L-Ribose isomerase (L-RI) catalyzes the reversible aldose-ketose isomerization between L-ribulose and L-ribose. L-ribose is an aldose which is not often found in nature. It can be used as a precursor material for antiviral and anticancer drugs. The gene of Actinotalea fermentans ATCC 43279 L-RI (Af-RI) was overexpressed in Escherichia coli BL21 Star (DE3). The purified recombinant enzyme demonstrated its optimal activity at 45oC and pH 8, and its half-life at 50oC was 58 min. To enhance the thermostability of Af-RI by protein engineering, four residues (L69, P78, V139, and F141) on the interface of enzyme tetramers and the mutation sites from the study of Geodermatophilus obscurus DSM43160 have been chosen for mutagenesis (D135, A221). In addition, the peptide derived from the acidic tail of a-synuclein (ATS) has been fused to the C-terminal of Af-RI to evaluate the effect of fusion on the thermostability of ATS-fused Af-RI (Af-RI-ATS). L69Q, P78K, P78R, P78T, V139T, F141Y, D135G, A221W, R132Q/D134G/D135G, P220Q/A221W, and Af-RI-ATS were successfully constructed and expressed in E. coli BL21 Star (DE3). The relative activities of the crude extracts containing wild-type, F141Y, R132Q/D134G/D135G, and P220Q/A221W were 100%, 77.6%, 58.4%, and 26.9%, respectively. However, other mutations and ATS fusion caused severe loss of enzyme activities. After incubating enzymes at 50oC for 10 min, the residual activities of wild-type and F141Y were 27% and 38.4%, respectively. The specific activity of purified F141Y Af-RI was 25.1 U/mg, which was comparable to 26.6 U/mg of wild-type enzyme; the half-life of F141Y Af-RI at 50oC was 66.6 min, which was 8 min longer than that of the wild-type enzyme. Although most of the mutations on the interface of Af-RI tetramers cause severe loss of activity, F141Y Af-RI possesses good activity and enhanced thermostability and has potential to be used in L-ribose production.

Protein engineering of recombinant L-ribose isomerase from Actinotalea fermentans ATCC 43279 to alter its thermostability

2019 Fall National Meeting

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