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 LsCR_M3, coevolved thermostability, and activity. Compared with LsCR_M3, the catalytic efficiency k_cat/K_M of LsCR_M3-E145A (LsCR_M4) was increased from 6.6 to 21.9 s⁻¹ mM⁻¹. Moreover, E145A prolonged the half-life t_1/2 at 40°C from 4.1 to 117 h, $T_m$ was increased by 5°C, $T_{50}^{30}$ was increased by 14.6°C, and T_opt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCR_M4 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% ee_P, with a space-time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCR_M3, the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCR_WT, the substrate loading was increased by 6.5 times. In contrast, LsCR_M4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.     

Asymmetric synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using a self-sufficient biocatalyst based on carbonyl reductase and cofactor co-immobilization

Bioprocess and Biosystems Engineering - tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate [(3R,5S)-CDHH] is the key chiral intermediate to synthesize the side chain of the lipid-lowering drug...     

Optimization of media composition and culture conditions for acarbose production by <Emphasis Type="Italic">Actinoplanes utahensis</Emphasis> ZJB-08196

Acarbose is a clinically useful drug for the treatment of type II, insulin-independent diabetes as a hypoglycemic agent. An acarbose-overproducing strain ZJB-08196, indentified as Actinoplanes utahensis, was able to produce 4,210 mg l⁻¹ of acarbose at 591 mOsm kg⁻¹ with the optimized conditions at bench scale. Shake flask fermentation showed that maltose, glycerol and monosodium glutamate were supportive for acarbose production; soybean meal had higher bioavailability than corn steep liquor. Moreover, acarbose formation was not parallel with mycelial growth and the pattern of acarbose production by A. utahensis ZJB-08196 was the type of mixed-growth associated.     

Structural-guided design to improve the catalytic performance of aldo-keto reductase KdAKR

Aldo-keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH-dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t-butyl 6-chloro (5S)-hydroxy-3-oxohexanoate ((5S)-CHOH) were improved by mutating its residues in the loop regions around the substrate-binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67-fold higher catalytic efficiency compared to the wild-type (WT) KdAKR, and improved R-selectivity toward (5S)-CHOH (de_p value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3-fold increase in half-life (t_1/2) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)-CHOH to diastereomeric pure t-butyl 6-chloro-(3R, 5S)-dihydroxyhexanoate ((3R, 5S)-CDHH) within 8.0 h, with a space-time yield of 300.7 g/L/day. Our results deepen the understandings of the structure−function relationship of AKRs, providing a certain guidance for the modification of other AKRs.     

棘白菌素B脱酰基酶的研究

棘白菌素B脱酰基酶是合成阿尼芬净重要中间产物棘白菌素B母核的关键酶。本文综述了近年来国内外学者对该酶的成熟机制、酶学性质、重组表达及生物催化应用等方面的相关研究进展。