Characterization of lyophilized equine colostrum.

Foals require maternal colostrum in the first hours of life to prevent failure of transfer of passive immunity (FTIP). Innovative storage methods such as lyophilization may enable conservation of colostrum immunoglobulins by a differentiated process of dehydration. The current study aimed to compare the quality of equine colostrum after freezing and after the lyophilization process. Thirty-one pregnant Quarter Horse mares were used. The IgG concentration of frozen and lyophilized colostrum was determined by simple radial immunodiffusion (SRID) and Brix refractometry. The physical-chemical composition (pH, total protein (TP), fat, lactose, salts, total solids (TS), and density) of the samples was evaluated and the lyophilized colostrum reconstitution test was performed. There were no significant differences (P > 0.05) in the variables IgG, fat, lactose, salts, TS, density, and pH between samples measured before and after lyophilization. There was a significant difference (P < 0.05) between the Brix average and the TP of the frozen and lyophilized colostrum samples. Lyophilization resulted in a small reduction (6.55%) in the IgG concentration measured by SRID. A strong positive correlation was observed between colostrum density and IgG concentration by SRID (r = 0.76) and between Brix and IgG concentration by SRID (r = 0.77). In the reconstitution test, the lyophilized colostrum was easily rehydrated in water, with full dilution, and remained stable. Lyophilization could be an alternative for the conservation of mare colostrum, since it is a very efficient process for retaining the physicochemical characteristics of the product, with minimal loss, particularly of IgG.

Optimization strategy for inulinase production by Aspergillus niger URM5741 and its biochemical characterization, kinetic/thermodynamic study, and application on inulin and sucrose hydrolysis.

Inulinases are enzymes of great interest in the food industry, especially due to their application in the synthesis of fructose and fructo-oligosaccharides. Moreover, some inulinases (I) also present invertase activity (S), making them useful for sucrose hydrolysis processes. In the present study, the production of inulinase by Aspergillus niger URM5741 was evaluated and optimized using two statistical approaches. First, the composition of the cultivation medium was determined through a simplex centroid mixture design, followed by the selection of optimal fermentation conditions using the Box-Behnken design. Based on these experimental designs, the maximum activities of inulinase (16.68 U mL-1) and invertase (27.80 U mL-1) were achieved using a mixture of wheat, soy, and oat brans (5 g), along with 2.5% inulin and 40% moisture. The inulinase exhibited optimum temperature and pH of 60 °C and 4.0, respectively, displayed a high affinity for both substrates, as evidenced by very-low Michaelis constant values (1.07-1.54 mM). A relative thermostability was observed at 55-60 °C as indicated by half-lives values (I: 169.06-137.27 min; S: 173.29-141.52 min) and D-values (I: 561.61-456.00 min; S: 575.65-470.11 min) which were further confirmed by the high activation energy (123.01 and 143.29 kJ mol-1). The enzyme demonstrated favorable results in terms of inulin and sucrose hydrolysis, being a maximum release of reducing sugars of 6.04 and 15.80 g L-1, respectively. These results indicate that the sequential statistical approach proved to be beneficial to produce inulinase by A. niger URM5741, with the obtained enzyme considered promising for long-term industrial applications.

Extraction of collagenolytic proteases from Aspergillus heteromorphus URM 0269 in an aqueous two-phase system for application in collagen hydrolysis.

Collagenolytic proteases produced by Aspergillus heteromorphus URM0269 were extracted using a PEG/sulfate aqueous two-phase system (ATPS). A 23 factorial design was performed to analyze the independent variables: PEG molar mass (MPEG), PEG concentration (CPEG), and sulfate concentration (Csulf). The extracted proteases were also evaluated for their optimum pH and stability at different pH levels (4.0 - 11.0) after 20 h of incubation. Collagen was extracted from mutton snapper (Lutjanus analis) skin using acetic acid (0.5 mol L-1). The enzyme was preferentially partitioned to the PEG-rich phase (K > 1), whose highest purification factor and recovery (PF = 6.256 and Y = 404.432%) were obtained under specific conditions: MPEG 8000 g.mol-1, CPEG 30%, Csulf 10%. The ATPS extraction provided an enzymatic activity range of pH 7.0 - 11.0, exhibiting greater stability compared to the crude extract. Approximately 80% of protease activity was maintained after 20 hours of incubation at all analyzed pH levels, except pH 11.0. Collagen extraction from L. analis skin yielded 8.056%, and both crude extract samples and ATPS-derived samples successfully hydrolyzed the extracted collagen, reaching peak hydrolysis after 36 hours of treatment. These findings demonstrate the feasibility of extracting highly purified and active proteases capable of hydrolyzing L. analis collagen.

Occurrence of antimicrobial-resistant Staphylococcus aureus in a Brazilian veterinary hospital environment.

Antimicrobial resistance is a threat to public health. The emergence of antibiotic-resistant Staphylococcus aureus represents a priority for the implementation of preventive measures. The objective was to isolate S. aureus in humans, animals, and animal health care environment, and to characterize the genotypic and phenotypic profile of antimicrobial resistance in these isolates. We isolated S. aureus from staff, animals, and environment of a veterinary hospital, and identified their antimicrobial resistance profiles. Samples were collected from 20 humans, 13 animals, 14 surfaces, 8 mobile phones, and 7 veterinarians' stethoscopes by using sterile swabs. S. aureus was isolated by culturing on mannitol salt agar and preliminary identification was done by Gram staining and catalase test. Subsequently, a polymerase chain reaction was performed for species confirmation and investigating their antimicrobial-resistant genotypic profiles. Phenotypic profiles of resistant isolates were determined using the disk-diffusion technique. Ten S. aureus isolates were recovered from 5/20 humans (25%), it was also recovered from 2/13 animals (15.38%), including 1 dog and 1 cat, and from 1/14 of surfaces (7.14%). The oxacillin-susceptible mecA-positive Staphylococcus aureus phenotype was identified in a feline. Most of the isolates carried at least two resistance genes of different antimicrobial classes, with 90% (9/10) presenting the gene blaZ, with 10% (1/10) presenting the gene mecA, 20% (2/10) presenting tet38, 10% (1/10) presenting tetM, 90% (9/10) presenting norA, 50% (5/10) presenting norC, 10% (1/10) presenting ermA, and 60% (6/10) presenting ermB. In antibiograms, resistance to penicillin was identified in all the isolates, resistance to erythromycin was identified in 80% (8/10), and all the isolate's resistance to erythromycin presented erythromycin-induced resistance to clindamycin. Antimicrobial resistance in the veterinary hospital requires attention due to the risk of interspecies transmission, gene transfer between bacteria that colonize companion animals and humans and, can make antimicrobial therapy difficult.

Production, Kinetic/Thermodynamic Study, and Evaluation of the Influence of Static Magnetic Field on Kinetic Parameters of ß-Fructofuranosidase from Aspergillus tamarii Kita UCP 1279 Produced by Solid-State Fermentation.

ß-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and can be used in the production of invert sugar and fructo-oligosaccharides (FOS). This last is an important prebiotic extensively used in the food industry. In the present study, the FFase production by Aspergillus tamarii Kita UCP 1279 was assessed by solid-state fermentation using a mixture of wheat and soy brans as substrate. The FFase presents optimum pH and temperature at 5.0-7.0 and 60 °C, respectively. According to the kinetic/thermodynamic study, the FFase was relatively stable at 50 °C, a temperature frequently used in industrial FOS synthesis, using sucrose as substrate, evidenced by the parameters half-life (115.52 min) and D-value (383.76 min) and confirmed by thermodynamic parameters evaluated. The influence of static magnetic field with a 1450 G magnetic flux density presented a positive impact on FFase kinetic parameters evidenced by an increase of affinity of enzyme by substrate after exposition, observed by a decrease of 149.70 to 81.73 mM on Km. The results obtained indicate that FFases present suitable characteristics for further use in food industry applications. Moreover, the positive influence of a magnetic field is an indicator for further developments of bioprocesses with the presence of a magnetic field.

Production, Biochemical Characterization, and Kinetic/Thermodynamic Study of Inulinase from Aspergillus terreus URM4658.

Inulinases are enzymes involved in the hydrolysis of inulin, which can be used in the food industry to produce high-fructose syrups and fructo-oligosaccharides. For this purpose, different Aspergillus strains and substrates were tested for inulinase production by solid-state fermentation, among which Aspergillus terreus URM4658 grown on wheat bran showed the highest activity (15.08 U mL-1). The inulinase produced by this strain exhibited optimum activity at 60 °C and pH 4.0. A detailed kinetic/thermodynamic study was performed on the inulin hydrolysis reaction and enzyme thermal inactivation. Inulinase was shown to have a high affinity for substrate evidenced by very-low Michaelis constant values (0.78-2.02 mM), which together with a low activation energy (19.59 kJ mol-1), indicates good enzyme catalytic potential. Moreover, its long half-life (t1/2 = 519.86 min) and very high D-value (1726.94 min) at 60 °C suggested great thermostability, which was confirmed by the thermodynamic parameters of its thermal denaturation, namely the activation energy of thermal denaturation (E*d = 182.18 kJ mol-1) and Gibbs free energy (106.18 ≤ ΔG*d ≤ 111.56 kJ mol-1). These results indicate that A. terreus URM4658 inulinase is a promising and efficient biocatalyst, which could be fruitfully exploited in long-term industrial applications.

Extraction and purification of Aspergillus tamarii ß-fructofuranosidase with transfructosylating activity using aqueous biphasic systems (PEG/phosphate) and magnetic field.

ß-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and fructo-oligosaccharides' production which are of great interest for the food industry. FFase from Aspergillus tamarii URM4634 was extracted using PEG/Phosphate Aqueous Biphasic Systems (ABS), and the impact of magnetic field on the extraction behavior was evaluated. A 24-full experimental design was employed to study the influence of molar mass of PEG, concentrations of PEG and phosphate and pH on the selected response variables, i.e., partition coefficient (K), purification factor (PF), activity yield (Y) and selectivity (S). The influence of magnetic field during partition and NaCl concentration on the same responses was also studied. The best results of FFase extraction without magnetic field (K = 0.50, PF = 4.05, Y = 72.66% and S = 0.06) were observed at pH 8.0 using 12.5% (w/w) PEG 400 and 25% (w/w) NaH2PO4/K2HPO4. Application of the magnetic field allowed improving the performance, with the best results being obtained at the longest distance between magnets (lowest magnetic field) and absence of NaCl (K = 0.93, PF = 4.22, Y = 83.79% and S = 0.09). The outcomes obtained demonstrate that ABS combination with low intensity magnetic field can be used as an efficient FFase pre-purification method.

Extractive fermentation for process integration of protease production by Aspergillus tamarii Kita UCP1279 and purification by PEG-Citrate Aqueous Two-Phase System.

The present study evaluated the influence of the variables polyethylene glycol (PEG) molar mass, pH, PEG concentration and sodium citrate concentration in the integrated production of the protease from Aspergillus tamarii Kita UCP1279 by extractive fermentation, obtaining as a response the partition coefficient (K), activity yield (Y) and concentration factor (CF). The enzyme preferably partitioned to the top phase and obtained in the system formed by variables MPEG = 400 g mol-1, CPEG = 20% (w w-1), and CCIT = 20% (w w-1) and pH 6, in this condition were obtained CF = 1.90 and Y = 79.90%. The protease showed stability at a temperature of 60 °C for 180 min, with optimum temperature 40 °C and pH 8.0. For the ions and inhibitors effects, the protease activity increased when exposed to Fe2+, Ca2+ and Zn2 + and inhibited by EDTA, being classified as metalloprotease. The kinetic parameters Km (35.63 mg mL-1) and Vmax (1.205 mg mL-1 min-1) were also estimated. Thus, the protease showed desirable characteristics that enable future industrial applications, especially, for beer industry.

Kinetic/thermodynamic study of immobilized ß-fructofuranosidase from Aspergillus tamarii URM4634 in chitosan beads and application on invert sugar production in packed bed reactor.

ß-fructofuranosidase (FFase) from Aspergillus tamarii URM4634 was immobilized covalently in chitosan beads. It was characterized biochemically, studied in terms of kinetic and thermodynamic parameters, and applied on conversion of sucrose for invert sugar production in a packed bed reactor (PBR). The optimum reactional conditions were determined and obtained at pH 5.0 and 60 °C. FFase was thermostable at 50-55°C. At 50°C, the enzyme shows longer half-life (t1/2) (594.13 min) and a higher D-value (1,973.64 min). This indicates that immobilized FFase was stable at temperature commonly used in invert sugar production. The following thermodynamic parameters were obtained: activation energy (E*d = 301.57 kJ mol-1), enthalpy (298.76 ≤ ΔH*d ≤ 298.89 kJ mol-1), entropy (579.88 ≤ ΔS*d ≤ 589.27 J K-1 mol-1) and Gibbs free energy (100.29 ≤ ΔG*d ≤ 108.47 kJ mol-1). The high E*d, ΔH*d and ΔG*d values confirmed FFase thermostability. The high and positive values for ΔS*d indicate an increase in disorder due opening of the enzyme structure. The sucrose hydrolysis in PBR showed a maximum invert sugar yield (96.0%) at 15 min of operation. The hydrolysis process remained efficient up to 100 min (70.22%). The results obtained in the present study provide a good indication that immobilized FFase on chitosan beads in PBR is efficient to invert sugar production for food industry.

Purification of a lectin from Cratylia mollis crude extract seed by a single step PEG/phosphate aqueous two-phase system.

The partitioning and purification of lectins from the crude extract of Cratylia mollis seeds (Cramoll 1,4) was investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1500-8000 g/mol), PEG concentration (12.5-17.5% w/w), phosphate (10-15% w/w) concentration, and pH (6-8) on the differential partitioning, purification factor, and yield of the lectin. Polymer and salt concentration were the most important variables affecting partition of lectin and used to find optimum purification factor by experimental Box-Behnken design together with the response surface methodology (RSM). ATPS showed best conditions composed by 13.9% PEG1500, 15.3% phosphate buffer at pH 6, which ensured purification factor of 4.70. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of protein with 26.1 kDa. Furthermore, results demonstrated a thermostable lectin presenting activity until 60 °C and lost hemagglutinating activity at 80 °C. According to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of lectins.

Fructo-oligosaccharides production by an Aspergillus aculeatus commercial enzyme preparation with fructosyltransferase activity covalently immobilized on Fe3O4-chitosan-magnetic nanoparticles.

Pectinex Ultra SP-L, a commercial enzyme preparation with fructosyltransferase activity, was successfully immobilized by covalent binding to Fe3O4-chitosan- magnetic nanoparticles. Immobilization carried out according to a 23-full factorial design where glutaraldehyde concentration, activation time and time of contact between enzyme and support were selected as the independent variables and immobilization yield as the response. The highest immobilization yield (94.84%) was obtained using 3.0% (v/v) glutaraldehyde and activation and contact times of 180 and 30 min, respectively. The immobilized biocatalyst, which showed for both hydrolytic and transfructosylating activities optimum pH and temperature of 7.0 and 60 °C, respectively, retained 70 and 86% of them after 6 cycles of reuse. A kinetic/thermodynamic study focused on thermal inactivation of the immobilized construct indicated high thermostability at temperatures commonly used for fructo-oligosaccharides (FOS) production. Maximum FOS concentration obtained in lab-scale experiments was 101.56 g L-1, with predominant presence of 1-kestose in the reaction mixture. The results obtained in this study suggest that the immobilized-enzyme preparation may be effectively exploited for FOS production and easily recovered from the reaction mixture by action of a magnetic field.

Production, extraction, and thermodynamics protease partitioning from Aspergillus tamarii Kita UCP1279 using PEG/sodium citrate aqueous two-phase systems.

The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 24, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (MPEG), concentrations of PEG (CPEG) and citrate (CCIT), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG°m (-10.89 kJ mol-1), ΔHm (-5.0 kJ mol-1) and partition ΔSm (19.74 J mol-1 K-1) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40-50 °C and 9.0-11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.

Production of ß-fructofuranosidase with transfructosylating activity by Aspergillus tamarii URM4634 Solid-State Fermentation on agroindustrial by-products.

Eight different Aspergillus strains were tested for their ability to produce ß-fructofuranosidase (FFase) by Solid-State Fermentation. The Aspergillus tamarii URM4634 strain was selected as the most performant and tested on six different agroindustrial by-products. Soy, wheat and oat brans, which allowed for the highest hydrolytic (UH) and transfructosylating (UTF) activities, were tested individually or in mixtures according to a simplex-centroid mixture design in order to investigate their effects on FFase production at different times. The best results in terms of both enzyme activities were obtained with only soy bran. The influence of substrate, moisture and sucrose levels on FFase production was evaluated, and the highest UH and UTF activities were 229.43 ± 4.88 and 66.93 ± 3.02 U/mL, respectively. The obtained results indicate that A. tamarii FFase may be a biocatalyst with great potential for industrial applications such as sugar inversion and fructo-oligosaccharides production.

Biochemical characterization and kinetic/thermodynamic study of Aspergillus tamarii URM4634 ß-fructofuranosidase with transfructosylating activity.

This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 ß-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45-65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol-1 , and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3-290.4 kJ mol-1 , 568.7-571.0 J mol-1 K-1 , and 97.9-108.8 kJ mol-1 , respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.

Pectin hydrolysis in cashew apple juice by Aspergillus aculeatus URM4953 polygalacturonase covalently-immobilized on calcium alginate beads: A kinetic and thermodynamic study.

The kinetics and thermodynamics of pectin hydrolysis in cashew apple juice by polygalacturonase (PG) from Aspergillus aculeatus URM4953 covalently-immobilized on calcium alginate beads were investigated. Immobilized-PG activity in cashew apple juice was the highest at 20 °C, showing a maximum hydrolysis rate of 58.2 mg/mL·min, a catalytic constant of 166.2 s-1 and an affinity constant of 113.0 mg/mL. Since the enzyme exhibited an allosteric behavior, the hydrolysis rate was modeled, with excellent accuracy, by the Hill Equation as function of pectin concentration. The Hill coefficient increased from 3 to 5 with increasing temperature from 20 to 50 °C, evidencing a positive cooperativity mechanism. The reaction activation energy and the standard enthalpy variation of enzyme unfolding were 80.3 and 16.6 kJ/mol, respectively. Consistently with the kinetic results, PG-catalyzed pectin hydrolysis proceeded with maximum spontaneity at 20 °C, showing activation Gibbs free energy, enthalpy and entropy of 59.3 kJ/mol, 77.9 kJ/mol and 63.4 J/mol·K, respectively. Immobilized PG was successful in the hydrolysis of cashew apple juice pectin, requiring a low temperature to act optimally.

Biophysical, photochemical and biochemical characterization of a protease from Aspergillus tamarii URM4634.

Circular dichroism (CD) and fluorescence spectroscopy (FS) were used to monitor the pH-dependent conformational and structural stability changes induced by temperature and UV light on the protease from Aspergillus tamarii URM4634 at different pH values. The formation of photoproducts, such as N-formylkynurenine, dityrosine and kynurenine, were monitored with FS. The pH-dependent melting temperatures (Tm) were determined using CD and FS from 20 to 90 °C. Conformational changes were correlated with the pH-dependent biochemical activities. CD revealed that the protease is rich in α-helices. Thermal denaturation was irreversible at all pH range and displayed Tm values from 42.8 to 67.8 °C (CD) and from 38 to 60.3 °C (FS), which the highest Tm was observed at pH 6. The light and temperature induced to the formation of photoproducts was more intense at high pH value. Despite the biochemical data shows optimum pH 9, the highest stability was at pH 6, maintaining 100% of activity after 24 h. The acquired data permits to select the best physicochemical parameters to secure the optimal activity and stability when used in biotechnological applications. Furthermore, the conformal changes induced by temperature in the protein are directly correlated with its level of biochemical activity.

Purification and characterization of a novel extracellular serine-protease with collagenolytic activity from Aspergillus tamarii URM4634.

An extracellular serine-protease from Aspergillus tamarii URM4634 was purified and characterized. The possibility of using Aspergillus tamarii URM4634 protease in detergent formulations and collagenolytic activity was investigated. The protease demonstrated excellent stability at pH range 7.0-11.0, the optimum being at pH 9.0. The enzyme was stable at 40 °C for 180 min, enhanced by Mg++ and Ca++, but inhibited by Zn++, and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggested as serine-protease. The azocasein substrate result showed Km = 0.434 mg/mL and Vmax = 7.739 mg/mL/min. SDS-PAGE and azocasein zymography showed that the purified alkaline protease (2983.8 U/mg) had a molecular mass of 49.3 kDa. The enzyme was purified by column chromatography using Sephadex A50 resin. The proteolytic activity was activated by SDS (sodium dodecyl sulfate), Tween-80, Tween 20 and Triton-100. This study demonstrated that A. tamarii URM4634 protease has potent, stable and compatible collagenolytic activity to the desired level in local laundry detergent brands compared with similar enzymes produced by solid-state fermentation. This protease can thus be chosen as an option in both the food industry to tenderization meat and the detergent industry to washing process.

Thermodynamic and kinetic studies on pectinase extracted from Aspergillus aculeatus: Free and immobilized enzyme entrapped in alginate beads.

The kinetics and thermodynamics of Aspergillus aculeatus pectinase, either free or immobilized in alginate beads, were investigated. Pectinase immobilization ensured an enzyme immobilization yield of 59.71%. The irreversible denaturation of pectinase in both preparations was evaluated at temperatures ranging from 30 to 60 °C. When temperature was raised, the first-order thermal denaturation constant increased from 0.0011 to 0.0231 min-1 for the free enzyme and from 0.0017 to 0.0700 min-1 for the immobilized one, respectively. The results of residual activity tests enabled us to estimate, for denaturation of both free and immobilized pectinase, the activation energy (E⁎d = 85.1 and 101.6 kJ·mol-1), enthalpy (82.59 ≤ ΔH⁎d ≤ 82.34 kJ·mol-1 and 99.11 ≤ ΔH⁎d ≤ 98.86 kJ·mol-1), entropy (-63.26 ≤ ΔS⁎d ≤ -63.85 J·mol-1·K-1 and -5.50 ≤ ΔS⁎d ≤ -5.23 J·mol-1·K-1) and Gibbs free energy (101.8 ≤ ΔG⁎d ≤ 104.7 kJ·mol-1 and 100.6 ≤ ΔG⁎d ≤ 102.0 kJ·mol-1). The integral activity of a continuous system using the free and immobilized enzyme was also predicted, whose results indicated a satisfactory enzyme long-term thermostability in both preparations at temperatures commonly used to clarify juice. These results suggest that both free and immobilized pectinase from A. aculeatus may be profitably exploited in future food industrial applications, with special concern to the immobilized enzyme because of its reusability.

Purification, biochemical, and structural characterization of a novel fibrinolytic enzyme from Mucor subtilissimus UCP 1262.

Fibrinolytic proteases are enzymes that degrade fibrin. They provide a promising alternative to existing drugs for thrombolytic therapy. A protease isolated from the filamentous fungus Mucor subtilissimus UCP 1262 was purified in three steps by ammonium sulfate fractionation, ion exchange, and molecular exclusion chromatographies, and characterized biochemically and structurally. The purified protease exhibited a molecular mass of 20 kDa, an apparent isoelectric point of 4.94 and a secondary structure composed mainly of α-helices. Selectivity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate suggests that this enzyme is a chymotrypsin-like serine protease, whose activity was enhanced by the addition of Cu2+, Mg2+, and Fe2+. The enzyme showed a fibrinolytic activity of 22.53 U/mL at 40 °C and its contact with polyethylene glycol did not lead to any significant alteration of its secondary structure. This protein represents an important example of a novel fibrinolytic enzyme with potential use in the treatment of thromboembolic disorders such as strokes, pulmonary emboli, and deep vein thrombosis.

Purification of a fibrinolytic protease from Mucor subtilissimus UCP 1262 by aqueous two-phase systems (PEG/sulfate).

A fibrinolytic protease from M. subtilissimus UCP 1262 was recovered and partially purified by polyethylene glycol (PEG)/sodium sulfate aqueous two-phase systems (ATPS). The simultaneous influence of PEG molar mass, PEG concentration and sulfate concentration on the enzyme recovery was first investigated using a 2(3) full factorial design, and the Response Surface Methodology used to identify the optimum conditions for enzyme extraction by ATPS. Once the best PEG molar mass for the process had been selected (6000g/mol), a two-factor central composite rotary design was applied to better evaluate the effects of the other two independent variables. The fibrinolytic enzyme was shown to preferentially partition to the bottom phase with a partition coefficient (K) ranging from 0.2 to 0.7. The best results in terms of enzyme purification were obtained with the system formed by 30.0% (w/w) PEG 6000g/mol and 13.2% (w/w) sodium sulfate, which ensured a purification factor of 10.0, K of 0.2 and activity yield of 102.0%. SDS-PAGE and fibrin zymography showed that the purified protease has a molecular mass of 97kDa and an apparent isoelectric point of 5.4. When submitted to assays with different substrates and inhibitors, it showed selectivity for succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide and was almost completely inhibited by phenylmethylsulfonyl fluoride, behaving as a chymotrypsin-like protease. At the optimum temperature of 37°C, the enzyme residual activity was 94 and 68% of the initial one after 120 and 150min of incubation, respectively. This study demonstrated that M. subtilissimus protease has potent fibrinolytic activity compared with similar enzymes produced by solid-state fermentation, therefore it may be used as an agent for the prevention and therapy of thrombosis. Furthermore, it appears to have the advantages of low cost production and simple purification.