Date Published: June 8, 2016
Publisher: Springer Berlin Heidelberg
Author(s): Nooshin Mohandesi, Seyed Omid Ranaei Siadat, Kamahldin Haghbeen, Ardeshir Hesampour.
Invertase (EC.188.8.131.52) 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.
Invertases [β-d-fructofuranoside fructohydrolase (EC 184.108.40.206)] are disaccharidases which belong to the Gh32 family of glycoside hydrolases and catalyse the hydrolysis of sucrose into an equimolar mixture of d-glucose and d-fructose. To produce invert sugar at concentrations lower than 10 % sucrose that are more stable than pure sucrose syrups and with less carbohydrate are sweeter than sucrose and minimise crystallisation in comparison with sucrose at high (Gehlawat 2001; Boer et al. 2004; Mona et al. 2009; Patil et al. 2012) concentration. They have been reported to be produced by variety of higher plants, some animal cells and wide range of microorganisms including bacteria and fungi (Kim et al. 2000).
Although invertase has been produced by different microorganisms (Kim et al. 2000), however, the low expression level of invertase in the native hosts make these organisms unsuitable to produce heterologous invertase for biotechnological applications and because of the interest in use of biologically produced invertase in confectionary and dairy, pharmaceutical, prebiotics and bioethanol production, highly efficient and cost-effective process of enzyme production by recombinant micro-organisms have been developed (Gehlawat 2001).