Fusion of the peptide derived from the acidic tail of alpha-synuclein improves the thermostability and soluble expression of recombinant Agrobacterium sp. D-allulose 3-epimerase

Abstract

D-Allulose 3-epimerase (DAEase) catalyzes the epimerization between 6-fructose and D-allulose. The peptide derived from the acidic tail of alpha-synuclein (ATS) was fused to wild-type and mutant I33L/S213C Agrobacterium sp. ATCC 31749 DAEases (AsDAEases) in an attempt to evaluate the effects of ATS fusion on the thermostability and soluble expression of AsDAEases expressed in Escherichia coli. The half-lives at 65 degrees C for wildtype, ATS-fused, I33L/S213C, and I33L/S213C/ATS-fused (LCATS) AsDAEases are 4.37, 10.7, 55.9, and 81.5 min, respectively. The ATS peptide fusion to the wild-type and I33L/S213C AsDAEases has improved the thermostability by extending their half-lives at 65 degrees C to 2.45- and 1.46-fold, respectively, and also has increased their soluble expressions to 1.57- and 1.40-fold, respectively. During eight rounds of D-allulose production from D-fructose by repeatedly using calcium alginate beads with entrapped E. coli cells harboring wild-type and LCATS AsDAEases, respectively, the freshly prepared immobilized cells harboring LCATS AsDAEase have maintained around 33 % conversion, while those harboring wild-type enzyme have decreased conversion from 30 % to 18 % due to thermoinactivation. And, the immobilized cells maintained good stability after 150-day storage. Because of the best thermostability and similar specific activity among these recombinant enzymes, LCATS AsDAEase shows great potential to be applied in industry for the production of D-allulose.