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The β-fructofuranosidase (invertase) from the yeast Saccharomyces cerevisiae is one of the earliest enzymes to be isolated and characterised (Berthelot 1860). The work of Michaelis- Menten on enzyme kinetics was carried out using yeast invertase and the contribution of this enzyme to the study of biochemistry is significant (Michaelis et al. 2011; Michaelis and Menten 1913). Much is known about Saccharomyces cerevisiae β-fructofuranosidase but it is intriguing to note that the crystal structure of Suc2p was only recently solved. The work revealed a biological assembly consisting of a ‘tetramer of dimers’ (Sainz-Polo et al. 2013). The gene sequence for the enzyme was first determined by Taussig and Carlson (1983) and it has been shown that the same gene (SUC2) encodes both the intracellular and secreted forms, which are differentially regulated by glucose (Carlson and Botstein 1982). Suc2p is the quintessential β-fructofuranosidase known to hydrolyse sucrose to glucose and fructose. That this enzyme is able to produce FOS is a lesser-known fact (Bacon 1954). It has been shown to produce both inulin- and levan-type trisaccharides, 1-kestose and 6-kestose, respectively from sucrose. Suc2p has also been reported to produce neokestose (Hidaka et al. 1988). No higher DP oligosaccharides were identified following incubation of the enzyme with sucrose (Hidaka et al. 1988; Lafraya et al. 2011). Although Suc2p was incubated under conditions favouring FOS synthesis (reverse hydrolysis conditions with high substrate concentration; 600 g/l sucrose), the yields of FOS were very low, comprising less than 3% of total sugar. The report of Lafraya et al. (2011) is one of a few investigating the structural determinants of fructosyltransferase efficiency and product specificity of a fungal β-fructofuranosidase. Amino acid substitutions were made following identification of variable positions from alignments of fungal, bacterial and plant GH32 hydrolases and transferases, as well as bacterial GH68 transferases. Modifications to the β-fructosidase (WMNDPNG) and ECP motifs surrounding active site residues (underlined) affected increases in fructosyltransferase activity, raising the percent FOS of total sugar from 2.5 to 18.3% after an incubation period of 24-48 h. A decrease in structural rigidity of the active site was proposed to be key to modifying transfructosylating efficiency. Clear determinants of product specificity were not obtained but residues possibly involved were identified.

A fungal β-fructofuranosidase displaying high level FOS synthesis has been isolated from

A. japonicus. The fungal strain producing this enzyme was initially classified as A. niger ATCC

20611 and reports on this enzyme, named FopA, refer to it as an A. niger enzyme (Hidaka et al. 1988; Hirayama et al. 1989; Nishizawa et al. 2001; Yanai et al. 2001). The strain was later reclassified as A. japonicus ATCC 20611 (ATCC culture collection database;

http://www.lgcpromochem-atcc.com, Yuan et al. 2006). FopA is very well studied and it has been shown to be one of the most efficient FOS synthesising β-fructofuranosidases. The enzyme has been purified and the coding gene sequence is known (Yanai et al. 2001). It produces inulin-type FOS from sucrose with 1-kestose (GF2), nystose (GF3) and

β-fructofuranosylnystose (GF4) as major products. By virtue of a Ut:Uh ratio of 14.2,

A. japonicus ATCC 20611 was initially identified as harbouring an enzyme displaying good FOS

synthesis potential. The second best candidate in the screen for FOS synthesising microbes/enzymes was Aureobasidium pullulans with a Ut:Uh ratio of 9.8 (Hidaka et al. 1988).

The fructosyltransferase activity of FopA has been shown to dominate over hydrolytic activity when tested at high substrate concentration (reverse hydrolysis conditions; 500 g/l sucrose) and low concentrations (50 g/l sucrose). Even at 5 g/l sucrose, FOS synthesis is still observed while under the same conditions no FOS are produced by Suc2p of S. cerevisiae (Hidaka et al. 1988; Hirayama et al. 1989). Purified FopA catalyses the synthesis of FOS from sucrose with a yield of ~60% FOS of total sugar within 3 h (Nishizawa et al. 2001). It is this capacity that distinguishes FopA as an enzyme capable of high level FOS synthesis. Both FOS yield and the time span of a few hours to reach given yields are important distinguishing traits of enzymes displaying high level FOS synthesis. What sets FopA apart from other β-fructofuranosidases displaying dominant sucrose hydrolysis activity is not clear. Although strategic substitution of certain amino acids may improve fructosyltransferase activity of β-fructofuranosidases with dominant hydrolytic activity, it has not yet been demonstrated that it is possible to transform such a fungal enzyme into one displaying high level FOS synthesis of the same order as FopA. Although studies investigating the latter possibility in fungal enzymes are scarce, it is proposed that the determinants of high level FOS synthesis do not only rely on single amino acids at certain positions in the three dimensional enzyme structure. Rather, a particular spatial positioning of amino acids in the folded enzyme, as well as particular key amino acids are responsible for an observed enzyme activity. These determinants are at particular sub-localities within the enzyme’s native structure. As protein sequence is an important driver of three dimensional enzyme structure, it is theoretically possible to identify characteristic sequence motifs that give rise to the required positioning of amino acids that produce a certain enzyme activity. The three dimensional structure of A. japonicus fructosyltransferase has been solved and is the only crystal structure of an enzyme displaying high level FOS synthesis (Chuankhayan et al. 2010). The extracellular enzyme exists as a monomer. Superimposition of crystal structure models of closely related GH32 and GH68 enzymes with the structure of FopA revealed differentiation of the shape and size of the active site pockets. Despite different residues surrounding the active site pockets with varied shapes between the enzymes, sucrose substrate bound to the different enzymes could be well aligned. Chuankhayan et al. (2010) proposed that amino acid residues surrounding sugar moieties at the +2 subsite, i.e. further

away from active site residues, were responsible for the varied substrate specificities of the different enzymes. The β-fructosidase motif (WMNDPNG) was not conserved in FopA and other GH32 transferases. The motif sequence was QIGDPC in FopA which altered the shape of the active-site pocket due to interactions with other surrounding residues. Mutations at equivalent positions in this region of plant enzymes alter transfructosylation activity and product specificity (Ritsema et al. 2006; Ritsema et al. 2005; Schroeven et al. 2008). Thus indications are there that the β-fructosidase motif is involved in determination of transfructosylation activity. The synthesis of FOS by the Schwanniomyces occidentalis β-fructofuranosidase is also well studied. Although the enzyme displays predominantly sucrose hydrolysis activity, under reverse hydrolysis conditions it synthesises FOS, mostly the levan-type 6-kestose and smaller amounts of 1-kestose (Alvaro-Benito et al. 2007). Particular interest in the S. occidentalis β-fructofuranosidase relates to the observation that the 6-kestose yields at 16.9% of total sugars after approximately 20 h are the highest reported for enzymatic synthesis of 6-kestose. The three dimensional structure of the enzyme has been solved and it was revealed to exist as a homodimer (Alvaro-Benito et al. 2010b). Further study of the enzyme revealed that non- universal decoding of the leucine CUG codon by S. occidentalis results in a serine at position 196 of the wild type enzyme (Alvaro-Benito et al. 2010a). Both versions of the enzyme, FFase- Ser196 and FFase-Leu196 were compared by heterologous expression in S. cerevisiae. The catalytic efficiency of FFase-Leu196 was severely compromised, being 1000 times lower than that of FFase-Ser196. The temperature and pH optima were also decreased. However, FFase- Leu196 showed a 3-fold improved transferase activity and maximum yields of 21.1% FOS of total sugar were attained after 72 h. The synthesis of 1-kestose was also reduced with the 6-kestose:1-kestose ratio increased from 3:1 to 15:1. Mapping the leucine substitution to the crystal structure proposed that a local rearrangement was caused which influenced the positioning of residues impacting on two of the three catalytic triad residues. A further mutation introduced to the β-fructosidase motif caused significant improvements in production levels of both types of trisaccharides. Four rounds of directed evolution of the FFase-Leu196 protein, employing a random mutagenesis strategy, have improved the transfructosylation activity of the

S. occidentalis β-fructofuranosidase (de Abreu et al. 2013). High selectivity for 6-kestose

synthesis remained fairly constant and FOS levels were approximately doubled from 70.7 g/l FOS to 168.3 g/l and 158.2 g/l FOS for two variants, respectively. The synthesis of neokestose and a tetrasaccharide by the variants was also noted. The four mutations responsible for each variant’s activity could not be rationalised or were proposed to impact on active site residues. Further work on this enzyme has provided direct evidence for determinants of the ability to degrade long substrates (high DP inulin as well as longer FOS) relating to its dimeric assembly and the determinants of product specificity (Álvaro-Benito et al. 2012).