Premium
High‐level extracellular expression of inulin fructotransferase in Pichia pastoris for DFA III production
Author(s) -
Zhan Rongrong,
Mu Wanmeng,
Jiang Bo,
Li Yungao,
Zhou Liuming,
Zhang Tao
Publication year - 2015
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.6931
Subject(s) - pichia pastoris , inulin , extracellular , chemistry , food science , pichia , biochemistry , biology , recombinant dna , gene
BACKGROUND Inulin fructotransferase ( IFTase ) catalyzes inulin conversion to difructose anhydride ( DFA III ), which is a natural low‐calorie sweetener. Although heterologous expression of IFTase was achieved in Escherichia coli , the extracellular enzyme activity was very low, which limited the commercialization of IFTase . RESULTS Active IFTase of about 43 kDa molecular mass of subunit was extracellularly expressed by Pichia pastoris and was greatly regulated by the IFTase gene copy number integrated into the P. pastoris genome and by the methanol concentration in the induction phase. Under optimized culture conditions, multicopy P. pastoris exhibited a maximum extracellular IFTase activity of 105.4 U mL −1 in a 5 L fermenter, which was 8.9‐fold the activity in shake flasks and 5.3‐fold that obtained from wild‐type strain. CONCLUSION IFTase was expressed in a eukaryotic P. pastoris system for the first time and achieved high‐level extracellular expression using a high‐cell‐density fed‐batch cultivation strategy. This demonstrated that P. pastoris was a good candidate for potential DFA III production as a novel IFTase expression system. © 2014 Society of Chemical Industry