Three-dimensional Structure of Saccharomyces Invertase

Sainz-Polo, M.A.; Ramirez-Escudero, M.; Lafraya, Álvaro; González, Beatriz; Marín-Navarro, Julia; Polaina, Julio; Sanz‐Aparicio, Julia

doi:10.1074/jbc.m112.446435

Cited by 93 publications

(50 citation statements)

References 41 publications

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“…Reducing sugars were quantified by addition of dinitrosalicylic acid reagent and the reaction was terminated by heating in 100 °C boiling water bath for 10 min. Finally, absorbance was read at 540 nm in spectrophotometer (Sainz-Polo et al 2013). One unit of invertase was defined as the amount of enzyme that catalyzed the formation of l μmol of reducing sugar/min from sucrose at 50 °C and pH 4.8.…”

Section: Methodsmentioning

confidence: 99%

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

et al. 2016

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Invertase (EC.3.2.1.26) catalyzes the hydrolysis of sucrose to an equimolar mixture of d-glucose and d-fructose which is of interest for various industrial applications. In this research, Saccharomyces cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into the methylotrophic yeast Pichia pastoris under the control of the inducible AOX1 promoter. High level of the extracellular recombinant invertase (R-inv) production was achieved via methanol induction for 4 days and purified by His-Tag affinity chromatography which appeared to be a mixture of glycosylated proteins with various sizes of 85–95 kDa on SDS-PAGE. Deglycosylation of the proteins by Endo-H resulted in the proteins with average molecular weight of 60 kDa. The purified recombinant invertase biochemical properties and kinetic parameters determined a pH and temperature optimum at 4.8 and 60 °C, respectively, which in comparison with native S. cerevisiae invertase, thermal stability of recombinant invertase is highly increased in different heating treatment experiments. The purification of recombinant invertase resulted in an enzyme with specific activity of 178.56 U/mg with 3.83-fold of purification and the kinetic constants for enzyme were Km value of 19 mM and Vmax value of 300 μmol min−1 mg−1 With kinetic efficiency (Kcat/Km) of 13.15 s−1 mmol−1 it can be concluded that recombinant P. pastoris invertase can be more effective for industrial quality criteria. We conclude that recombinant P. pastoris enzyme with broad pH stability, substrate specificity and proper thermal stability can fulfil a series of predefined industrial quality criteria to be used in food, pharmaceutical and bio ethanol production industries.

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Section: Methodsmentioning

confidence: 99%

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

et al. 2016

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“…In this respect, and taking into account the fact The invertase, PDB code 4EQV [124], is shown with the catalytic triad (red) and the hydrogen bond network of the sucrose-binding box. In this respect, and taking into account the fact The invertase, PDB code 4EQV [124], is shown with the catalytic triad (red) and the hydrogen bond network of the sucrose-binding box.…”

Section: Sucrase-type Enzymesmentioning

confidence: 99%

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

Bissaro

Monsan

Fauré

et al. 2015

Biochemical Journal

141

152

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Carbohydrates are ubiquitous in Nature and play vital roles in many biological systems. Therefore the synthesis of carbohydrate-based compounds is of considerable interest for both research and commercial purposes. However, carbohydrates are challenging, due to the large number of sugar subunits and the multiple ways in which these can be linked together. Therefore, to tackle the challenge of glycosynthesis, chemists are increasingly turning their attention towards enzymes, which are exquisitely adapted to the intricacy of these biomolecules. In Nature, glycosidic linkages are mainly synthesized by Leloir glycosyltransferases, but can result from the action of non-Leloir transglycosylases or phosphorylases. Advantageously for chemists, non-Leloir transglycosylases are glycoside hydrolases, enzymes that are readily available and exhibit a wide range of substrate specificities. Nevertheless, non-Leloir transglycosylases are unusual glycoside hydrolases in as much that they efficiently catalyse the formation of glycosidic bonds, whereas most glycoside hydrolases favour the mechanistically related hydrolysis reaction. Unfortunately, because non-Leloir transglycosylases are almost indistinguishable from their hydrolytic counterparts, it is unclear how these enzymes overcome the ubiquity of water, thus avoiding the hydrolytic reaction. Without this knowledge, it is impossible to rationally design non-Leloir transglycosylases using the vast diversity of glycoside hydrolases as protein templates. In this critical review, a careful analysis of literature data describing non-Leloir transglycosylases and their relationship to glycoside hydrolase counterparts is used to clarify the state of the art knowledge and to establish a new rational basis for the engineering of glycoside hydrolases.

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“…So far, eleven X-ray structures of GH32 enzymes have been solved and reported. [18][19][20][21][22][23][24][25][26][27][28][31][32][33][34] Among them, the X-ray structure of B. longum β-d-fructofuranosidase (PDB code 3PIJ) 20 was chosen, as the sequence homology between the template and target proteins is 55% (35% identity). The optimized model is shown in Fig.…”

Section: Modeling Of Invertase and Mechanistical Implicationsmentioning

confidence: 99%

Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major

Belaz

Rattier

Lafite

et al. 2015

Carbohydrate Research

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Three-dimensional Structure of Saccharomyces Invertase

Cited by 93 publications

References 41 publications

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major

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