The β-Fructofuranosidase from Rhodotorula dairenensis: Molecular Cloning, Heterologous Expression, and Evaluation of Its Transferase Activity

Abstract

The β-fructofuranosidase from the yeast Rhodotorula dairenensis (RdINV) produces a mixture of potential prebiotic fructooligosaccharides (FOS) of the levan-, inulin- and neo-FOS series by transfructosylation of sucrose. In this work, the gene responsible for this activity was characterized and its functionality proved in Pichia pastoris. The amino acid sequence of the new protein contained most of the characteristic elements of β-fructofuranosidases included in the family 32 of the glycosyl hydrolases (GH32). The heterologous yeast produced a protein of about 170 kDa, where N-linked and O-linked carbohydrates constituted about 15% and 38% of the total protein mass, respectively. Biochemical and kinetic properties of the heterologous protein were similar to the native enzyme, including its ability to produce prebiotic sugars. The maximum concentration of FOS obtained was 82.2 g/L, of which 6-kestose represented about 59% (w/w) of the total products synthesized. The potential of RdINV to fructosylate 19 hydroxylated compounds was also explored, of which eight sugars and four alditols were modified. The flexibility to recognize diverse fructosyl acceptors makes this protein valuable to produce novel glycosyl-compounds with potential applications in food and pharmaceutical industries.

Insights into the Structure of the Highly Glycosylated Ffase from Rhodotorula dairenensis Enhance Its Biotechnological Potential

“…As was previously reported [ 1 ], RdINV shows broad substrate specificity, hydrolyzing sucrose, 1-kestose, nystose, raffinose and inulin. However, it has a clear preference for short substrates, showing a catalytic efficiency 20–30 times superior on sucrose as compared to that on 1-kestose.…”

“…Invertases or β-fructofuranosidases (EC 3.2.1.26) are biotechnologically important enzymes that catalyze the release of β-fructose from the non-reducing termini of various β-D-fructofuranoside substrates. These enzymes may also produce short-chain fructooligosaccharides (FOS) by transfructosylation, in which one to three fructosyl moieties are linked to a sucrose unit [ 1 , 2 ]. FOS are well recognized for their beneficial effects on the organism, such as stimulating the growth of beneficial bacteria in the gut while inhibiting pathogenic bacteria, also known as a prebiotic effect [ 3 ].…”

“…The large β–fructofuranosidase from Rhodotorula dairenensis RdINV (170 kDa) produces mainly 6-kestose but also a varied mixture of the three series of FOS [ 1 , 32 ], which makes it of biotechnological interest. Additionally, RdINV is also able to fructosylate a variety of carbohydrates, such as disaccharides, alditols and monosaccharides [ 1 ].…”

“…The large β–fructofuranosidase from Rhodotorula dairenensis RdINV (170 kDa) produces mainly 6-kestose but also a varied mixture of the three series of FOS [ 1 , 32 ], which makes it of biotechnological interest. Additionally, RdINV is also able to fructosylate a variety of carbohydrates, such as disaccharides, alditols and monosaccharides [ 1 ]. However, although RdINV has already been biochemically characterized [ 32 ] and heterologously expressed in Komagataella phaffii [ 1 ], the structural determinants responsible for its interaction with substrates and selective production of FOS are unclear.…”

“…Additionally, RdINV is also able to fructosylate a variety of carbohydrates, such as disaccharides, alditols and monosaccharides [ 1 ]. However, although RdINV has already been biochemically characterized [ 32 ] and heterologously expressed in Komagataella phaffii [ 1 ], the structural determinants responsible for its interaction with substrates and selective production of FOS are unclear. Furthermore, both native and recombinant RdINV showed a molecular mass of ~170 kDa, which is far from the theoretical ~70 kDa calculated on the basis of the deduced sequence of RdINV, and enzymatic treatment with different deglycosylating enzymes revealed that glycans are responsible for 50% of the total molecular mass, with only 15% attributed to N-glycosylation.…”