Biochemical properties of an extracellular trehalase from Malbranchea pulchella var. Sulfurea

Abstract

The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680 U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0-5.5 and 55° C, respectively. The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver ions. The apparent K(M) values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70 ± 0.29 and 2.58 ± 0.13 mM, respectively. Manganese ions affected only the apparent V(max), increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also from trehalases from other thermophilic fungi.

A trehalase from Zunongwangia sp.: characterization and improving catalytic efficiency by directed evolution

“…The activity of TreZ was determined by a modified method reported by Pereira et al [ 38 ]. The amount of reducing D-glucose was determined by the 3,5-dinitrosalicylic acid (DNS) method and GOD-POD method [ 39 , 40 ].…”

A novel thermostable xylanase GH10 from Malbranchea pulchella expressed in Aspergillus nidulans with potential applications in biotechnology

“…Malbranchea pulchella is a thermophilic fungus isolated from soil and decaying vegetation. Several studies have described enzymes produced by this fungus, such as the purification and characterization of a trehalase [ 6 ], and the demonstration of high level production of thermostable cellulolytic and xylanolytic enzymes [ 7 ]. Furthermore, other species from the Malbranchea genus have been shown to be good enzyme producers, especially of proteases [ 8 , 9 ], and the production of alpha-amylase, beta-xylosidase, chitinase, and xyloglucanase has also been reported [ 10 – 13 ].…”

Trehalases: A neglected carbon metabolism regulator?

“…[12][13][14][15][16][17][18][19] In addition, trehalase contrastingly different sub-cellular localizations have been reported, including the cytoplasm, the vacuole, the apoplast, or being bound to the plasma membrane and oriented to the apoplast. 16,[20][21][22][23] In fungi, trehalase activity and localization have been directly involved in regulating the trehalose content and its mobilization into the cytoplasm and during the symbiotic interaction of the ectomycorrhizal fungi several genes for trehalose metabolism are strongly upregulated. 23,24 In insects the regulation of trehalose degradation by trehalase is essential to supply the energetic requirements for the flight and the direct regulation for chitin biosynthesis during the insect development.…”