Some biochemical, catalytic, thermodynamic and kinetic properties of purified fructosyltransferase from wild and improved mutant-type Aureobasidium pullulans NAC8

Abstract

A low molecular weight intracellular and extracellular fructosyltransferase was purified from the wild (W_t) and improved mutant-type (M_t) Aureobasidium pullulans and its characteristics and thermodynamic properties determined. The W_t had been previously genetically modified by chemical mutagenesis. The purified fructosyltransferases from the W_t and M_t had subunit molecular weights between 13.4 and 17.0 kDa, respectively. The pH of the purified fructosyltransferase from the W_t and M_t were within 4.0–5.5. From the K_M values, the fructosyltransferase from M_t showed higher affinity for sucrose than W_t fructosyltransferase. The optimum temperature obtained for the M_t intracellular and extracellular fructosyltransferase were 80 and 70 °C, respectively, while the W_t extracellular and intracellular fructosyltransferase was 60 °C. Most metals enhanced fructosyltransferase activity in a concentration-dependent manner except for mercury. Triton X-100 and Tween-80 enhanced the fructosyltransferase activity. Organic solvents such as acetone, ethanol, methanol, toluene and dichloromethane enhanced the enzyme activity while dimethylformamide inhibited the enzyme. From the thermodynamic and kinetic parameters (${\mathrm{t}}_{1/2},$ ${\Delta \mathrm{H}}^{\mathrm{*}},\mathrm{ }{\Delta \mathrm{S}}^{\mathrm{*}},\mathrm{ }{\Delta \mathrm{G}}^{\mathrm{*}})$ M_t fructosyltransferase was more stable to thermal inactivation than the W_t fructosyltransferase. Hence, it can be concluded that, after strain improvement of the W_t, the purified M_t fructosyltransferase was more stable to organic solvents, surfactants, and thermal denaturation, etc. compared to the W_t. This makes M_t fructosyltransferase more useful mostly in the food industry than the W_t.

Keywords:

• Fructosyltransferase
• kinetics
• purification
• characterization
• sucrose
• Aureobasidium pullulans NAC8

Disclosure statement

No potential conflict of interest was reported by the authors.