Abstract
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.
Highlights
Invertases [b-D-fructofuranoside fructohydrolase (EC 3.2.1.26)] 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 Dfructose
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
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
Summary
Invertases [b-D-fructofuranoside fructohydrolase (EC 3.2.1.26)] 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 Dfructose. Recombinant protein expression in cost effective cultivable and handling host can provide higher productivity in short period of time with low costs Regarding this effect on industry, increasing the level of invertase expression is considerable. Despite the success in using Saccharomyces cerevisiae as a host for expression of eukaryotic genes, some problems like hyperglycosylation, less secretion of recombinant protein and low level of expression reduce its potential in biotechnological industry For these reasons, instead of S. cerevisiae, methylotrophic yeast systems such as Pichia pastoris because of correct glycosylation profile, cost effective bulk recombinant protein production, high level expression, secretion of heterologous protein to media and easy scale up expression which is important for biotechnological industry, have been used (Cereghino and Cregg 2000). Expression of the optimized SUC2 gene in P. pastoris yields a secreted active recombinant protein with a little different molecular weight to the invertase secreted by S. cerevisiae, after purification of enzyme, biochemical properties and kinetic parameters of purified recombinant invertase was determined
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