RESUMO
The aim was to evaluate the kinetic parameters, total color differences (∆E*) and browning index differences (∆BI) of air flow pineapple drying. The experiments were performed on air temperatures at 60 and 70 °C, and air velocities at 1.5 and 2.0 m/s. The kinetic parameter (k) increased when air temperature was increased for all air velocity. The effective diffusivity coefficient (Deff) increased as high as the temperature of the heating medium. The variation of Deff of swirling flow was ranging from 6.72 × 10-9 to 10.23 × 10-9 m2/s, while the variation of Deff of non-swirling flow was ranging from 6.40 × 10-9 to 9.42 × 10-9 m2/s. The drying time of swirling flow was shorter than non-swirling flow in each drying condition. Moreover, the ∆E* and ∆BI of pineapple in swirling flow were lower than that obtained from non-swirling flow. Therefore, the convective drying using swirling flow can be minimized for drying time and color deterioration.
RESUMO
A gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4mg enzyme per L of growth culture with a specific activity of 159Umg(-1). By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 68.5kDa. The pH optimum for the oxidation of galactose was in the range of pH 6-7.5. Optimum temperature for the enzyme activity was 35°C, with a half-life of 11.2min, 5.3min, and 2.7min for incubation at 40°C, 50°C, and 60°C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-ß-galactopyranoside (226Umg(-1)) and the highest catalytic efficiency (kcat/Km) for melibiose (2700mM(-1)s(-1)). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN.
Assuntos
Escherichia coli/metabolismo , Proteínas Fúngicas , Fusarium/enzimologia , Galactose Oxidase , Expressão Gênica , Escherichia coli/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Fusarium/genética , Galactose Oxidase/biossíntese , Galactose Oxidase/química , Galactose Oxidase/genética , Galactose Oxidase/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
A gene coding for galactose 6-oxidase from Fusarium oxysporum G12 was cloned together with its native preprosequence and a C-terminal His-tag, and successfully expressed both in Escherichia coli and Pichia pastoris. The enzyme was subsequently purified and characterized. Among all tested substrates, the highest catalytic efficiency (kcat/Km) was found with 1-methyl-ß-D-galactopyranoside (2.2 mM(-1) s(-1)). The Michaelis constant (Km) for D-galactose was determined to be 47 mM. Optimal pH and temperature for the enzyme activity were 7.0 and 40°C, respectively, and the enzyme was thermoinactivated at temperatures above 50°C. GalOx contains a unique metalloradical complex consisting of a copper atom and a tyrosine residue covalently attached to the sulphur of a cysteine. The correct formation of this thioether bond during the heterologous expression in E. coli and P. pastoris could be unequivocally confirmed by MALDI mass spectrometry, which offers a convenient alternative to prove this Tyr-Cys crosslink, which is essential for the catalytic activity of GalOx.
Assuntos
Escherichia coli/genética , Fusarium/enzimologia , Galactose Oxidase/genética , Galactose Oxidase/metabolismo , Pichia/genética , Sequência de Aminoácidos , Clonagem Molecular , Éteres/química , Fusarium/genética , Galactose Oxidase/química , Galactose Oxidase/isolamento & purificação , Expressão Gênica , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The gene gaoA encoding the copper-dependent enzyme galactose oxidase (GAO) from Fusarium graminearum PH-1 was cloned and successfully overexpressed in E. coli. Culture conditions for cultivations in shaken flasks were optimized, and optimal conditions were found to be double-strength LB medium, 0.5% lactose as inducer, and induction at the reduced temperature of 25°C. When using these cultivation conditions ~24 mg of active GAO could be produced in shaken flasks per litre medium. Addition of copper to the fermentation medium decreased the enzyme production significantly. The His-tagged recombinant enzyme could be purified conveniently with a single affinity chromatography step. The purified enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 66 kDa and had kinetic properties similar to those of the fungal wild-type enzyme.
