Structural differences of commercial and recombinant lipase B from Candida antarctica: An important implication on enzymes thermostability.

Lipase B from Candida antarctica (CalB) is the most widely used lipase, including in many industrial sectors, such as in biodiesel and pharmaceuticals production. CalB has been produced by heterologous expression using Pichia pastoris under PGK constitutive promoter (named LipB). Here, we have studied the structural features of commercial CalB and LipB enzymes using circular dichroism and fluorescence under different conditions. In the presence of denaturing agents CalB was more stable than LipB, in contrast, at increasing temperatures, LipB was more thermostable than CalB. Mass spectrometry data indicates that both enzymes have an insertion of amino acids related to α-factor yeast signal, however LipB enzyme showed the addition of nine residues at the N-terminal while CalB showed only four residues. Molecular modeling of LipB showed the formation of an amphipathic α-helix in N-terminal region that was not observed in CalB. This data suggests that this new α-helix possess could be involved in LipB thermostability. These results associated with new structural studies may provide information to the design of novel biocatalysts.

Assessing a mixture of biosurfactant and enzyme pools in the anaerobic biological treatment of wastewater with a high-fat content.

The use of rhamnolipid-type biosurfactant produced by Pseudomonas aeruginosa was evaluated for solubilization of fat present in effluent from a poultry processing plant, followed by anaerobic biological treatment. The rhamnolipid was used in combination with enzyme pools produced by solid medium fermentation of the fungi Penicillium simplicissimum and Penicillium brevicompactum. In experiments with raw effluent, the accumulation of fat caused the specific methane production (SMP) to be much less than with pretreated effluent (0.074 vs. 0.167 L CH4/g chemical oxygen demand (COD) removed). In experiments with pretreated effluent, the SMP ranged from 0.105 to 0.207 L CH4/g CODremoved. A statistical analysis of the results of four sequential batches found that all variables had a significant effect on the SMP in the fourth batch. A fifth batch was initiated for three conditions, and it led to the highest SMP when compared with the control, which showed similar behaviour for the SMP over time, ending up with values three times greater than the SMP in the control conditions. The residual oil and grease analysis revealed removals from 51% to 90% with pretreated effluent and only 1% in the control conditions with raw effluent. Thus, the best synergistic effect of fat release/hydrolysis of effluent components from a poultry processing plant was found with a 0.5% P. brevicompactum pool and rhamnolipid at half the critical micelle concentration (24 mg/L).

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds.

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Profiles of fatty acids and triacylglycerols and their influence on the anaerobic biodegradability of effluents from poultry slaughterhouse.

The hydrolysis of effluent from a poultry slaughterhouse containing 800 mg oil and grease (O&G)/L was conducted with 1% (w/v) of an enzymatic pool obtained by solid-state fermentation with the fungus Penicillium restrictum. The chromatographic evaluation of the lipid profile during hydrolysis indicated a higher concentration of acids after 4h of reaction (2954 mg/L), with a predominance of oleic, palmitic, and linoleic acids. Effluent aliquots were collected after 4, 8, and 24h of hydrolysis and tested for anaerobic biodegradation in sequential batches. An adaptation of the biomass was observed, both in the control experiment (with non-hydrolyzed raw effluent) and in the experiments with enzymatically pre-treated effluent. The specific methane production in the control experiment was 0.248 L CH(4)/g COD(consumed), and in the experiment with effluent pre-treated for 4h, this production was 0.393 L CH(4)/g COD(consumed), indicating a higher methane production after enzymatic hydrolysis.

Evaluation of different pre-hydrolysis times and enzyme pool concentrations on the biodegradability of poultry slaughterhouse wastewater with a high fat content.

The effect of different hydrolysis times (4, 8 and 24 h) of a lipase-rich enzymatic preparation (0.1, 0.5 and 1.0% w/v) produced by fungus Penicillium sp. in solid-state fermentation was evaluated on the anaerobic biodegradability of a poultry slaughterhouse wastewater with 800 mg oil and grease [O&G]/L in three sequential batches. The enzymatic pre-treatment of O&G in the experiments with no acclimated sludge had no discernible effect because regardless of the conditions adopted COD removal efficiencies of 95.3% to 98.7% were obtained. However, when the sludge was reused (once or twice) the COD removal efficiencies in the control experiments (69.8% and 53.4%) were considerably lower than in the experiments with hydrolyzed effluent (of 93.8% to 98.4%). Higher values of specific methane production were obtained with 0.1% SEP and 4 h of hydrolysis. After acclimation of the sludge, 19.9% fat was still adhered to the sludge in the control experiment, while the sludge in the experiment with enzymatically pre-treated effluent contained only 8.6% fat, confirming the accumulation of fat when the enzyme pool was not used.

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces.

AIMS: To investigate the effect of the biosurfactants surfactin and rhamnolipids on the adhesion of the food pathogens Listeria monocytogenes, Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces. METHODS AND RESULTS: Quantification of bacterial adhesion was performed using the crystal violet staining technique. Preconditioning of surfaces with surfactin caused a reduction on the number of adhered cells of Ent. sakazakii and L. monocytogenes on stainless steel. The most significant result was obtained with L. monocytogenes where number of adhered cells was reduced by 10(2) CFU cm(-2). On polypropylene, surfactin showed a significant decrease on the adhesion of all strains. The adsorption of surfactin on polystyrene also reduces the adhesion of L. monocytogenes and Salm. Enteritidis growing cells. For short contact periods using nongrowing cells or longer contact periods with growing cells, surfactin was able to delay bacterial adhesion. CONCLUSIONS: The prior adsorption of surfactin to solid surfaces contributes on reducing colonization of the pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work investigating the effect of surfactin on the adhesion of the food pathogens L. monocytogenes, Ent. sakazakii and Salm. Enteritidis to polypropylene and stainless steel surfaces.

A low-cost fermentation medium for thermophilic protease production by Streptomyces sp. 594 using feather meal and corn steep liquor.

Protease production by Streptomyces sp. 594 was obtained after submerged fermentation (SF) and solid-state fermentation (SSF) using feather meal (FM) and corn steep liquor (CSL) as sole sources of carbon and nitrogen. Enzyme productions were 13.4 U ml(-1) in SF and 21.5 U g(-1) in SSF; these values were approximately 86% and 39% higher, respectively, than those obtained previously when yeast extract was used in place of CSL. The proteases, which belong to the serine and metalloproteinase classes, were active at high temperatures (55 degrees C to 90 degrees C) and over a wide range of pH values (5.0 to 10.0). Thus, these thermophilic proteases have shown interesting properties for industrial purposes. As far as we are concerned, this is the first contribution toward the microbial production of thermophilic proteases by a streptomycete using a low-cost medium composed of industrial poultry (FM) and corn processing by-products (CSL).

New group-specific 16S rDNA primers for monitoring foaming mycolata during saline waste-water treatment.

Newly designed group-specific PCR primers for denaturing gradient gel electrophoresis (DGGE) were used to investigate foaming mycolata from a bioreactor treating an industrial saline waste-water. Genetic profiles on DGGE gels were different with NaCl at 1.65 and 8.24 g l(-1), demonstrating that mycolata community was affected by salinity. A semi-nested PCR strategy resulted in more bands in community genetic profiles than direct amplification. DNA sequencing of bands confirmed the efficacy of the novel primers with sequences recovered being most similar to foam producing mycolata. The new group-specific primers/DGGE approach is a new step toward a more complete understanding of functionally important groups of bacteria involved in biological treatment of waste-water.

A review on hydrolytic enzymes in the treatment of wastewater with high oil and grease content.

Wastewater from dairies and slaughterhouses contains high levels of fats and proteins that present low biodegradability. A large number of pretreatment systems are employed to remove oil and grease (O&G) to prevent a host of problems that may otherwise arise in the biological process, and reduce the efficiency of the treatment station. Problems caused by excessive O&G include a reduction in the cell-aqueous phase transfer rates, a sedimentation hindrance due to the development of filamentous microorganisms, development and flotation of sludge with poor activity, clogging and the emergence of unpleasant odors. Therefore the application of a pretreatment to hydrolyze and dissolve lipids may improve the biological degradation of fatty wastewaters, accelerating the process and improving time efficiency. However thus far, only a few studies describing the degradation of fats and oils by alkaline/acid/enzymatic hydrolysis have been reported; the treatment of effluents from several origins is a new and promising application for lipases. Among the strains that produce the hydrolytic enzymes studied, the fungus Penicillium restrictum is a particularly promising one. When cultivated in low-cost solid medium composed of agro-industrial waste, P. restrictum produces a pool of hydrolases capable of degrading the most complex organic compounds. This degradation enables a considerable increase in organic matter removal efficiency to be realized, which results in the attainment of a high-quality effluent in the subsequent biological treatment stage. Consequently, there is presently a wide variety of ongoing scientific investigation in the field of developing enzymatic hydrolysis processes to precede traditional biological treatment.

Thermophilic protease production by Streptomyces sp. 594 in submerged and solid-state fermentations using feather meal.

AIMS: Protease production by Streptomyces sp. 594 in submerged (SF) and solid-state fermentation (SSF) using feather meal, an industrial poultry residue, and partial characterization of the crude enzyme. METHODS AND RESULTS: Streptomyces sp. 594 produced proteases in SF (7.2 +/- 0.2 U ml(-1)) and SSF (15.5 +/- 0.41 U g(-1)), with pH increase in both media. Considering protease activity, values obtained in the liquid extract after SSF (6.3 +/- 0.17 U ml(-1)) were lower than those from SF. The proteases, which belong to serine and metalloproteinase classes, were active over a wide range of pH (5.0-10.0) and high temperatures (55-80 degrees C). Strain 594 was also able to degrade feather in agar and liquid media. Keratinase activity (80 U l(-1)) also confirmed the keratin degrading capacity of this streptomycete. CONCLUSIONS: Proteases produced using residues from poultry industry have shown interesting properties for industrial purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as we are concerned, this is the first contribution towards the production of thermophilic protease by a streptomycete in SSF using a keratinous waste.

Production of poly(3-hydroxybutyrate) by solid-state fermentation with Ralstonia eutropha.

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g(-1) medium on soy cake in 36 h, and 0.7 mg g(-1) medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g(-1) medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.