## 1 Introduction  
The \(\beta\)-fructofuranosidases (EC 3.2.1.26) catalyze the release of fructose from the non-reducing termini of different-size fractrans and sucrose (invertases). As well as hydrolyning sucrose, \(\beta\)-fructofuranosidases may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to a sucrose unit. Different FOS series can be distinguished depending on the linkage type between the monosaccharide residues: \({}^{1}\)F-FOS, containing \(\beta\)-(2-1)-linked fructose units (inulin-type structure, e.g., 1-kestose); \({}^{6}\)F-FOS, containing \(\beta\)-(2-6)-linked fructose units (levan-type structure, e.g., 6-kestose); and \({}^{6}\)G-FOS, where a \(\beta\)-(2-6) linkage connects fructose to the glucosyl moiety of sucrose (neo-FOS, e.g., neokestose). FOS selectively stimulate the growth and activity of health-promoting microbial species in the gut, the so-called prebiotic effect [1, 2], stimulating or improving the immune and cardiovascular systems, mineral absorption, and even mental health [3, 4, 5, 6, 7]. Although the three series of FOS show beneficial effects, \(\beta\)-(2-6)-linked FOS appear to have improved prebiotic properties and increased chemical stability compared to the traditionally commercialized \(\beta\)-(2-1)-linked FOS [8, 9]. In addition, neo-FOS also showed a potential inhibitory activity against melanoma cells and colon cancer [10, 11]. Enzymatic synthesis of FOS has been reported using different microbial systems. Among others, \(\beta\)-fructofuranosidases from _Aspergillus_ (main industrial producers) provide a mixture of sugars included in the \({}^{1}\)F-FOS series, whereas those from yeasts _Saccharomyces cerevisiae_ and _Schvenninomyces occidentalis_ produce mainly 6-kestose and, from _Xanthophyllomyces dendorhous_, neokestose [12; 13; 14; 15; 16; 17]. Although structural determinants responsible for the selective production of FOS by these proteins are not yet clear, the relevance of particular residues in substrate binding and product specificity has been demonstrated by mutational analyses [18; 19; 20; 21]. The ability to produce FOS of \(\beta\)-fructofuranosidases from some species of the genus _Rhodotorula_ has also been analyzed with different results. Curiously, enzymes from _Rhodotorula_ sp.-LEB-V10 and _Rhodotorula mucilaginous_ only generated sugars included in the \({}^{1}\)F-FOS series, whereas that from _Rhodotula glutinis_, a yeast synthesizing numerous valuable compounds (carotenoids and lipids among others) with a wide industrial usage, did not produce any type of FOS [22; 23; 24; 25]. However, the large protein RdINV from _Rhodotorula diarenensis_ (\(\sim\)170 kDa, of which N-linked carbohydrates constituted \(\sim\)16% of the total mass) produced mainly 6-kestose, but also a varied mixture of FOS of the three referenced series [26], which makes it of biotechnological interest. Based on structural features, \(\beta\)-fructofuranosidases are classified into the family 32 of the glycosyl hydrolases (GH32) (CAZY; [[http://www.cazy.org](http://www.cazy.org)]([http://www.cazy.org](http://www.cazy.org)), accessed on 29 March 2021), which contains a characteristic five-blade \(\beta\)-propeller N-terminal module, in which the \(\beta\)-sheets are arranged around a central pocket that accommodates the active site [27; 28]. Three conserved sequences, each containing a key acidic residue implicated in substrate binding and hydrolysis, are located in the protein active site: WMNDPNG (D acting as nucleophilic), FRDP (D acting as stabilizer of transient state) and ECP (E acting as acid-base catalyst) [29]. Proteins of GH32 include an additional C-terminal \(\beta\)-sandwich domain implicated in their oligomerization and substrate recognition [18; 19; 20]. In this context, and although several genomes from yeasts included in the genus _Rhodotorula_ have been sequenced [30; 31; 32], functionality of none of the DNA sequences a priori codifying for proteins included in the family GH32 was proven.