BIOTECHNOLOGY LETTERS
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2022
·Vol. 44
·期 10
·1163-1173
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IF 2.716
Zhang Ke,Pan Tingze,Wang Liuzhu,Wang Hualei,Ren Yuhong,Wei Dongzhi
Purpose We screened nitrilases with significant nitrile hydratase activity to exploit their potential in benzylic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on benzylic amide production by nitrilase. Methods A sequence-based screening method using previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as “amide-formation hotspots”) as molecular probes to identify putative amide-forming nitrilases. Results Based on the previously reported “amide-formation hotspots,” we identified a nitrilase NitPG from Paraburkholderia graminis DSM 17151 that could produce a significant amount of mandelamide toward mandelonitrile and exhibited general hydration activity toward various benzylic nitriles. The time-course experiment with NitPG demonstrated that amide was also a true reaction product of nitrilase, suggesting that the nitrile catalysis by amide-forming nitrilase could be a post-transition state bifurcation-mediated enzymatic reaction. Further research demonstrated that low temperature, metal ion addition, and specific substrate structure could profoundly improve the amide formation capability of nitrilase. Conclusions NitPG with broad hydration activity is a potential candidate for the enzymatic synthesis of benzylic amides for biotechnological applications. Studying the effect of nitrilase hydration activity could promote our understanding of the factors that influence amide and acid distribution.
研究领域:
生物技术
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酶学
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生物化学
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BIOTECHNOLOGY AND BIOENGINEERING
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2025
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IF 3.6
Wenzhe Ma,Wanqing Wei,Jian Wen,Cong Gao,Guipeng Hu,Xiaomin Li,Jia Liu,Jing Wu,Wei Song
The synthesis of tertiary amides is crucial in pharmaceutical manufacturing, but achieving this efficiently through enzymatic methods remains challenging. Here, we describe the screening, evolution, and application of a lactamase for the synthesis of 2-(2-oxopyrrolidin-1-yl)-butanoic acid (2-OYBA) in aqueous phase. Through database mining, we identified a lactamase from Paraburkholderia xenovorans ( Px AmpC) with the highest amide synthesis activity in this study at 31.3 U/g. Using protein crystallization, molecular dynamics simulations, and quantum mechanics calculations, we proposed and verified three potential synthesis mechanisms for 2-OYBA. We identified a mechanism for synthesis of 2-OYBA involving adenylation and lactamization, with the carboxyl group in the intermediate 2-2 (INT2-2) acting as a proton shuttle. Guided by this mechanism, rational design efforts enhanced amide synthesis activity by 16.6-fold. The Px AmpC mutants also exhibited a broader substrate scope for amide bond synthesis compared to the wild type. Our findings advance the green synthesis of complex amide compounds.
研究领域:
生物化学
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酶工程
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制药工业
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JOURNAL OF HAZARDOUS MATERIALS
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2023
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IF 13.6
Jinghua Li,Yiwen Ou,Lijuan Wang,Yue Zheng,Weiyun Xu,Jianbiao Peng,Xin Zhang,Zhiguo Cao,Junpei Ye
For the bioremediation of mixed-contamination sites, studies on polycyclic aromatic hydrocarbon (PAH) degradation or Cd (II) tolerance in bacteria are commonly implemented in nutrient-rich media. In contrast, in the field, inocula usually encounter harsh oligotrophic habitats. In this study, the environmental strain Paraburkholderia fungorum JT-M8 was used to explore the overlooked Cd (II) defense mechanism during PAH dissipation under P-limited oligotrophic condition. The results showed that the growth and PAH degradation ability of JT-M8 under Cd (II) stress were correlated with phosphate contents and exhibited self-regulating properties. Phosphates mainly affected the Cd (II) content in solution, while the cellular distribution of Cd (II) depended on Cd (II) levels; Cd (II) was mainly located in the cytoplasm when exposed to less Cd (II), and vice versa. The unique Cd (II) detoxification pathways could be classified into three aspects: (i) Cd (II) ionic equilibrium and dose–response effects regulated by environmental matrices (phosphate contents); (ii) bacterial physiological self-regulation, e.g., cell surface-binding, protein secretion and active transport systems; and (iii) specific adaptive responses (flagellum aggregation). This study emphasizes the importance of considering culture conditions when assessing the metal tolerance and provides new insight into the bacterial detoxification process of complex PAH-Cd (II) pollutants.
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ORGANIC PROCESS RESEARCH & DEVELOPMENT
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2022
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IF 3.858
Ling He,Wenjie Ye,Youyu Xie,Qinghai Liu,Hualei Wang,Dongzhi Wei
Bioreductions catalyzed by ketoreductases play an important role in the synthesis of chiral alcohols. However, the synthesis of (R)-2-chloro-1-(3,4-difluorophenyl)ethanol (24b), an important chiral intermediate for the synthesis of the anticoagulant ticagrelor, poses significant challenges in terms of high substrate concentration requirements that limit its production. In this study, a novel NADH-dependent, short-chain dehydrogenase, PpKR8, from Paraburkholderia phymatum STM815, exhibited excellent enantioselectivity and high activity for the production of (R)-CFPL (24b) through the reduction of 2-chloro-1-(3,4-difluorophenyl)ethanone (24a). The coexpression of PpKR8 and glucose dehydrogenase from Bacillus subtilis in Escherichia coli allowed up to 300 g/L (1.57 M) CFPO (24a) to be completely converted into 24b with 99.9% enantiomeric excess and a high space–time yield (728 g/(L day)). Substrate specificity assays demonstrated a broad substrate spectrum for PpKR8, which included 35 α/β-ketoesters, aromatic ketones, and heterocyclic ketones. Moreover, three additional optically pure chiral alcohols that are used as important drug intermediates were synthesized at high substrate concentrations (150–330 g/L), demonstrating the excellent industrial potential of PpKR8-mediated bioreductions.
研究领域:
生物化学
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酶工程
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制药工业
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ISME Journal
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2023
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IF 11
Liu Xiaohan,Li Ping,Wang Helin,Han Li-Li,Yang Kai,Wang Yanhong,Jiang Zhou,Cui Li,Kao Shuh-Ji
Biological nitrogen fixation (BNF), the conversion of N 2 into bioavailable nitrogen (N), is the main process for replenishing N loss in the biosphere. However, BNF in groundwater systems remains poorly understood. In this study, we examined the activity, abundance, and community composition of diazotrophs in groundwater in the Hetao Plain of Inner Mongolia using 15 N tracing methods, reverse transcription qPCR (RT-qPCR), and metagenomic/metatranscriptomic analyses. 15 N 2 tracing incubation of near in situ groundwater (9.5–585.4 nmol N L −1 h −1 ) and N 2 -fixer enrichment and isolates (13.2–1728.4 nmol N g −1 h −1 , as directly verified by single-cell resonance Raman spectroscopy), suggested that BNF is a non-negligible source of N in groundwater in this region. The expression of nifH genes ranged from 3.4 × 10 3 to 1.2 × 10 6 copies L −1 and was tightly correlated with dissolved oxygen (DO), Fe(II), and NH 4 + . Diazotrophs in groundwater were chiefly aerobes or facultative anaerobes, dominated by Stutzerimonas , Pseudomonas , Paraburkholderia , Klebsiella , Rhodopseudomonas , Azoarcus , and additional uncultured populations. Active diazotrophs, which prefer reducing conditions, were more metabolically diverse and potentially associated with nitrification, sulfur/arsenic mobilization, Fe(II) transport, and CH 4 oxidation. Our results highlight the importance of diazotrophs in subsurface geochemical cycles.
研究领域:
环境微生物学
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地球化学
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水文地质学
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微生物生态学
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