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1.
BioTech (Basel) ; 12(1)2023 Mar 03.
Article in English | MEDLINE | ID: mdl-36975311

ABSTRACT

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.

2.
Prep Biochem Biotechnol ; 52(4): 478-486, 2022.
Article in English | MEDLINE | ID: mdl-34428129

ABSTRACT

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.


Subject(s)
Phosphates , beta-Fructofuranosidase , Aspergillus , Magnetic Fields , Polyethylene Glycols , Sodium Chloride , Water
3.
Prep Biochem Biotechnol ; 52(1): 30-37, 2022.
Article in English | MEDLINE | ID: mdl-33787455

ABSTRACT

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.


Subject(s)
Aspergillus/metabolism , Citric Acid/chemistry , Fungal Proteins/metabolism , Peptide Hydrolases/metabolism , Polyethylene Glycols/chemistry , Enzyme Stability , Fermentation , Fungal Proteins/isolation & purification , Hydrogen-Ion Concentration , Industrial Microbiology , Peptide Hydrolases/isolation & purification , Temperature
4.
Article in English | MEDLINE | ID: mdl-27183214

ABSTRACT

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.


Subject(s)
Fungal Proteins/isolation & purification , Mucor/enzymology , Peptide Hydrolases/isolation & purification , Enzyme Stability , Fungal Proteins/analysis , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Mucor/chemistry , Peptide Hydrolases/analysis , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Polyethylene Glycols , Sulfates , Temperature
5.
Appl Biochem Biotechnol ; 172(5): 2682-93, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24425299

ABSTRACT

Parkia pendula seed lectin was used to treat cutaneous wounds of normal and immunocompromised mice, inducing cicatrization. Methotrexate (0.8 mg/kg/week) was used as immunosuppressive drug. Wounds were produced in the dorsal region (1 cm(2)) of female albino Swiss mice (Mus musculus), health and immunocompromised. Wounds were daily topically treated with 100 µL of the following solutions: (1) control (NaCl 0.15 M), (2) control Im (0.15 M NaCl), (3) P. pendula seed lectin (100 µg/mL), and (4) P. pendula seed lectin Im (100 µg/mL). Clinical evaluation was performed during 12 days. Biopsies for histopathology analysis and microbiological examinations were carried out in the second, seventh, and 12th days. The presence of edema and hyperemia was observed in all groups during inflammatory period. The first crust was detected from the second day, only in the groups treated with P. pendula seed lectin. Microbiological analysis of wounds from day 0 to day 2 did not show bacterium at P. pendula seed lectin group; however, Staphylococcus sp. was detected every day in the other groups. The lectin markedly induced a total wound closing at P. pendula seed lectin and P. pendula seed lectin Im groups on 11th day of evolution. The present study suggests that P. pendula seed lectin is a biomaterial potential to show pharmacological effect in the repair process of cutaneous wounds.


Subject(s)
Fabaceae/chemistry , Immunocompromised Host , Plant Lectins/pharmacology , Skin/injuries , Staphylococcal Skin Infections/immunology , Wound Healing/drug effects , Animals , Female , Immunosuppressive Agents/adverse effects , Methotrexate/adverse effects , Mice , Plant Lectins/isolation & purification , Seeds/chemistry , Skin/immunology , Skin/microbiology , Staphylococcal Skin Infections/drug therapy , Staphylococcal Skin Infections/microbiology , Staphylococcus/drug effects , Staphylococcus/growth & development , Wound Healing/immunology
6.
Int J Pharm ; 408(1-2): 113-9, 2011 Apr 15.
Article in English | MEDLINE | ID: mdl-21335081

ABSTRACT

Cramoll 1,4 is a lectin extracted from seeds of Cratylia mollis Mart. Many assays have shown the cytokine release activity and pro-inflammatory profile of this lectin. Here, we used Cramoll 1,4 in the treatment of cutaneous wounds in normal and immunocompromised mice for available your cicatricial power. Surgical wounds were treated daily with a topical administration of Cramoll 1,4 and parameters as edema, hyperemia, scab, granulation and scar tissues as well as contraction of wounds were analyzed. Cramoll 1,4 wounds showed higher edema and arrival of more polimorphonuclear cells at the site of lesions. Granulation tissue and collagen fiber deposition were observed with higher intensity in all Cramoll 1,4 treated wounds and promoted excellent closing and repair of lesions in less time than other groups. Results showed that Cramoll 1,4 lectin was effective in the repair of experimental lesions in mice and can be used as a future cicatricial compound.


Subject(s)
Immunocompromised Host , Plant Lectins/therapeutic use , Wound Healing/drug effects , Wound Healing/immunology , Wounds, Penetrating/drug therapy , Animals , Disease Models, Animal , Fabaceae/chemistry , Female , Mice , Neutrophil Infiltration/drug effects , Neutrophil Infiltration/immunology , Neutrophils/cytology , Neutrophils/drug effects , Neutrophils/immunology , Plant Lectins/isolation & purification , Seeds/chemistry , Skin/drug effects , Skin/immunology , Skin/injuries , Skin/pathology , Wounds, Penetrating/immunology , Wounds, Penetrating/pathology
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