Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties.
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-1mg-1With kinetic efficiency (Kcat/Km) of 13.15 s-1mmol-1it 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.
National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Islamic Republic of Iran.
Nooshin
N
Mohandesi
Protein Research Centre, Shahid Beheshti University, GC, Tehran, Islamic Republic of Iran. o_ranaei@sbu.ac.ir.
Seyed Omid Ranaei
SO
Siadat
National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Islamic Republic of Iran.
Kamahldin
K
Haghbeen
Department of Biology, Central Tehran Branch, Islamic Azad University, Tehran, Islamic Republic of Iran.
Ardeshir
A
Hesampour
Cloning; Pichia pastoris; Recombinant invertase; SUC2 gene; Saccharomyces cerevisiae