Systems Microbiology and Biomanufacturing
·
2022
·
IF 0
Zhu Jiufeng,Bai Yajun,Fan Tai-Ping,Zheng Xiaohui,Cai Yujie
Ethyl ( R )-4-chloro-3-hydroxybutyrate (( R )-CHBE), as a chiral intermediate, is widely used in the synthesis of various chiral drugs. In this study, we screened two aldo–keto reductases (LP-AKRs) from the probiotic Lactobacillus plantarum DSM20174, both with a molecular weight of approximately 31 kDa. Both enzymes could reduce 4-chloroacetoacetic acid ethyl ester (COBE) to produce ( R )-CHBE with an enantioselectivity value of 99%. When determining the kinetic parameter, the K m , K cat , and V max of LP-AKR5 and LP-AKR9 were 9.5 mM, 7.6 U/mg, 3.96 s −1 and 8.7 mM, 8.59 U/mg, 4.47 s −1 , respectively. Both LP-AKR5 and LP-AKR9 had an optimal reaction pH of 6 and could maintain a high level of stability at pH 6, allowing them to perform well in an acidic environment. LP-AKR5 and LP-AKR9 had optimal reaction temperatures of 30 °C and 40 °C, respectively. Metal ions had minimal influence on LP-AKR5 and LP-AKR9 enzyme activities. This series of enzymatic properties showed that LP-AKR5 and LP-AKR9 mined from Lactobacillus plantarum DSM20174 could asymmetrically catalyze the synthesis of ( R )-CHBE under weakly acidic circumstances, which could maintain product stability and provide a good foundation for industrial production.
研究领域:
生物化学
·
分子生物学
·
工业微生物学
✓ 已验证
DOI
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Li Qiao,Bianqin Ma,Yongjian Jiang,Xiaoting Pan,Zhili Mao,Yi Zhang,Roger A. Sheldon,Anming Wang
Keto reductases are crucial NAD( P )H-dependent enzymes used for the enantioselective synthesis of alcohols from prochiral ketones. Typically, the NADPH cofactor is regenerated through a second enzyme and/or substrate. However, photocatalytic cofactor regeneration using water as a sacrificial electron and hydrogen donor presents a promising alternative, albeit a challenging one. Herein we fabricated several nitrogen-doped carbon dots (CDs) with visible light absorption properties, good water solubility and biocompatibility for photocatalytic regeneration of NADPH. The CD with a smaller size and suitable redox potential gave the highest NADPH yield (55.7 %). Based on this, NADPH-dependent aldo-keto reductase crosslinked aggregates (AKR-CLEs) were initially applied as a stable biocatalyst to reduce the prochiral ketone. ( S )-1-(2-Chlorophenyl) ethanol, an intermediate for LPA1R antagonists, was obtained in 65.3 % yield and 99.99 % enantiomeric excess (ee) under visible light irradiation. The isotope tracer experiment confirmed that water is the hydrogen donor in this light-driven, photo-enzymatic asymmetric hydrogenation system. This method is useful for the sustainable synthesis of chiral alcohols. Moreover, the general principle of utilizing water as the sacrificial hydrogen and electron donor holds potential for application in other redox cofactor regeneration systems.
研究领域:
生物化学
·
催化化学
·
酶工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Lingzhi Zhang,Rui Zhou,Dekai Liu,Meinan Zhu,Guangya Zhang,Lijuan Zhang,Shu-Feng Zhou,Wei Jiang
Given their outstanding efficiency and selectivity, enzymes are integral in various domains such as drug synthesis, the food industry, and environmental management. However, the inherent instability of natural enzymes limits their widespread industrial application. In this study, we underscore the efficacy of enhancing protein thermal stability through comprehensive protein design strategies, encompassing elements such as the free energy of protein folding, internal forces within proteins, and the overall structural design. We also demonstrate the efficiency and precision of combinatorial screening in the thermal stability design of aldo-keto reductase (AKR7-2-1). In our research, three single-point mutations and five combinatorial mutations were strategically introduced into AKR7-2-1, using multiple computational techniques. Notably, the E12I/S235I mutant showed a significant increase of 25.4 °C in its melting temperature ( Tm ). Furthermore, the optimal mutant, E12V/S235I, maintained 80 % of its activity while realizing a 16.8 °C elevation in Tm . Remarkably, its half-life at 50 °C was increased to twenty times that of the wild type. Structural analysis indicates that this enhanced thermal stability primarily arises from reduced oscillation in the loop region and increased internal hydrogen bonding. The promising results achieved with AKR7-2-1 demonstrate that our strategy could serve as a valuable reference for enhancing the thermal stability of other industrial enzymes.
研究领域:
生物化学
·
分子生物学
·
工业酶工程
✓ 已验证
DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2025
·
IF 13.2
Shuling Zhang,Yuqing Zhang,Weixi Kong,Wanting Guo,Quan Yuan,Yanjun Jiang,Yunting Liu
Enzymatic reduction of (hetero)aryl ketones is an important reaction for the synthesis of industrially relevant chiral alcohols, but its preparative application remains restricted due to enzyme unavailability and substrate inhibition. Herein, we reported the identification of a ketoreductase and its mutant from Metabacillus lacus ( Ml KREDs) with high activity and wide substrate scope (50 examples) for the reduction of (hetero)aryl ketones by gene mining and protein engineering. During process development, we constructed a deep eutectic solvent (DES)-buffer system to relieve substrate inhibition, achieving the efficient conversion of high-concentration (up to 1.2 M) (hetero)aryl ketones to the corresponding chiral alcohols, some of which are key intermediates for pharmaceuticals. In addition, molecular dynamics simulations provided deeper insights into the substrate inhibition mechanism as well as the DES-induced enzyme activation and stabilization mechanisms. This work provides a green and reliable strategy for the preparative synthesis of pharmaceutical-related chiral (hetero)aryl alcohols.
研究领域:
生物化学
·
制药工程
·
合成生物学
✓ 已验证
产品关联
DOI
高影响因子
Catalysts
·
2022
·Vol. 12
·期 3
·304
·
IF 4.501
Xinxing Gao,Qianqian Pei,Nianqing Zhu,Yi Mou,Jilei Liang,Xin Zhang,Shoushuai Feng
(S)-N-Boc-3-hydroxypiperidine is an important intermediate of the anticancer drug ibrutinib and is mainly synthesized by the asymmetric reduction catalyzed by ketoreductase coupled with glucose dehydrogenase at present. In this study, the coexpression recombinant strainsE. coli/pET28-K-rbs-G with single promoter andE. coli/pETDuet-K-G with double promoters were first constructed for the coexpression of ketoreductase and glucose dehydrogenase in the same cell. Then, the catalytic efficiency ofE. coli/pET28-K-rbs-G for synthesizing (S)-N-Boc-3-hydroxypiperidine was found to be higher than that ofE. coli/pETDuet-K-G due to the more balanced activity ratio and higher catalytic activity. On this basis, the catalytic conditions ofE. coli/pET28-K-rbs-G were further optimized, and finally both the conversion of the reaction and the optical purity of the product were higher than 99%. In the end, the cell-free extract was proved to be a better catalyst than the whole cell with the improved catalytic efficiency of different recombinant strains. This study developed a better coexpression strategy for ketoreductase and glucose dehydrogenase by investigating the effect of activity ratios and forms of the biocatalysts on the catalytic efficiency deeply, which provided a research basis for the efficient synthesis of chiral compounds.Keywords:(S)-N-Boc-3-hydroxypiperidine;coexpression strategy;activity ratio;ketoreductase;glucose dehydrogenase
研究领域:
生物化学
·
生物技术
·
药物化学
✓ 已验证
产品关联
DOI
高影响因子
Journal of Chemical Information and Modeling
·
2024
·
IF 5.6
Ru-De Lin,Xiu Xing,Yuan Yu,Wen-Dian Li,Dan-Dan Chang,Fei-Yan Tao,Na Wang
Lactobacillus kefir alcohol dehydrogenase (LkADH) and ketoreductase from Chryseobacterium sp. CA49 (ChKRED12) exhibit different chemoselectivity and stereoselectivity toward a substrate with both keto and aldehyde carbonyl groups. LkADH selectively reduces the keto carbonyl group while retaining the aldehyde carbonyl group, producing optically pure R-alcohols. In contrast, ChKRED12 selectively reduces the aldehyde group and exhibits low reactivity toward ketone carbonyls. This study investigated the structural basis for these differences and the role of specific residues in the active site. Molecular dynamics (MD) simulations and quantum chemical calculations were used to investigate the interactions between the substrate and the enzymes and the essential cause of this phenomenon. The present study has revealed that LkADH and ChKRED12 exhibit significant differences in the structure of their respective active pockets, which is a crucial determinant of their distinct chemoselectivity toward the same substrate. Moreover, residues N89, N113, and E144 within LkADH as well as Q151 and D190 within ChKRED12 have been identified as key contributors to substrate stabilization within the active pocket through electrostatic interactions and van der Waals forces, followed by hydride transfer utilizing the coenzyme NADPH. Furthermore, the enantioselectivity mechanism of LkADH has been elucidated using quantum chemical methods. Overall, these findings not only provide fundamental insights into the underlying reasons for the observed differences in selectivity but also offer a detailed mechanistic understanding of the catalytic reaction.
研究领域:
生物化学
·
酶学
·
计算生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ORGANIC PROCESS RESEARCH & DEVELOPMENT
·
2020
·
IF 3.023
Hong-Yi Wang,Jia-Wei Tang,Peng Peng,Hai-Jun Yan,Fu-Li Zhang,Shao-Xin Chen
We first developed a novel and efficient chemoenzymatic process to prepare (S)-1-(pyridin-4-yl)-1,3-propanediol, a vital HepDirect prodrug intermediate, from inexpensive and commercially available isonicotinic acid. Through this process, we provide a creative way to obtain the key chiral intermediate, β-hydroxyester, with ketoreductase (KRED) EA. After optimization of the process, we performed the reaction on a 100 g scale with a substrate concentration of up to 150 g/L, a yield of 93%, and an ee value of up to 99.9%. Additionally, we used a simple and effective NaBH4/MgCl2 reduction system to obtain (S)-1-(pyridin-4-yl)-1,3-propanediol with >99.9% ee and an 80% yield. This novel chemoenzymatic process has the potential to be a cost-effective and environmentally friendly process suitable for industrial use.
研究领域:
生物化学
·
制药工程
✓ 已验证
DOI
高影响因子
NEW JOURNAL OF CHEMISTRY
·
2022
·Vol. 46
·期 13
·6274-6282
·
IF 3.925
Xiu Xing,Yan Liu,Ming-Liang Shi,Kun Li,Xin-Yue Fan,Zhong-Liu Wu,Na Wang,Xiao-Qi Yu
Controllable and mild photoenzymatic production of chiral alcohols was realized by coupling a versatile photochemical NAD(P)H regeneration system with (R)- or (S)-selective ketoreductases. The efficiency of NAD(P)H regeneration was improved using a Rhodium functionalized metal organic framework, namely Rh-UiO-67, to adjust and control electron transport and electron utilization. Furthermore, six different ketoreductases could be successfully immobilized on Rh-UiO-67 and combined with the light-driven NAD(P)H regeneration system to produce chiral aryl alcohols. Various chiral alcohols with complementary (R)- and (S)-conformations can be constructed by this method with high yields (97%) and excellent stereoselectivity (>99% ee).
研究领域:
化学
·
生物化学
·
催化
·
有机合成
✓ 已验证
产品关联
DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2025
·
IF 6.2
Bin Ma,Jiafeng Niu,Hao Zhu,Huibing Chi,Zhaoxin Lu,Fengxia Lu,Ping Zhu
Aldo-keto reductase is responsible for the formation of nontoxic 3-epi-deoxynivalenol (3-epi-DON) from 3-keto-DON, which is an enzymatic detoxification manner to completely eliminate the toxicity of mycotoxin DON against health and environmental threats. Therefore, unlocking the facilitated substrate specificity of aldo-keto reductase for 3-keto-DON has become a critical challenge for advanced catalytic performance. In this endeavor, a loop-based engineering strategy was developed for aldo-keto reductase AKR13B3 from Devosia A6–243 to catalyze 3-keto-DON. A 31.9-fold switch from a disfavored substrate to the preferred one was produced along with a significant 37.9-fold increase in catalytic efficiency. Kinetic parameter determinations, structural analyses, and molecular simulations were employed to elucidate the molecular mechanisms underlying these enhancements in catalytic activity and substrate specificity. Overall, our work presents a feasible scheme for designing aldo-keto reductases with exceptional substrate specificity and catalytic activity, holding great promise for developing enzymatic detoxification agents.
研究领域:
生物化学
·
分子生物学
·
环境科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
·
2022
·
IF 4.885
Wenhao Qu,Yajun Bai,Tai-Ping Fan,Xiaohui Zheng,Yujie Cai
Lactobacillus reuteri DSM20016 has several aldo-keto reductases (AKRs), and they were heterologously expressed in E. coli BL21 (DE3) in this study. Subsequently, these AKRs were purified, and their enzymatic properties were investigated. It was found that these enzymes used NADPH as coenzyme and did not depend on metal ions. The optimum pH of these AKRs was a slightly acidic (AKR-1 and AKR-6: 5.5; AKR-3 and AKR-8: 6; AKR-2 and AKR-4: 6.5). These enzymes had a broad substrate spectrum and could convert a variety of aldehydes to alcohols; however, they did not catalyze the reverse reactions. All results showed that Lactobacillus reuteri had the ability to produce various volatile compounds, which allowed Lactobacillus reuteri to impart complex aroma to something.
研究领域:
微生物学
·
生物化学
·
酶学
·
食品科学
✓ 已验证
DOI
高影响因子
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
·
2014
·
IF 3.043
Chenxi Ning,Erzheng Su,Dongzhi Wei
Lodderomyces elongisporus LH703 isolated from soil samples contained three novel aldo–keto reductases (AKRs) (LEAKR 48, LEAKR 49, and LEAKR 50). The three enzymes were cloned, expressed, and purified to homogeneity for characterization. These three AKRs shared <40% amino acid identity with each other. LEAKR 50 was identified as a member of AKR3 family, whereas the other two LEAKRs were identified as members of two novel AKR families, respectively. All the three AKRs required nicotinamide adenine dinucleotide phosphate as a cofactor. However, they showed diverse characteristics, including optimum catalyzing conditions, resistance to adverse reaction conditions, and substrate specificity. LEAKR 50 was estimated to be a promising biocatalyst that could reduce ethyl 4-chloroacetoacetate with high enantiomeric excess (98% e. e.) and high activity residue under adverse conditions.
研究领域:
生物化学
·
酶学
·
微生物学
✓ 已验证
DOI
PubMed
高影响因子
BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
·
2021
·Vol. 69
·期 4
·1428-1437
·
IF 2.431
Di Liu,Linbo Gou,Yajun Bai,Tai-Ping Fan,Xiaohui Zheng,Yujie Cai
In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl ( S )-4-chloro-3-hydroxybutyrate (( S )-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency ( k cat / K m ) for COBE was 51.36 s –1 ·mM –1 . This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.
研究领域:
生物化学
·
酶学
·
生物催化
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）