Engineered thermostable β–fructosidase from Thermotoga maritima with enhanced fructooligosaccharides synthesis
Introduction
Invertases or β–fructosidases (exo–β–fructofuranosidase EC 3.2.1.26) catalyze the release of the terminal non–reducing fructosyl moiety of various β–D–fructofuranoside substrates, such as sucrose, raffinose, 1–kestotriose, inulin, and levan. The products of the enzyme reaction at high sucrose concentrations are glucose, fructose, and low levels of short–chain fructooligosaccharides (FOS). Due to their intrinsic exo–fructanase activity, invertases frequently degrade the fructosylated products when sucrose is depleted [[1], [2], [3]].
Invertase is ubiquitous in plants and also exists in a wide range of saccharolytic microorganisms [[4], [5], [6]]. No matter the enzyme origin, sucrose hydrolysis is the predominant reaction even at high substrate loadings. Invertases from different sources often show variations in the ratio of the transferase versus hydrolase activities and the structure of the fructosylated products. The major FOS produced by plant invertases is 1–kestotriose [1,7], while yeast β–fructofuranosidases synthesize a more proportional mixture of 1–kestotriose, 6–kestotriose, and 6G–kestotriose (neokestose) [2,3,[8], [9], [10]]. Some bacterial levansucrases and inulosucrases yield β–(2→1) and β–(2→6)–linked FOS respectively in addition to fructan polymers [[11], [12], [13], [14], [15], [16]]. There are no reports describing the yield, linkage type and degree of polymerization (DP) of the FOS synthesized by a bacterial invertase.
Short–chain FOS are soluble non–digestible prebiotics with proven health–promoting effects in humans and animals [17,18]. Among the traditionally commercialized inulin–type FOS, 1–kestotriose (1 K) exhibits the highest sweetness level and stimulates the growth of probiotic bacteria faster than 1,1–kestotetraose and 1,1,1–kestopentaose [19,20]. Not only the DP but also the linkage type appears to influence the efficiency of FOS fermentation by the gut microbiota. Short–chain FOS containing β–(2→6) linkages as 6–kestotriose (6 K) and neokestose (NK) are claimed to have superior prebiotic properties in comparison to inulin–type FOS [21,22]. Neither 6 K nor NK is produced at a commercial scale for application as food ingredients.
Fungal β–fructofuranosidase (EC 3.2.1.26) or fructosyltransferase (EC 2.4.1.100) currently used for the high–scale production of inulin–type FOS from sucrose show thermal instability at temperatures above 50 °C, hampering its application in continuous processes. A relatively low improvement in thermostability of β–fructofuranosidases from Aspergillus japonicus [23] and Microbacterium saccharophilum K – 1 [24] was achieved by random mutagenesis and direct evolution. The alternative approach of engineering a naturally thermostable invertase to enhance FOS synthesis remains poorly investigated.
The thermostable β–fructosidase (BfrA) from the hyperthermophilic bacterium Thermotoga maritima synthesizes low levels of still structurally uncharacterized short–chain FOS in reactions at high substrate concentrations and temperatures between 60–70 °C [25,26]. The gene was expressed in Pichia pastoris with no evidence of changes in the enzyme catalytic properties due to N–glycosylation [26]. This non–saccharolytic yeast holds the GRAS–status and it is thus an appropriate host for the recombinant production of sucrose–converting enzymes with intended application in the food industry. The yeast–produced BfrA has been immobilized and efficiently reused as biocatalyst for the continuous production of inverted sucrose syrup at pasteurization temperatures [27,28].
The crystal structure of BfrA was determined in the apo form and in complex with raffinose using a recombinant protein produced in Escherichia coli [29,30]. As other enzymes of the glycoside hydrolase (GH) family 32 defined in the CAZy database (http://www.cazy.org/), BfrA displays a bimodular fold involving an N–terminal five–bladed β–propeller domain connected to a shorter C–terminal β–sandwich domain. The catalytic triad consisting of the nucleophilic D17, the transition state stabilizer D138, and the acid–base catalyst E190 within the corresponding conserved motifs 14WMNDPNG, 137RDP, and 190EC is localized at the bottom of the active site pocket in the β–propeller domain. In comparison to mesophilic GH32 enzymes, the shorter size of the BfrA loops particularly in the β–sandwich domain may contribute to preserve the protein conformational rigidity at temperatures as high as 80 °C [31].
Sequence divergences between hydrolases and fructosyltransferases belonging to the GH32 family have been the basis for the rational design of mutations aimed to improve the transfructosylating capacity of plant and yeast invertases. In several cases, site–directed replacement of only few divergent amino acids was sufficient to increase FOS synthesis although sucrose hydrolysis remained by far the most dominant activity [1,3,7,[32], [33], [34], [35], [36]].
In the present work, three rationally–designed BfrA mutants (W14Y–W256Y, W14Y–N16S and W14Y) were generated and constitutively expressed in P. pastoris. The secreted recombinant enzymes were characterized in terms of post–translational modifications, optimal activity, thermal stability, substrate/product specificity, kinetic properties, and FOS production. The introduced mutations enhanced FOS yield and varied the proportion of the products 1–kestotriose, 6–kestotriose, and neokestose. This is the first report of improved transferase activity through rational design in a naturally thermostable GH32 enzyme.
Section snippets
Microorganisms, media, and substrates
Escherichia coli strain DH5α (Novagen, USA) was used as a cloning host and for plasmid propagation. Pichia pastoris strain X–33 (Invitrogen, USA) was used as the expression host. E. coli was grown at 37 °C in low–salt LB medium [1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) NaCl, pH 7.5] supplemented or not with zeocin (25 μg mL−1). P. pastoris was grown at 30 °C in YPG medium [1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (v/v) glycerol, pH 6.5] supplemented or not with zeocin
Constitutive expression of site–directed BfrA mutants in Pichia pastoris
A codon–optimized gene encoding the intact β–fructosidase protein (BfrA) from Thermotoga maritima strain MSB8 was used as template to replace three non–catalytic residues (W14, N16 and W256) strictly conserved in the active site of GH32 hydrolases by their equivalent substitute in plant fructosyltransferases (Fig. 1). Wild–type BfrA and the three constructed mutants (W14Y, W14Y–N16S and W14Y–W256Y) were fused to the Saccharomyces cerevisiae α–factor signal peptide and constitutively expressed
Discussion
The thermostable β–fructosidase (BfrA) from the bacterium Thermotoga maritima efficiently hydrolyses sucrose during continuous incubations at elevated temperatures (60–70 °C). The reaction at the high substrate concentration of 1.75 M also yielded 1–kestotriose (1 K), 6–kestotriose (6 K), and neokestose (NK) in the proportion 0.3:1:0.5, accounting for 12.6% (w/w) of the total carbohydrates in the reaction mixture. Similarly, 6 K and NK have been reported as the main FOS produced by mesophilic
Conclusion
The naturally thermostable β–fructosidase (BfrA) from Thermotoga maritima was engineered to enhance FOS synthesis. The constructed single or double mutants (W14Y, W14Y–N16S and W14Y–W256Y) were constitutively expressed in the non–saccharolytic yeast Pichia pastoris, purified from culture supernatants, and biochemically characterized. None of the mutations altered the enzyme thermophilicity and thermostability. The three mutants and wild–type BfrA synthesized a FOS mixture with the dominant
Acknowledgements
Authors wish to acknowledge Maria Elena Rodriguez–Alegría and Fernando Gonzalez Muñoz for analytical support.
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