The transketolase from Geobacillus stearothermophilus (TK ) is a thermostable enzyme with notable high activity and stability at elevated temperatures, but it accepts non-α-hydroxylated aldehydes only with low efficiency. Here we report a protein engineering study of TK based on double-site saturation mutagenesis either at Leu191 or at Phe435 in combination with Asp470; these are the residues responsible for substrate binding in the active site. Screening of the mutagenesis libraries resulted in several positive variants with activity towards propanal up to 7.4 times higher than that of the wild type. Variants F435L/D470E and L191V/D470I exhibited improved (73 % ee, 3S) and inverted (74 % ee, 3R) stereoselectivity, respectively, for propanal. L191V, L382F/E, F435L, and D470/D470I were concluded to be positive mutations at Leu191, Leu382, Phe435, and Asp470 both for activity and for stereoselectivity improvement. These results should benefit further engineering of TK for various applications in asymmetric carboligation.

The inside back cover picture shows two positive variants of thermostable transketolase from Geobacillus stearothermophilus (TKGst) obtained by saturation mutagenesis at the 191, 435, and 470 positions “discussing” their roughly fivefold improved activity towards non‐α‐hydroxy aldehyde acceptors, as well as the inversed stereoconfiguration of their 4‐deoxyketose products, which have more than 70 % ee starting from lithium hydroxypyruvate as donor and non‐α‐hydroxy aldehydes as acceptors. More information can be found in the communication by D. Yi et al. on page 455 in Issue 5, 2017 (DOI: 10.1002/cbic.201600609).