Abstract
Purposed-psicose-3-epimerase (DPEase) catalyses the isomerisation of d-fructose to d-psicose, a rare sugar in nature with unique nutritional and biological functions. An effective industrial-scale method is needed for d-psicose production. Herein, the expression of a neutral and a slightly acidic pH DPEase in Bacillus subtilis was evaluated.MethodsTwo DPEase genes from Clostridium bolteae and Dorea sp. were separately expressed in B. subtilis via plasmid pSTOP1622, and an extra P43 promoter was employed to the expression cassette. The fermentation conditions of the engineered B. subtilis strains were also optimised, to facilitate both cell growth and enzyme production.ResultThe introduction of P43 promoter to the two DPEase genes increased enzyme production by about 20%. Optimisation of fermentation conditions increased DPEase production to 21.90 U/g at 55 °C and 24.01 U/g at 70 °C in B. subtilis expressing C. bolteae or Dorea sp. DPEase, equating to a 94.67% and 369.94% increase, respectively, relative to controls.ConclusionEnhanced DPEase production was achieved in B. subtilis expressing C. bolteae or Dorea sp. DPEase genes.
Highlights
D-psicose, the C-3 epimer of D-fructose, is rarely found in nature
Enhanced DPEase production was achieved in B. subtilis expressing C. bolteae or Dorea sp
Optimisation of fermentation conditions We examined the efficiency of DPEase assays using whole-cell reactions to determine optimal carbon and nitrogen sources
Summary
D-psicose, the C-3 epimer of D-fructose, is rarely found in nature. It has 70% relative sweetness but 0.3% energy of sucrose and is suggested as an ideal substitute for sucrose in foodstuffs due to its low energy and safety (generally recognised as safe (GRAS)) (Zhang et al 2017b). D-psicose has attracted increasing attention for its ability reduce blood glucose levels and intra-abdominal fat accumulation, protect against atherosclerosis, and scavenge reactive oxygen species (ROS) (Chen et al 2019; Shintani et al 2017). D-psicose is only found in limited quantities in some natural plants such as cane and wheat (Mu et al 2012). It is necessary to develop a method for D-psicose production to meet the increasing market demand
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