In previous studies, we isolated a novel beta-glucosidase fromPenicillium oxalicum16 (16BGL), which is useful for producing cellulosic ethanol. However, 16BGL has a relatively low enzyme activity and product tolerance; besides, a huge gap exists between the optimum temperature of 16BGL (70 degrees C) and the fermentation temperature for producing cellulosic ethanol (40 degrees C). Here, we present a directed evolution-based study, which combines one-round error-prone PCR with three rounds of high-throughput screening, for coevolving multiple enzymatic characteristics of 16BGL. We identified an improved variant Y-1-B1 with a triple mutant (G414S/D421V/T441S). Y-1-B1 had an optimum temperature of 50 degrees C, much closer to the fermentation temperature. The catalytic efficiency of Y-1-B1 for hydrolyzing pNPG was 1355 mM(-1) s(-1)at 50 degrees C and pH 5, significantly higher than that of 16BGL (807 mM(-1) s(-1)). Y-1-B1 also achieved a slightly reduced strength of product inhibition of 1.1 at a glucose concentration of 20 mM, compared with the ratio of 1.3 for 16BGL. A maximum titer of 6.9 g/L for ethanol production was achieved in the reaction with Y-1-B1, which was 22% higher than that achieved with 16BGL. Structure modeling revealed that the mutations are distant from the active-site pocket. Therefore, we performed molecular dynamics (MD) simulations to understand why these mutations can improve catalytic efficiency. MD simulation revealed that the nucleophilic residue Asp261 had a much closer contact with the glucosidic center of pNPG in the simulation with Y-1-B1 than that with 16BGL, suggesting that the mutant is more favorable for catalysis. Key points center dot Multiple enzymatic properties of Penicillium oxalicum 16 BGL were coevolved. center dot A catalytically efficient triple mutant G414S/D421V/T441S was reported. center dot Molecular dynamics simulation supported the enhanced catalytic activity.