APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2015
·Vol. 100
·期 7
·3101-3111
·
IF 3.337
Chen Yujie,Yi Dong,Jiang Shuiqin,Wei Dongzhi
ω-Transaminases (ω-TAs) are one of the most popular candidate enzymes in the biosynthesis of chiral amines. Determination of yet unidentified ω-TAs is important to broaden their potential for synthetic application. Taurine–pyruvate TA (TPTA, EC 2.6.1.77) is an ω-TA belonging to class III of TAs. In this study, we cloned a novel thermostable TPTA from Geobacillus thermodenitrificans (TPTA gth ) and overexpressed it in Escherichia coli . The enzyme showed the highest activity at pH 9.0 and 65 °C, with remarkable thermostability and tolerance toward organic solvents. Its K
M and v
max values for taurine were 5.3 mM and 0.28 μmol s −1 mg −1 , respectively. Determination of substrate tolerance indicated its broad donor and acceptor ranges for unnatural substrates. Notably, the enzyme showed relatively good activity toward ketoses, suggesting its potential for catalyzing the asymmetric synthesis of chiral amino alcohols. The active site of TPTA gth was identified by performing protein sequence alignment, three-dimensional structure simulation, and coenzyme pyridoxamine phosphate docking. The protein sequence and structure of TPTA gth were similar to those of TAs belonging to the 3N5M subfamily. Its active site was found to be its special large pocket and substrate tunnel. In addition, TPTA gth showed a unique mechanism of sulfonate/α-carboxylate recognition contributed by Arg163 and Gln160. We also determined the protein sequence fingerprint of TPTAs in the 3N5M subfamily, which involved Arg163 and Gln160 and seven additional residues from 413 to 419 and lacked Phe/Tyr22, Phe85, and Arg409.
研究领域:
生物催化
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酶学
·
手性化学
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蛋白质工程
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PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2024
·
IF 3.4
Puhong Yi,Mengdan Liu,Yuhua Hao,Ziwen Wang,Hanlin Liu,Xue Cai,Feng Cheng,Zhiqiang Liu,Yaping Xue,Liqun Jin,Yuguo Zheng
L-phosphinothricin (L-PPT) is the most popular broad-spectrum and highly effective herbicide. Transaminases (TAs) play a pivotal role in asymmetric synthesis of L-PPT, yet encounter the challenge of unfavorable reaction equilibrium. In this study, the novel dual transaminases cascade system (DTCS) was introduced to facilitate the synthesis of L-PPT. The specific amine transaminase BdATA, originating from Bradyrhizobium diazoefficiens ZJY088, was screened and identified. It exhibited remarkable activity, good stability, and required only 2.5 equivalents of isopropylamine to transform pyruvate effectively. By coupling BdATA with previously reported SeTA to construct the DTCS for pyruvate removal in situ , the L-PPT yield escalated from 37.37% to 85.35%. Three advantages of the DTCS were presented: the removal of pyruvate alleviated by-product inhibition, the use of isopropylamine reduced reliance on excess L-alanine, and no demand for expensive cofactors like NAD(P)H. It demonstrated an innovative idea for addressing the challenges associated with transaminase-mediated synthesis of L-PPT.
研究领域:
生物催化
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酶工程
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化学生物学
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药物化学
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PubMed
高影响因子
ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY
·
2017
·
IF 2.313
Xiaoyan Zhao,Liqing Wang,Hao Zhang,Duoduo Zhang,Zhihao Zhang,Jie Zhang
The liver disease related to chronic alcohol consumption is one of the leading causes of death for alcoholics. The efficient drug to ameliorate the alcoholic liver injury was needed urgently. The present study was performed to investigate whether artemisinin possessed the protective effect against chronic alcohol consumption. 50 male Kunming mice were divided into 5 groups: control group (C): 10 ml/kg saline + 10 ml/kg saline, alcohol group (A): 10 ml/kg 56%(v/v) alcohol + 10 ml/kg saline, low dose group of artemisinin (L): 10 ml/kg 56%(v/v) alcohol + 30 mg/kg/day artemisinin, medium dose group of artemisinin (M): 10 ml/kg 56%(v/v) alcohol + 60 mg/kg/day artemisinin, high dose group of artemisinin (H): 10 ml/kg 56%(v/v) alcohol + 120 mg/kg/day artemisinin. Drugs were given orally every day. The general state of mice was observed and the levels of serum activities of AST and ALT were detected after treatment with drugs for 30 days. Besides, the liver weight index was calculated and histopathological analysis was performed. We successfully demonstrated that treatment with high dose of artemisinin significantly decreased the elevated levels of AST ( p < 0.05 ) and ALT ( p < 0.01 ) in plasma, as well as the liver weight index ( p < 0.01 ). The loss of body weight, tissue injury, oedema and inflammatory cell infiltration in the hepatocytes were found in the A group. These symptoms were remarkably alleviated in animals treated with artemisinin. Artemisinin can inhibit the activation of NF-кB and the expression of inflammatory cytokines inducible nitric oxide synthase. Besides, it can also enhance the stability of liver cell membrane, and reduce the damage of liver cell membrane and liver cell. Artemisinin showed a protective effect against chronic alcohol poisoning and it has a great potential for the clinical application to treat the liver injury induced by alcohol.
研究领域:
药理学
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肝病学
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分子生物学
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PubMed
高影响因子
MOLECULES
·
2023
·Vol. 28
·期 10
·4131
·
IF 4.6
Guohua Wu,Lei Jiang,Jianjun Guo,Wushuang Li,Lin Ma,Bozhi Tang,Charles C. Liu
Alanine transaminase (ALT) is an important amino acid-metabolizing enzyme in silkwormBombyx moriL., and is mainly involved in transferring glutamate to alanine (serving as an essential precursor in silk protein synthesis) through transamination. Therefore, it is generally believed that silk protein synthesis in the silk gland and the cocoon quantity increase with the increase in ALT activity to a certain extent. Here, a novel analytical method was developed to determine the ALT activity in several key tissues ofBombyx moriL. including the posterior silk gland, midgut, fat body, middle silk gland, trachea and hemolymph, by combining the direct-analysis-in-real-time (DART) ion source with a triple-quadrupole mass spectrometer. In addition, a traditional ALT activity assay, the Reitman–Frankel method, was also used to measure ALT activity for comparison. The ALT activity results obtained via the DART-MS method are in good agreement with those obtained via the Reitman–Frankel method. However, the present DART-MS method provides a more convenient, rapid and environmentally friendly quantitative method for ALT measurement. Especially, this method can also monitor ALT activity in different tissues ofBombyx moriL. in real time.Keywords:ALT;DART-MS;Bombyx mori;biochemical analysis
研究领域:
昆虫学
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生物化学
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PubMed
高影响因子
Metabolites
·
2025
·Vol. 15
·期 3
·175
·
IF 3.7
Liang Chen,Jiaxin Li,Chengwei Fang,Jiepeng Wang
Background/Objectives: Epilepsy is a common chronic and recurrent neurological disorder that poses a threat to human health, andAcorus tatarinowiiSchott (ATS), a traditional Chinese medicine, is used to treat it. This study aimed to determine its effects on plasma metabolites. Moreover, the possible mechanism of its intervention in epilepsy was preliminarily explored, combined with network pharmacology. Methods: An epileptic model of rats was established using pentylenetetrazol. The potential targets and pathways of ATS were predicted by network pharmacology. Ultra Performance Liquid Chromatography–Quadrupole–Time of Flight Mass Spectrometrynce Liquid Chromatography–Quadrupole–Time of Flight Mass Spectrometryance Liquid Chromatography–Quadrupole–Time of Flight Mass Spectrometry and statistical analyses were used to profile plasma metabolites and identify ATS’s effects on epilepsy. Results: Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that ATS was involved in regulating multiple signaling pathways, mainly including the neuroactive ligand–receptor interaction and GABAerGamma-aminobutyrate transaminaseAminobutyrate Transaminaseapse signaling pathway. ATS treatment restored 19 metabolites in epiGamma-aminobutyrate transaminaseminobutyrate Transaminase rats, affecting lysine, histidine, and purine metabolism. GABA-T was found as a new key target for treating epilepsy with ATS. The IC50of ATS for inhibiting GABA-T activity was 57.9 μg/mL. Through metabolomic analysis, we detected changes in the levels of certain metabolites related to the GABAergic system. These metabolite changes can be correlated with the targets and pathways predicted by network pharmacology. One of the limitations of this study is that the correlation analysis between altered metabolites and seizure severity remains unfinished, which restricts a more in-depth exploration of the underlying biological mechanisms. In the future, our research will focus on conducting a more in-depth exploration of the correlation analysis between altered metabolites and seizure severity. Conclusions: These results improved our understanding of epilepsy and ATS treatment, potentially leading to better therapies. The identification of key metabolites and their associated pathways in this study offers potential novel therapeutic targets for epilepsy. By modulating these metabolites, future therapies could be designed to better manage the disorder. Moreover, the insights from network pharmacology can guide the development of more effective antiepileptic drugs, paving the way for improved clinical outcomes for patients.
研究领域:
神经科学
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中药学
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代谢组学
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PubMed
高影响因子
Molecular Catalysis
·
2023
·
IF 4.6
Nan Jiang,Xiaotong Du,Liangyu Zheng
In this study, a series of chiral lactams were successfully prepared by one-pot two-step reactions. It included a transamination reaction catalyzed by a ω -transaminase from Bacillus megaterium ( ω -TA Bm ), and subsequent spontaneous cyclisation. The different amine donors were firstly screened with ethyl levulinate as an amine acceptor, and both the excellent conversion and enantiomeric excess ( e.e. p ) of >99% could be achieved with 4-bromophenylethylamine as an amine donor, however, the long reaction time of 24 h was unfavorable for its application. After the amino acid residue Thr120 situated in the small pocket was mutated to Ser, the T120S variant could exhibit 10.6-fold higher activity comparing to that of wild type, and achieve the reaction with both conversion and e.e. p of >99% within 3 h. The T120S variant could also be applied to prepare the other chiral γ- and δ-lactams with excellent conversion and e.e. p . Furthermore, the inclusion bodies of ω -TA Bm and its variants could be aggregated as “native-like structures” to continuously prepare the chiral lactam for 18 h. The aim of the study is to provide a simply and efficiently biocatalytic route for preparing chiral lactams.
研究领域:
生物化学
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酶工程
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有机合成
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DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2022
·Vol. 88
·期 9
·
IF 5.005
Dong-Xu Jia,Fan Wang,Rui Zhao,Bo-Di Gu,Chen Peng,Li-Qun Jin,Zhi-Qiang Liu,Yu-Guo Zheng
d-Alanine belongs to nonessential amino acids that have diverse applications in the fields of food and health care. (R)-transaminase [(R)-TA]-catalyzed asymmetric amination of pyruvate is a feasible alternative for the synthesis of d-alanine, but low catalytic efficiency and thermostability limit enzymatic utilization. In this work, several potential (R)-TAs were discovered using NCBI database mining synchronously with enzymatic structure-function analysis, among which Capronia epimyces TA (CeTA) showed the highest activity for amination of pyruvate using (R)-α-methylbenzylamine as the donor. Furthermore, enzymatic residues surrounding a large catalysis pocket were subjected to saturation and combinatorial mutagenesis, and positive mutant F113T showed dramatic improvement in activity and thermostability. Molecular modeling indicated that the substitution of phenylalanine with threonine afforded alleviation of steric hindrance in the pocket and induced formation of additional hydrogen bonds with neighboring residues. Finally, using recombinant cells containing F113T as a biocatalyst, the conversion yield of amination of 100 mM pyruvate to d-alanine achieved up to 95.2%, which seemed to be the highest level in the literature regarding synthesis of d-alanine using TAs. The inherent characteristics rendered CeTA F113T a promising platform for efficient preparation of d-alanine operating with high productivity.IMPORTANCE d-Alanine is an important compound with many valuable applications. Its asymmetric synthesis employing (R)-ω-TA is considered an attractive choice. According to the stereoselectivity, ω-TAs have either (R)- or (S)-enantiopreference. There has been a variety of literature regarding screening, characterizing, and molecular modification of (S)-ω-TAs; in contrast, the research about (R)-ω-TA has lagged behind. In this work, we identify several (R)-ω-TAs and succeeded in creating mutant F113T, which showed not only better efficiency toward pyruvate but also higher thermostability compared with the original enzyme. The obtained original enzymes and positive mutants displayed important application value for pushing symmetric synthesis of d-alanine to a higher level.
研究领域:
生物技术
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酶工程
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化学生物学
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PubMed
高影响因子
JOURNAL OF BIOTECHNOLOGY
·
2019
·
IF 3.163
Xiao-Ling Tang,Nan-Nan Zhang,Guo-Yan Ye,Yu-Guo Zheng
( R )-3-amino-1-butanol is a key intermediate of Dolutegravir for the treatment of HIV/AIDS and its green and efficient biosynthesis has attracted a great deal of attention. Transaminases are currently used as promising biocatalyst for the synthesis of chiral amines. However, many transaminases have ( S )-specificity and ( R )-selective transaminases were less exploited and studied, making the production of ( R )-amines remain challenging. In this study, a novel transaminase from Actinobacteria sp. ( As -TA) was obtained and applied for the biosynthesis of ( R )-3-amino-1-butanol by transferring the amino group from isopropylamine to 4-hydroxy-2-butanone. After optimization of the reaction condition and using a substrate fed-batch strategy, the conversion of 100, 200, 300, 400 and 500 mM 4-hydroxy-2-butanone reached 100%, 94.9%, 86.1%, 76.1% and 70.9%, respectively. ( R )-3-amino-1-butanol with a maximum yield of 29.6 g/L and 99.9% e.e. value was obtained. This was the first time demonstrating the successful biosynthesis of ( R )-3-amino-1-butanol with transaminase as biocatalyst and the obtained As -TA enriched the enzyme pool of transaminase with ( R )-specificity.
研究领域:
生物化学
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酶工程
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药物合成
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PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2023
·Vol. 415
·期 9
·1733-1740
·
IF 4.3
Tang Kexin,Dong Jiacheng,Zheng Zhengheng,Zhang Ting,Pan Huayi,Jia Honghua,Li Yan,Wei Ping
ω-Transaminases (ω-TAs) are widely available for the production of chiral amines and unnatural amino acids. Herein, a rapid spectrophotometric method was developed for screening ω-TAs based on the colored products that can be generated from transamination reactions between aliphatic α-diketones and amino donors catalyzed by ω-TAs. The possible mechanism of the formation of the colored product was investigated according to LC-Q-TOF–MS analysis. Among seven diketones, 2,3-butanedione was selected as the most suitable amino acceptor for colorimetric screening of ω-TAs with high efficiency, high sensitivity, and low background interference. Meanwhile, the absorbance of the colored product generated by 2,3-butanedione catalyzed by ω-TAs in this method was linearly correlated with the results by HPLC analysis. This method was also confirmed to effectively screen ω-TA mutants with high activity towards isopropylamine. Graphical Abstract
研究领域:
生物化学
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酶工程
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DOI
PubMed
高影响因子
ACS Sustainable Chemistry & Engineering
·
2025
·
IF 7.3
Chaoqun Huang,Xuerui Jin,Yeyi Kan,Can Cui,Longchen Wei,Huayi Liu,Dawen Niu,Yunzi Luo
Nonnatural amino acids hold the promise of enhancing enzyme activity and bolstering the efficacy of drugs. Enzymatic synthesis emerges as a valuable adjunct to traditional synthetic methodologies, providing an alternative pathway for the generation of nonnatural amino acids. This study introduces a one-pot enzymatic strategy for the synthesis of aryl and heteroaryl γ-hydroxy-α-amino acids. The process involved the utilization of a newly identified aldolase from Streptomyces to catalyze the formation of C–C bond from the cost-effective fluorinated pyridine aldehyde substrate, and an α-transaminase sourced from Saccharomyces cerevisiae to transfer an amino group. After optimization, a 94% conversion efficiency was achieved, along with a product diastereoselectivity ratio of up to 13:87. Furthermore, the one-pot reaction system demonstrated efficacy across a broad spectrum of substrates, including fluorinated pyridines, quinolines, azabiphenyls, thiazoles, and phenyl derivatives. This study significantly broadened the scope of nonnatural amino acids containing heteroaromatic rings and diversified the array of enzymes accessible for the production of functionally diverse bioactive fluorinated compounds.
研究领域:
生物化学
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合成生物学
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酶工程
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DOI
高影响因子
BIOORGANIC CHEMISTRY
·
2024
·
IF 4.5
He Liu,Shixi Wang,Meng Xu,Kaiyue Zhang,Qian Gao,Hualei Wang,Dongzhi Wei
( R )-selective transaminases show promise as catalysts for the asymmetric synthesis of chiral amines, which are building blocks of various small molecule drugs. However, their application is limited by poor substrate acceptance and low catalytic efficiency. Here, a potential ( R )-selective transaminase from Fodinicurvata sediminis (FsTA) was identified through a substrate truncating strategy, and used as starting point for enzyme engineering toward catalysis of 4-hydroxy-2-butanone, a substrate that poses challenges in catalysis. Molecular docking and dynamics simulations revealed Y90 as the key residue responsible for poor substrate binding. Starting from the variant (Y90F, mut1) with initial activity, FsTA was systematically modified to improve substrate-binding through active site reshaping and consensus sequence strategy, yielding three variants (H30R, V152K, and Y156F) with improved activity. A quadruple mutation variant H30R/Y90F/V152K/Y156F (mut4) was also found to show a 7.95-fold greater catalytic efficiency ( k cat / K M ) than the initial variant mut1. Furthermore, mut4 also enhanced the thermostability of enzyme significantly, with the T m value increasing by 10 °C. This variant also exhibited significantly improved activity toward a series of ketones that are either not accepted or poorly accepted by the wild-type. This study provides a basis for the rational design of an active to creating variants that can accommodate novel substrates.
研究领域:
生物化学
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酶工程
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PubMed
高影响因子
JOURNAL OF BIOTECHNOLOGY
·
2025
·
IF 3.9
Yuwen Wei,Fulong Li,Yukun Zheng,Youxiang Liang,Yan Du,Huimin Yu
Transaminases have important applications in the synthesis of drug intermediates such as chiral amines. However, natural transaminases exhibit suboptimal thermal stability, limiting their further applications. Building upon an Rhodobacter sp. -derived ( R )-selective transaminase ( Rb TA), we report a dual-region coupling engineering approach to improve thermostability of Rb TA by strengthening the core hydrogen-bond networks and rigidifying the flexible surface loop. Through single strategy, we identified 4 thermostability improved single mutations, among which I249Q demonstrated the most substantial improvement, achieving a 18-fold increase in half-life ( t 1 / 2 40 ) and a 11.2 ℃ increase in T 50 10 . Then in strategic coupling, the synergistic effect of dual-region modification was observed in both thermal stability and activity enhancement, as mutant with the best high-temperature catalytic performance, R136P/F228Y, had its T 50 10 improved by 7.1℃ and exhibited a 4.2-fold increase in k cat / K m towards ( R )-3-amino-1-butanol. Finally, R136P/F228Y achieved a 20.5 % improvement in conversion over WT in an analytical-scale synthesis in 72 h at a 5 ℃ elevated catalytic temperature. Molecular dynamics simulations demonstrated that the synergy of the formation of new hydrogen bonds and decrease in flexibility accounted for the thermostability improvements. This study provides guidance for enhancing thermostability of similar fold-type enzymes without impairing enzymatic activity in an efficient manner.
研究领域:
生物化学
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蛋白质工程
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）