María J. Llama

Industrial applications of pectic enzymes: a review

Abstract Although pectic enzymes have long been used to increase the yield and clarity of fruit juices, it is only recently that technological innovations, such as the use of immobilization supports and continuous-flow systems, have been considered to optimize these fruit processing procedures. To our knowledge, this is the first review to focus on the benefits brought to the field by these new technologies and their potential for commercial applications.

Aerobic chromate reduction by Bacillus subtilis

We have studied the reduction of hexavalent chromium (chromate) to the less toxic trivalent form by using cell suspensions and cell-free extracts from the common soil bacterium, Bacillus subtilis. B. subtilis was able to grow and reduce chromate at concentrations ranging from 0.1 to 1 mM K2CrO4. Chromate reduction was not affected by a 20-fold excess of nitrate-compound that serves as alternate electron acceptor and antagonizes chromate reduction by anaerobic bacteria. Metabolic poisons including sodium azide and sodium cyanide inhibited chromate reduction. Reduction was effected by a constitutive system associated with the soluble protein fraction and not with the membrane fraction. The reducing activity was heat labile and showed a Km of 188 μm CrO42-. The reductase can mediate the transfer of electrons from NAD(P)H to chromate. The results suggest that chromate is reduced via a detoxification system rather than dissimilatory electron transport.

Immobilization of Candida rugosa lipase and some properties of the immobilized enzyme

Lipase (triacylglycerol ester hydrolase, E.C.3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene. The enzyme was rapidly adsorbed on the support, and more than 60% of the soluble activity disappeared from the medium after 1 min of incubation at room temperature. A recovery of immobilized activity of 21% was obtained when the wet preparation was immediately assayed with olive oil at the end of the immobilization protocol. The activity of the immobilized enzyme drastically decreased with the loss of water of the preparation. Pretreatment of the support with organic solvents significantly increased the recovered immobilized activity. Our results strongly suggest that the soluble lipase could exist in different aggregation forms depending on the pH of the medium. At acidic pH, the relative proportion of high-molecular-weight forms of the enzyme is higher than at pH 7.0, suggesting that the lipase would be also immobilized in different aggregation forms depending on the pH used in the immobilization procedure. Crosslinking of the adsorbed enzyme with glutaraldehyde diminished its activity but increased the stability of the lipase against the washing-out effect of Triton X-100. Data on the most relevant catalytic properties of the soluble and immobilized enzyme, such as optimum pH and temperature as well as ranges of stability, kinetic parameters, and activation energy for the hydrolysis of olive oil and p-nitrophenyl acetate, are reported.

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.

Biosorption of heavy metals to immobilised Phormidium laminosum biomass

The capacity to biosorb Cu(II), Fe(II), Ni(II) and Zn(II) by non-viable biomass of the cyanobacterium Phormidium laminosum entrapped in polysulfone and epoxy resin beads was investigated. The biosorption process depended on the wetting of biomass beads, the rate of metal biosorption decreasing when dry biomass beads were used. A decrease in the immobilised biomass bead size led to an increase in the rate of metal biosorption. The amount of metal biosorbed increased with the biomass and the amount of metal available. The biosorbed metal was completely desorbed from the biomass beads by washing with 0.1 M HCl. Polysulfone biomass beads can be reused for, at least, ten consecutive biosorption/desorption cycles without apparent loss of efficiency after its reconditioning with 0.1 M NaOH.

Nitrate removal from water by Scenedesmus obliquus immobilized in polymeric foams

Abstract Scenedesmus obliquus cells were immobilized by adsorption in preformed polyurethane and polyvinyl foams as well as by entrapment using urethane prepolymer. Adsorption on polymeric foams appeared to be the most convenient immobilization method, mainly when cells were initially adsorbed in a nitrogen-free medium. Nitrogen starvation of cells appeared to enhance their adsorption to the polymeric foams. The net O 2 evolution activity and nitrate removal capacity of free-living and polyvinyl-adsorbed cells were similar, indicating that immobilization did not significantly affect the physiology of microalgae. The N removal capacity of polyvinyl-adsorbed cells was studied in batch and in a lab-scale continuous-flow bioreactor. Nitrogen starvation greatly increased the N uptake rate of immobilized cells. The effects of illumination (intensity and photoperiod), residence time, and CO 2 supply were investigated with respect to the N removal efficiency of the bioreactor. It was concluded that S. obliquus cells adsorbed in hydrophilic, commercially available polymeric foams are of potential value for biologic N depollution of drinking waters contaminated with nitrogenous fertilizers.

Formaldehyde removal in synthetic and industrial wastewater by Rhodococcus erythropolis UPV-1.

Abstract. Rhodococcus erythropolis strain UPV-1 is able to grow on phenol as the only carbon and energy source and to remove formaldehyde completely from both synthetic and industrial wastewater. The rate of formaldehyde removal is independent of either initial biomass or formaldehyde concentration. The presence of viable, intact cells is strictly necessary for this removal to take place. Discontinuous and continuous formaldehyde-feed systems were successfully tested with synthetic wastewater in shaken flasks. Once biodegradation was well established in model synthetic wastewater, a real wastewater sample was obtained from a local phenolic and melamine resin-manufacturing company. Incubation of biomass with this wastewater at subtoxic concentrations of formaldehyde resulted in the complete removal of the pollutant. Parameters, such as chemical oxygen demand and toxicity, were assessed as indicators of wastewater cleanup progress.

Hydrolysis of animal fats by immobilized Candida rugosa lipase

Lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) from Candida rugosa was immobilized by adsorption on a commercially available microporous polypropylene support of 200- to 400-μm particle size. A contact period of 90 min allowed the highest degrees of hydrolysis to be achieved, particularly in the second and third hydrolysis reactions. The optimal hydrolysis conditions were 0.10 kg enzyme per kilogram fat, 50% (w/v) fat, and 40°C for 24 h. The immobilized enzyme can be repeatedly used and hydrolysis degrees of 90% or higher can be achieved. Of the three animal fats studied, edible pork lard consistently yielded the highest degrees of hydrolysis (95%) in the first hydrolysis reaction and inedible beef tallow the lowest (65%). The immobilized enzyme lost its activity above 45°C. The support could be easily recovered and reused up to 5 times.

Purification and some properties of the pectin lyase from Penicillium italicum

For the first time, a pectin lyase (poly(methoxygalacturonide)lyase; EC 4.2.2.10) from a member of the generus Penicillium was isolated, purified to homogeneity and characterized. The monomeric enzyme from Penicillium italicum CECT 2294 culture filtrates showed a molecular mass of 34 kDa after SDS‐electrophoresis in polyacrylamide gradient gels, and the isoelectric point was 8.6 as determined by isoelectric focusing. The optimum pH (9.0), the high pH and temperature stabilities, the ability to degrade pectins from different sources and with a wide range of degrees of esterification (from 37% to 86%) as well as the importance of this type of biocatalysts in the food industry make this enzyme an interesting subject of study.

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.