We study the structure and function of enzymes with potential biotechnological applications. To this end we use a multidisciplinary approach that includes structural analysis, to solve the protein three-dimensional structure; random and rationally driven gene mutation, to modify enzyme properties; in silico mining of enzyme with specific properties from protein sequence databases. Enzymes with selected properties can be used as such or incorporated into zymoactive materials or genetically modified organisms.
Invertase cleaves sucrose to yield glucose and fructose. Saccharomyces cerevisiae invertase has been mutated, reversing its hydrolytic activity, making it capable of synthesizing fructooligosaccharides (prebiotic sugars) with high efficiency (Lafraya et al. 2011). Invertase played a great role as a model enzyme in early biochemical studies. Indeed, the whole theory of enzyme kinetics (Michaelis-Menten equation) was based on experimental results obtained with yeast invertase. The resolution of the enzyme structure (Sainz-Polo et al. 2013) provides a rational basis for understanding its biochemical properties and allows the designing of new versions of the enzyme with novel properties.
Representation of the octameric structure of S. cerevisiae invertase, top (A) and side (B) views. The eight monomers composing the structure are labelled A-H.
A Saccharomyces cerevisiae strain has been modified genetically to express a mutated version of invertase that produces high yield of prebiotic 6-kestose, using sucrose as substrate. A batch culture of the modified strain can produce ca. 200 grams of 6-kestose per kilogram of sucrose (Marín-Navarro et al. 2015).
References
Lafraya Á, Sanz-Aparicio J, Polaina J, Marín-Navarro J. Fructo-oligosaccharide synthesis by mutant versions of Saccharomyces cerevisiae invertase. Appl Environ Microbiol. 2011 Sep;77(17):6148-57. doi: 10.1128/AEM.05032-11.
Sainz-Polo MA, Ramírez-Escudero M, Lafraya A, González B, Marín-Navarro J, Polaina J, Sanz-Aparicio J. Three-dimensional structure of Saccharomyces invertase: role of a non-catalytic domain in oligomerization and substrate specificity. J Biol Chem. 2013 Apr 5;288(14):9755-9766. doi: 10.1074/jbc.M112.446435.
Marín-Navarro J, Talens-Perales D, Polaina J. One-pot production of fructooligosaccharides by a Saccharomyces cerevisiae strain expressing an engineered invertase. Appl Microbiol Biotechnol. 2015 Mar;99(6):2549-55. doi: 10.1007/s00253-014-6312-4.
