Coevolving stability and activity of LsCR by a single point mutation and constructing neat substrate bioreaction system.

Carbonyl reductase (CR)-catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCRM3 , coevolved thermostability, and activity. Compared with LsCRM3 , the catalytic efficiency kcat /KM of LsCRM3 -E145A (LsCRM4 ) was increased from 6.6 to 21.9 s-1 mM-1 . Moreover, E145A prolonged the half-life t1/2 at 40°C from 4.1 to 117 h, T m ${T}_{m}$ was increased by 5°C, T 50 30 ${T}_{50}^{30}$ was increased by 14.6°C, and Topt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCRM4 completely reduced up to 600 g/L 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) in 99.5% eeP , with a space-time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCRM3 , the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCRWT , the substrate loading was increased by 6.5 times. In contrast, LsCRM4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.

Impacts of smoking status on the clinical outcomes of coronary non-target lesions in patients with coronary heart disease: a single-center angiographic study.

BACKGROUND: Coronary atherosclerotic plaque could go through rapid progression and induce adverse cardiac events. This study aimed to evaluate the impacts of smoking status on clinical outcomes of coronary non-target lesions. METHODS: Consecutive patients with coronary heart disease who underwent two serial coronary angiographies were included. All coronary non-target lesions were recorded at first coronary angiography and analyzed using quantitative coronary angiography at both procedures. Patients were grouped into non-smokers, quitters, and smokers according to their smoking status. Clinical outcomes including rapid lesion progression, lesion re-vascularization, and myocardial infarction were recorded at second coronary angiography. Multivariable Cox regression analysis was used to investigate the association between smoking status and clinical outcomes. RESULTS: A total of 1255 patients and 1670 lesions were included. Smokers were younger and more likely to be male compared with non-smokers. Increase in percent diameter stenosis was significantly lower (2.7 [0.6, 7.1] % vs. 3.5 [0.9, 8.9]%) and 3.4 [1.1, 7.7]%, P = 0.020) in quitters than those in smokers and non-smokers. Quitters tended to have a decreased incidence of rapid lesions progression (15.8% [76/482] vs. 21.6% [74/342] and 20.6% [89/431], P = 0.062), lesion re-vascularization (13.1% [63/482] vs. 15.5% [53/432] and 15.5% [67/431], P = 0.448), lesion-related myocardial infarction (0.8% [4/482] vs. 2.6% [9/342] and 1.4% [6/431], P = 0.110) and all-cause myocardial infarction (1.9% [9/482] vs. 4.1% [14/342] and 2.3% [10/431], P = 0.128) compared with smokers and non-smokers. In multivariable analysis, smoking status was not an independent predictor for rapid lesion progression, lesion re-vascularization, and lesion-related myocardial infarction except that a higher risk of all-cause myocardial infarction was observed in smokers than non-smokers (hazards ratio: 3.00, 95% confidence interval: 1.04-8.62, P = 0.042). CONCLUSION: Smoking cessation mitigates the increase in percent diameter stenosis of coronary non-target lesions, meanwhile, smokers are associated with increased risk for all-cause myocardial infarction compared with non-smokers.