Engineered thermostable β–fructosidase from Thermotoga maritima with enhanced fructooligosaccharides synthesis

• Published in: Enzyme and microbial technology 2019-06, Vol.125, p.53-62
• Main Authors: Menéndez, Carmen, Martínez, Duniesky, Pérez, Enrique R., Musacchio, Alexis, Ramírez, Ricardo, López-Munguía, Agustín, Hernández, Lázaro
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; beta-Fructofuranosidase - genetics; beta-Fructofuranosidase - metabolism; Catalytic Domain; FOS; Fructooligosaccharides; Fructosidase; Gene Expression; Hydrogen-Ion Concentration; Invertase; Kinetics; Molecular Docking Simulation; Mutagenesis, Site-Directed; Mutation; Oligosaccharides - biosynthesis; Oligosaccharides - chemistry; Pichia - genetics; Pichia - metabolism; Pichia pastoris; Protein Engineering; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Substrate Specificity; Sucrose - metabolism; Temperature; Thermotoga maritima; Thermotoga maritima - enzymology; Thermotoga maritima - genetics
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2019.02.002

•First report of improved transferase activity through rational design in a naturally thermostable GH32 enzyme.•Wild–type BfrA synthesized a FOS mixture with the predominant presence of 6–kestotriose in reactions at high substrate and elevated temperatures.•The mutated BfrA enhanced FOS yield without altering the enzyme thermophilicity and thermostability.•Comparative docking analysis revealed the gain and loss of relevant enzyme–ligand interactions in the BfrA mutants. The thermostable β–fructosidase (BfrA) from the bacterium Thermotoga maritima converts sucrose into glucose, fructose, and low levels of short–chain fructooligosaccharides (FOS) at high substrate concentration (1.75 M) and elevated temperatures (60–70 °C). In this research, FOS produced by BfrA were characterized by HPAE–PAD analysis as a mixture of 1–kestotriose, 6G–kestotriose (neokestose), and to a major extent 6–kestotriose. In order to increase the FOS yield, three BfrA mutants (W14Y, W14Y–N16S and W14Y–W256Y), designed from sequence divergence between hydrolases and transferases, were constructed and constitutively expressed in the non–saccharolytic yeast Pichia pastoris. The secreted recombinant glycoproteins were purified and characterized. The three mutants synthesized 6–kestotriose as the major component of a FOS mixture that includes minor amounts of tetra– and pentasaccharides. In all cases, sucrose hydrolysis was the predominant reaction. All mutants reached a similar overall FOS yield, with the average value 37.6% (w/w) being 3–fold higher than that of the wild–type enzyme (12.6%, w/w). None of the mutations altered the enzyme thermophilicity and thermostability. The single mutant W14Y, with specific activity of 841 U mg−1, represents an attractive candidate for the continuous production of FOS–containing invert syrup at pasteurization temperatures.