO)]. Based on the strong tendency of the enzyme to interact with hydrophobic materials, a low-cost method which allows the rapid and easy purification and immobilization of the enzyme has been developed. Thus, the extracellular P(3HO) depolymerase present in the culture broth of cells of P. fluorescens GK13 grown on mineral medium supplemented with P(3HO) as the sole carbon and energy source has been tightly adsorbed onto a commercially available polypropylene support (Accurel MP-1000) with high yield and specificity. The activity of the pure enzyme was enhanced by the presence of detergents and organic solvents, and it was retained after treatment with an SDS-denaturing cocktail under both reducing and nonreducing conditions. The time course of the P(3HO) hydrolysis catalyzed by the soluble and immobilized enzyme has been assessed, and the resulting products have been identified. After 24 h of hydrolysis, the dimeric ester of 3-HO [(R)-3-HO-HO] was obtained as the main product of the soluble enzyme. However, the immobilized enzyme catalyzes almost the complete hydrolysis of P(3HO) polymer to (R)-3-HO monomers under the same conditions.

Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1.

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His(6)PheA1 and His(6)PheA2 were purified and its catalytic activity characterized. His(6)PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His(6)PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His(6)PheA1 and His(6)PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.

Purification, characterization and cloning of aldehyde dehydrogenase from Rhodococcus erythropolis UPV-1.

The enzyme responsible for formaldehyde removal in industrial wastewaters by cells of Rhodococcus erythropolis UPV-1 was identified as a broad-specific aldehyde dehydrogenase (EC 1.2.1.3). The enzyme was purified to electrophoretic homogeneity from ethanol-grown cells with a specific activity of 19.5 U mg-1 protein and an activity recovery of 56%. The enzyme showed an isoelectric point (pI) of 5.3 and was a trimer of 162 kDa consisting of three identical 54-kDa subunits. It was specific for NAD+ and showed hyperbolic kinetics for this coenzyme (Km=90 microM), but sigmoidal kinetics for the aliphatic aldehydes used as substrates. The enzyme affinity for aldehydes increased with their hydrocarbon chain length, ranging from 333 microM for formaldehyde to 85 nM for n-octanal. The corresponding calculated Hill coefficients were in the 1.55-2.77 range. With n-propanal as substrate, the optimum pH and temperature for activity were 9.5-10.0 and 47.5 degrees C, respectively, with an Ea for catalysis of 28.6 kJ mol-1. NAD+ protected the enzyme against thermal inactivation, but aldehydes were ineffective. The activity was severely inhibited by p-hydroxymercuribenzoate, indicating that a thiol was essential for catalysis. The 1,524-bp aldhR gene encoding a 507-amino-acid protein was expressed in cells of Escherichia coli M15 as a hexahistidine-tagged protein.

Purification and properties of NrtC and NrtD, the ATP-binding subunits of the ABC nitrate/nitrite transporter of Phormidium laminosum.

A genomic region from the thermophilic, filamentous, nondiazotrophic cyanobacterium Phormidium laminosum including nrtC and nrtD was cloned and sequenced. These genes encode NrtC and NrtD, the ATP-binding subunits of the ABC bispecific transporter of nitrate/nitrite NRT. We report a different nrtC sequence from the one previously reported (Merchán et al., Plant Mol. Biol. 28:759-766, 1995) and we identified the presence of nrtD gene downstream nrtC in the nirA operon. Each gene was expressed in E. coli cells as a hexahistidine-tagged fusion protein. The recombinant proteins (His(6)NrtC and His(6)NrtD) were purified, and their ability to catalyze the hydrolysis of ATP and other nucleosides triphosphate was characterized. Both subunits showed its maximum ATPase activity at 45-50 degrees C and pH 8.0, and similar K(m) (0.49 and 0.43 mM) and V(max) (0.085 and 0.114 U mg(-1) protein, respectively) values were calculated. The native NrtC subunit purified from nitrogen-starved cells of P. laminosum also hydrolyzed ATP in vitro in the absence of other components of NRT. These findings indicated that NrtC and NrtD are responsible for ATP-hydrolysis to energize the active transporter NRT. The effect of some activators (Mg(2+)) and inhibitors (ADP) on the ATPase activity of the subunits was assessed as well as the effect of some potential regulatory metabolites on His(6)NrtC. The existence in vitro of homodimers of either NrtC or NrtD but not heterodimers of both subunits was confirmed by matrix assisted laser desorption ionization-time of flight mass spectrometry and/or electrophoresis in non-denaturing conditions. Finally, the existence in vivo of NrtC-NrtD heterodimers is discussed.

The nitrate/nitrite ABC transporter of Phormidium laminosum: phosphorylation state of NrtA is not involved in its substrate binding activity.

Most cyanobacteria take up nitrate or nitrite through a multisubunit ABC transporter (ATP-binding cassette) located in the cytoplasmic membrane. Nitrate and nitrite transport activity is instantaneously blocked by the presence of ammonium in the medium. Previous biochemical studies reported the existence of phosphorylation/dephosphorylation events of the nitrate transporter (NRT) related to the presence of ammonium-sensitive kinase/phosphatase activities in plasma membranes of the cyanobacterium Synechococcus elongatus PCC 6301. In this work, we have analyzed the biochemical properties of the periplasmic nitrate/nitrite-binding subunit (NrtA) of NRT from the thermophilic nondiazotrophic cyanobacterium Phormidium laminosum. Our results show that cyanobacterial NrtA is phosphorylated in vivo. However, substrate binding activity in vitro is not affected by the phosphorylation state of the protein, ruling out the possibility that phosphorylation/dephosphorylation of NrtA is involved in the regulation of the nitrate/nitrite uptake by NRT transporter. Moreover, NrtA is present as multiple isoforms showing the same molecular mass but different isoelectric points ranging from pI 5 to 6. Mass spectrometric characterization of NrtA isoforms shows that the protein is phosphorylated at residue Tyr203, and contains several methionine sulphoxide residues which account for the observed isoforms. Both phosphorylated and non-phosphorylated forms of NrtA are active in vitro, showing comparable binding affinity for nitrate and nitrite. Both substrates behave as pure competitive inhibitors with a binding stoichiometry of one molecule of anion per NrtA monomer.

Characterization of the N-terminal domain of NrtC, the ATP-binding subunit of ABC-type nitrate transporter of the cyanobacterium Phormidium laminosum.

The N-terminal domain of NrtC, the ATP-binding subunit of nitrate/nitrite ABC-transporter in the cyanobacterium Phormidium laminosum, has been expressed in Escherichia coli as a histidine-tagged fusion protein (His(6)NrtC1). Binding of ATP to the pure His(6)NrtC1 was characterized using the nucleotide analogue TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate]. Fluorescence assays showed that His(6)NrtC1 specifically binds Mg(2+) TNP-ATP with high affinity, binding being dependent on protein concentration. The presence of ATP prevents the covalent modification of His(6)NrtC1 by fluorescein 5'-isothiocyanate (FITC), suggesting that this probe reacts at the nucleotide-binding site of NrtC. The active form of the truncated NrtC is a dimer that shows high affinity for TNP-ATP (K(d)=0.76+/-0.1 microM). Evidence for the presence of two nucleotide-binding sites per dimer protein is given. Our results indicate that nucleotide binding is strongly dependent on the dimerization of NrtC and that the N-terminal domain of the protein contains the binding site for ATP. No ATPase activity catalyzed in vitro by the truncated subunit was detected.

Degradation of phenol by Rhodococcus erythropolis UPV-1 immobilized on Biolite in a packed-bed reactor.

A strain of Rhodococcus erythropolis has been isolated and identified by 16S rRNA sequencing. Cells acclimated to phenol can be adsorbed on the external surface of beads of the ceramic support Biolite where they grow forming a network of large filaments. Exponentially-growing cells were adsorbed faster than their stationary-phase counterparts. Immobilization resulted in a remarkable enhancement of the respiratory activity of cells and a shorter lag phase preceding the active phenol degradation. Under optimum operation conditions, the immobilized cells in a laboratory-scale column reactor packed with support beads were able to degrade completely phenol in defined mineral medium at a maximum rate of 18 kg phenol m(-3) per day. The performance of the bioreactor in long-term continuous operation was characterized by pumping defined mineral medium which contained different concentrations of phenol at different flow-rates. Once phenol biodegradation in defined mineral medium was well established, an industrial wastewater from a resin manufacturing company, which contained both phenol and formaldehyde, was tested. In this case, after wastewater conditioning (i.e. pH, nitrogen source and micronutrient amendments) the immobilized cells were able to remove completely formaldehyde and to partly biodegrade phenols at a rate of 1 kg phenol m(-3) per day.

Changes in intracellular amino acids and organic acids induced by nitrogen starvation and nitrate or ammonium resupply in the cyanobacterium Phormidium laminosum.

In Phormidium laminosum cells, nitrogen starvation caused a decrease in the intracellular levels of all amino acids, except glutamate, and an increase in the total level of the analyzed organic acids. The addition of nitrate or ammonium to N-starved cells resulted in substantial increases in the pool size of most amino acids. Upon addition of ammonium the total level of organic acids diminished, whereas it increased upon addition of nitrate, after a transient decay during the first minutes. Nitrogen resupply stimulated amino acid synthesis, the effect being faster and higher when ammonium was assimilated. The data indicate that nitrate and ammonium assimilation induced an enhancement of carbon flow through the glycolytic and the tricarboxylic-acid pathways to amino acid biosynthesis, with a concurrent decrease in the carbohydrate reserves. The results suggest that the availability of carbon skeletons limited the rate of ammonium assimilation, whereas the availability of reducing equivalents limited the rate of nitrate assimilation.