Journal of Biotechnology & Biomaterials
Open Access

Abstract

Invertase (EC.3.2.1.26) catalyzes hydrolysis of sucrose to an equimolar mixture of D-glucose and D-fructose. This is a single step reaction which is of interest for various industrial applications. Saccharomyces cerevisiae is the organism of choice for invertase production with the ability to synthesize two forms of invertase. In this research, S. cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into 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. R-inv was purified by His-Tag affinity chromatography and appeared to be a mixture of glycosylated proteins with various sizes of 85 to 95 kDa on SDS-PAGE. Deglycosylation of the proteins by Endo-H resulted in the proteins with average molecular weight of 60 kDa. The optimal pH value for the R-inv activity was 4.8. The purified recombinant invertase biochemical properties and kinetic parameters determined a pH optimum at 4.8 and temperature optimum at 60° C which in compare 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 units/mg with 3.83 fold of purification and the kinetic constants for enzyme were Km value of 19 mM which can be concluded that recombinant P. pastoris invertase can be more effectively for industrial quality criteria. We conclude that recombinant P. pastoris enzyme with broad pH stability, substrate specify and proper thermal stability can be fulfil a serried of predefined industrial quality criteria to be used in food, confectionary, pharmaceutical and bio ethanol production industries.

Biography

Nooshin Mohandesi has completed her PhD from NIGEB (National Institue of Genetic Engineering and Biotechnology), Department of Biotechnology and she is working as Assistant Professor in IAU (Islamic Azad University). Her research focused on industrial enzymes biotechnology and engineering specially the enzymes are useful in food industry. Our scientific teams have produced several recombinant industrial enzymes in bulk production and now she is working on invertase engineering to fulfil industrial criteria and improvement of enzyme kinetic efficiency for enzyme applications in food and confectionary industries. Also our team have published several papers in food industrial enzymes field like phytase and amylase in reputed journals.