ENZYME AND MICROBIAL TECHNOLOGY
·
2024
·
IF 3.4
Lili Zhang,Yu Hong,Jiapeng Lu,Yi Wang,Wei Luo
Transaminases (EC 2.6.1.X, TAs) are important biocatalysts in the synthesis of chiral amines, and have significant value in the field of medicine. However, TAs suffer from low enzyme activity and poor catalytic efficiency in the synthesis of chiral amines or non-natural amino acids, which hinders their industrial applications. In this study, a novel TA derived from Paracoccus pantotrophus ( pp TA) that was investigated in our previous study was employed with a semi-rational design strategy to improve its enzyme activity to 2-ketobutyrate. By using homology modeling and molecular docking, four surrounding sites in the substrate-binding S pocket were selected as potential mutational sites. Through alanine scanning and saturation mutagenesis, the optimal mutant V153A with significantly improved enzyme activity was finally obtained, which was 578 % higher than that of the wild-type pp TA (WT). Furthermore, the mutant enzyme pp TA-V153A also exhibited slightly improved temperature and pH stability compared to WT. Subsequently, the mutant was used to convert 2-ketobutyrate for the preparation of L-2-aminobutyric acid (L-ABA). The mutant can tolerate 300 mM 2-ketobutyrate with a conversion rate of 74 %, which lays a solid foundation for the preparation of chiral amines.
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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DOI
PubMed
高影响因子
CATALYSIS LETTERS
·
2017
·Vol. 147
·期 4
·837-844
·
IF 2.799
An Zhengfang,Gu Xiaoxu,Liu Yue,Ge Jingyan,Zhu Qing
Abstract
l -2-Aminobutyric acid ( l -ABA), an unnatural amino acid, is a key intermediate of several important drugs. Although some methods have been developed to prepare pure chiral l -ABA, there are still many drawbacks, including low catalytic efficiency, cumbersome steps and high cost due to the addition of some expensive catalysts or coenzymes. Herein, with chemical and biological approaches together, we discovered a newly isolated Aspergillus tamarii ZJUT ZQ013 strain containing a microbial lipase which could be employed to resolve racemic methyl N -Boc-2-aminobutyrate to produce l -ABA with high enantioselectivity (e.e. s > 99.9%, E = 257). Moreover, the subsequent gram scale experiment confrimed that A. tamarii ZJUT ZQ013 could be an attractive biocatalyst for the efficient preparation of optically pure acid. Graphical Abstract
研究领域:
生物化学
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制药工程
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微生物学
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DOI
高影响因子
BIORESOURCE TECHNOLOGY
·
2021
·
IF 9.642
Jiajie Chen,Rongshuai Zhu,Junping Zhou,Taowei Yang,Xian Zhang,Meijuan Xu,Zhiming Rao
Leucine dehydrogenase (LDH) is widely used in the preparation of L-2-aminobutyric acid (L-2-ABA), however its wide application is limited by 2-ketobutyric acid (2-OBA) inhibition. Firstly, a novel high-throughput screening method of LDH was established, specific enzyme activity and 2-OBA tolerance of Lys72Ala mutant were 33.3% higher than those of the wild type. Subsequently, we constructed a single cell comprised of ivlA , Esldh K72A , fdh and optimized expression through fine-tuning RBS intensity, so that the yield of E. coli BL21/pET28a-R3 ivlA-Esldh K72A -fdh was 2.6 times higher than that of the original strain. As a result, 150 g L-threonine was transformed to 121 g L-2-ABA in 5 L fermenter with 95% molar conversion rate, and a productivity of 5.04 g·L -1 ·h −1 , which is the highest productivity of L-2-ABA currently reported by single-cell biotransformation. In summary, our research provided a green synthesis for L-2-ABA, which has potential for industrial production of drug precursors.
研究领域:
生物技术
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生物化学
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微生物学
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DOI
PubMed
高影响因子
Microbial Cell Factories
·
2019
·Vol. 18
·期 1
·1-10
·
IF 4.402
Xu Jian-Miao,Li Jian-Qiang,Zhang Bo,Liu Zhi-Qiang,Zheng Yu-Guo
Background l -2-aminobutyric acid ( l -ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important pharmaceuticals. To make the biosynthesis of l -ABA environmental friendly and more suitable for the industrial-scale production. We expand the nature metabolic network of Escherichia coli using metabolic engineering approach for the production of l -ABA. Results In this study, Escherichia coli THR strain with a modified pathway for threonine-hyperproduction was engineered via deletion of the rhtA gene from the chromosome. To redirect carbon flux from 2-ketobutyrate (2-KB) to l -ABA, the ilvIH gene was deleted to block the l -isoleucine pathway. Furthermore, the ilvA gene from Escherichia coli W3110 and the leuDH gene from Thermoactinomyces intermedius were amplified and co-overexpressed. The promoter was altered to regulate the expression strength of ilvA* and leuDH . The final engineered strain E. coli THR Δ rhtA Δ ilvIH /Gap- ilvA *-Pbs- leuDH was able to produce 9.33 g/L of l -ABA with a yield of 0.19 g/L/h by fed-batch fermentation in a 5 L bioreactor. Conclusions This novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of l -ABA.
研究领域:
生物技术
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代谢工程
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微生物生物技术
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DOI
PubMed
高影响因子
BMC BIOTECHNOLOGY
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2021
·Vol. 21
·期 1
·1-9
·
IF 2.563
Zhang Zhiwei,Liu Yang,Zhao Jing,Li Wenqiang,Hu Ruiwen,Li Xia,Li Aitao,Wang Yaping,Ma Lixin
Background The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering.Results Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g.Conclusion This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.
研究领域:
生物化学
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蛋白质工程
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酶学
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DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2016
·Vol. 182
·期 3
·898-909
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IF 1.606
Xu Jian-Miao,Cheng Feng,Fu Fang-Tian,Hu Hai-Feng,Zheng Yu-Guo
L -2-aminobutyric acid ( L -ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD + regeneration was developed for L -ABA production on a large scale. Previously, the L -ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method. Recently, an HTS assay was developed by us, which enables researchers to engineer LeuDH in a relatively short period of time. Herein, a semirational engineering was performed on LeuDH to increase the catalytic efficiency of BcLeuDH. Firstly, the structure of wild-type (WT) BcLeuDH was modeled and seven potentially beneficial positions were selected for mutation. Five beneficial variants were then identified from the seven site-saturation mutagenesis (SSM) libraries by HTS and confirmed by rescreening via amino acid analyzer. The “best” variant M5 (WT + Q358N) showed 44.5-fold higher catalytic efficiency ( k
cat / K
M ) than BcLeuDH WT, which suggested that BcLeuDH M5 is an attractive candidate for L -ABA production on a large scale. Furthermore, the structure-functional relationship was investigated based on the docking and kinetic results.
研究领域:
生物技术
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生物化工
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酶学
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药学
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DOI
PubMed
高影响因子
Molecular Catalysis
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2021
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IF 5.062
Wei Luo,Jiagui Hu,Jiapeng Lu,Huili Zhang,Xiaoping Wang,Yuantao Liu,Liqing Dong,Xiaobin Yu
ω-transaminase can mediate the asymmetric synthesis of chiral amines from aldehydes and ketones , which has important value in the synthesis of pharmaceutical intermediates. A novel ω-transaminase derived from Paracoccus pantotrophus ( Pp TA) was obtained and cloned in E. coli BL21(DE3) for expression. The enzyme has high activity for 2-ketobutyric acid and benzylamine , as well as for aromatic compounds with side chains longer than ethyl aliphatic hydrocarbons . Molecular simulation showed that the S-pocket in the active center is larger than those of other ω-transaminases. Thereafter, a whole-cell catalytic system was designed to prepare l -2-aminobutyric acid by cascading Pp TA and other enzymes. By using several strategies (regulation of RBS intensity, by-product decomposition and cofactor self-sufficiency), whole-cell cascade biocatalysis showed a high ee value (> 99%) and high yield (71%) in one pot reaction . This study therefore proposes an efficient biocatalyst for the synthesis of unnatural amino acids with the participation of ω-transaminase.
研究领域:
生物催化
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酶工程
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药物化学
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分子生物学
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DOI
高影响因子
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
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2024
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IF 3.1
Wenchan Deng,Rongrong Huang,Yuanjiang Pan,Cuirong Sun
The metabolism process of amino acids is closely related to the growth of normal and cancer cells. It is still not clear how L/D-configuration amino acids participate in the metabolism of colorectal cell. Herein, intra- and extra-cellular metabolic distribution of L/D-amino acids in colorectal cell (HCT116) and human normal intestinal epithelial cell (NCM460) were profiled utilizing HPLC-MS/MS coupled with a chiral probe. The results displayed the differential metabolic portrayal for the two cell lines. Compared with NCM460 cell, 13 kinds of significant differential amino acids were founded in a lower concentration within HCT116 cell, and L-Gln was even not detected for intra-cell; as for extra-cell culture medium, the HCT116 cell consumed more L-Gln, D-Phe and D-Leu, while L-Met was low ingested in HCT116 cell. L-Ala and Gly were excretion in both two cell lines, excepted L-Cit which was uptake in HCT116 and excretion in NCM460 cell. Furthermore, the dynamic changes of chiral amino acids displayed that phenylalanine, tyrosine and tryptophan biosynthesis and arginine biosynthesis is the major pathway for intra-cellular metabolites and extra-cellular metabolites, respectively. Moreover, with additional D-amino acids in culture medium, the results exhibited that high concentration of D-amino acids have no significant effect on the proliferation of NCM460 cell, but could influence the profiling of amino acids metabolites, and further affect the proliferation of HCT116 cell. This present work enhances the understanding of these differential amino acids metabolic network and depicts a dynamic process of metabolic dysregulation of HCT116 and NCM460 cell.
研究领域:
生物化学
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肿瘤学
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DOI
PubMed
高影响因子
Polymers
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2020
·Vol. 12
·期 9
·2053
·
IF 3.426
Chen Qi,Wenke Yang,Fuyan He,Jinshui Yao
Eight kinds of chiral diacid monomers were prepared with amino acids with different side groups or configurations. Polyester-imides (PEIs) were synthesized from these diacid monomers and diphenol monomers through polycondensation reaction, and the performances and properties were compared with the chiral polyamide-imides (PAIs) previously synthesized by our work group. Their thermal properties were analyzed by thermo gravimetric analysis (TGA) and dynamic thermomechanical analysis (DMA), and it was found that the glass transition temperature (Tg) of PEI was mainly affected by the volume of side groups. Their degradability was studied through buffer degradation experiments, and the changes in their water contact angle, molecular weight, structure and appearance during the degradation process were characterized by contact angle tester, gel permeation chromatography (GPC), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). With degradation, the hydrophilicity of PEI was improved, and when amino acids with larger side groups or D configuration were introduced into the backbone of PEI, the degradability decreased.Keywords:chiral amino acids;polyester-imides (PEIs);degradation;thermal property
研究领域:
材料科学
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生物医学工程
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DOI
PubMed
高影响因子
Catalysis Science & Technology
·
2021
·Vol. 11
·期 12
·4208-4215
·
IF 6.119
Liuzhu Wang,Shiqing Diao,Yangyang Sun,Shuiqin Jiang,Yan Liu,Hualei Wang,Dongzhi Wei
L-Homoalanine, a useful building block for the synthesis of several chiral drugs, is generally synthesized through biocascades using natural amino acids as cheap starting reactants. However, the addition of expensive external cofactors and the low efficiency of leucine dehydrogenases towards the intermediate 2-ketobutyric acid are two major challenges in industrial applications. Herein, a dual cofactor-dependent glutamate dehydrogenase from Acinetobacter tandoii (AtGluDH) was identified to help make full use of the intracellular pool of cofactors when using whole-cell catalysis. Through reconstruction of the hydrophobic network between the enzyme and the terminal methyl group of the substrate 2-ketobutyric acid, the strict substrate specificity of AtGluDH towards α-ketoglutarate was successfully changed, and the activity obtained by the most effective mutant (K76L/T180C) was 17.2 times higher than that of the wild-type protein. A three-enzyme co-expression system was successfully constructed in order to help release the mass transfer restriction. Using 1 M L-threonine, which is close to the solubility limit, we obtained a 99.9% yield of L-homoalanine in only 3.5 h without adding external coenzymes to the cascade, giving 99.9% ee and a 29.2 g L−1 h−1 space–time yield. Additionally, the activities of the engineered AtGluDH towards some other hydrophobic amino acids were also improved to 1.1–11.2 fold. Therefore, the engineering design of some dual cofactor-dependent GluDHs could not only eliminate the low catalytic activity of unnatural substrates but also enhance the cofactor utilization efficiency of these enzymes in industrial applications.
研究领域:
生物化学
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生物技术
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酶工程
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有机化学
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产品关联
DOI
高影响因子
PHYSICAL CHEMISTRY CHEMICAL PHYSICS
·
2023
·Vol. 25
·期 45
·31444-31456
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IF 3.3
Yiwei Zhu,Jian Kang,Die Gao,Bingtong Chen,Yi Nie,Hui Wang,Xiaowen Wu
Ionic liquid based technology is promising in the pretreatment of lignocelluloses. More efforts are still being made to intensify the separation of the main components in this biomass and to inhibit biopolymer degradation, especially in the fabrication of functional materials where excellent mechanical properties are often requisite. In this study, additives with amino and/or hydroxyl groups were proposed to improve the dissolution of lignocellulosic biomass in ionic liquids and to inhibit the degradation of cellulose. Among the tested additives (i.e., urea, L-2-aminobutyric acid, DL-aminopropanol, 3-aminopropanol and ethanolamine), 3-aminopropanol showed the best performance in enhancing wheat straw dissolution and cellulose recovery in 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac). Further study revealed that this additive could also inhibit cellulose degradation in [EMIM]Ac. The interactions between the ionic liquid and additive were revealed by NMR and IR analysis. It was found that the formation of hydrogen bonds between 3-aminopropanol and [EMIM]Ac changed the interactions between ionic liquids and biomass, resulting in improved dissolution efficiency and inhibition of cellulose degradation. Optimization investigation showed that when using the 3-aminopropanol/[EMIM]Ac composite system as the solvent and pine as the raw biomass, the cellulose content in the recovered cellulose-rich material was increased from 33.3% (for the raw pine) to 66.9%. Correspondingly, the regenerated cellulose spinning in the composite system exhibited improved mechanical properties, with the elongation at break reaching 15.6% and the tensile fracture strength of 184.1 N per tex (in comparison with 9.6% for elongation at break and 99.7 N per tex for tensile fracture strength for the sample obtained in neat [EMIM]Ac).
研究领域:
生物化学
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材料科学
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DOI
PubMed
高影响因子
BIOMATERIALS
·
2023
·
IF 14
Han Yan,Pengchao Xu,He Ma,Yanan Li,Runfeng Zhang,Hailin Cong,Bing Yu,Youqing Shen
In recent years, nano-drug delivery systems have made considerable progress in the direction of tumor treatment, but the low permeability of drugs has restricted the development of nano drugs. To solve this problem, we constructed a nano-drug delivery system with the dual effects of γ-glutamyltransferase (GGT) reaction and high nuclear targeting in tumor microenvironment to promote the deep penetration of drugs. Over-expression of GGT in tumor cells can specifically recognize γ-glutamyl substrate and release amino group from the hydrolysis reaction , which makes the whole system change from negative or neutral to positive charge system. The conjugated complex with positive charge rapidly endocytosis through electrostatic interaction , enhancing its permeability in tumor parenchyma. At the same time, the cell penetrating TAT contains a large amount of lysine, which can be identified by the nuclear pore complexes (NPCs) on the surface of the nuclear membrane , showing excellent nuclear localization function. The active DOX is released in the nucleus, which inhibits the mitosis of cancer cells and enhances the active transport ability of drugs in tumor cells. Therefore, this drug delivery system actively transports adriamycin into the tumor to achieve deep penetration of drugs through enzyme response and nuclear targeting, showing high anti-tumor activity and can be effectively applied to the treatment of liver cancer.
研究领域:
药学
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癌症研究
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纳米技术
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药物递送系统
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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）