Molecular Catalysis
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2023
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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
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2023
·Vol. 415
·期 9
·1733-1740
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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
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2016
·Vol. 181
·期 3
·972-985
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IF 1.606
Wu Hua-Lei,Zhang Jian-Dong,Zhang Chao-Feng,Fan Xiao-Jun,Chang Hong-Hong,Wei Wen-Long
Four uncharacterized ω-transaminases (ωTAs) from Pseudomonas putida NBRC 14164 have been identified and cloned from the pool of fully sequenced genomes. The genes were functionally expressed in Escherichia coli BL21, and the enzymes were purified and characterized. Four TAs showed highly ( S )-selective ωTA activity and converted ( S )-α-methylbenzylamine and pyruvate to acetophenone and l -Ala. The maximum activity of cloned enzymes was in the pH range of 8.0– 8.5 (Pp36420), 8.5 –9. 5 (Pp21050), 9 .0– 9.5 (PpspuC), and 9.5 –1 0 . 5 (PpbauA), and the optimal temperatures were at 35 °C (Pp36420, Pp21050, and PpspuC) and 50 °C (PpbauA), respectively, with K
M of 161.3 mM (Pp21050), 136.7 mM (PpbauA), 398.5 mM (Pp36420), and 130.9 mM (PpspuC) and yielding a catalytic efficiency k
cat / K
M of 0.015, 0.003, 0.012, and 0.023 mM −1 s −1 . Several racemic amines and amino alcohols were resolved by the cloned ωTAs; perfect conversions (48–50 %) were obtained by at least one enzyme, and the residual substrates were left with 97–99 % ee. Kinetic resolution of racemic phenylglycinol was done with PpspuC in a 100-mL scale. Enaniomeric excess of ( S )-phenylglycinol reached 99 % with 45 % isolated yield. The high enantioselectivity and large substrate spectra of the cloned PpTAs showed an attractive potency for biotechnology application in production of chiral amines and amino alcohols.
研究领域:
生物化学
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酶工程
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生物技术
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DOI
PubMed
高影响因子
CATALYSIS LETTERS
·
2020
·Vol. 151
·期 6
·1834-1841
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IF 2.482
Feng Xiaoqing,Zhang Lin,Zhu Xuan,Xia Yan,Ma Cuiluan,Liang Jianguang,He Yu-Cai
Furfurylamine is an important furfural-upgrading biobased chemical for the production of pharmaceuticals, biofuels, fibers, additives, polymers, etc. In one-pot reaction system, biomass was tandemly catalyzed to furfurylamine with aluminium-based alkaline-treated graphite (Al-AG) catalyst and recombinant ω-transaminase biocatalyst. Al-AG (3.6 wt%) catalyzed corncob (75.0 g/L) to 110.0 mM furfural at 56.8% yield (based on xylan in corncob) in γ-valerolactone–water (1:4, v:v; pH 1.0) for 40 min at 180 °C. The pH-adjusted corncob-slurry (pH 7.5) containing furfural were catalyzed to furfurylamine at high yield with γ-valerolactone-tolerant Aspergillus terreus ω-transaminase biocatalyst at 35 °C using isopropylamine (3 mol isopropylamine/mol furfural) as amine donor. Such an efficient and sustainable approach for catalytic conversion of biomass to high-value-added biobased furfurylamine was successfully established in tandem reaction with Al-AG and ω-transaminase biocatalyst. Graphic Abstract
研究领域:
生物化学
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催化化学
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可再生资源
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DOI
高影响因子
ANALYTICAL BIOCHEMISTRY
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2022
·
IF 3.191
Guozeng Wang,Zhihao Jiang,Qing Xiao,Chang Jiang,Xian'ai Shi
Omega-transaminases (ω-TAs) have attracted considerable interest for their use in asymmetric synthesis of chiral amines with a high degree of optical purity and yield. The rapid evaluation of the characteristics of newly identified or engineered ω-TAs is important for industrial applications. In this study, a visible spectrophotometric assay was developed for rapid quantitative determination of ω-TA activities based on the transamination of 2-(4-nitrophenyl)ethan-1-amine to generate a red product ( E )- N -(4-nitrophenethyl)-2-(4-nitrophenyl)ethen-1-amine. After various co-solvents were evaluated, dimethyl sulfoxide (DMSO) was considered optimal because the red product exhibits good solubility and retains its original color. The red product dissolved in DMSO has its highest absorbance at 465 nm, and its concentration has a good linear relationship with the absorbance. A spectrophotometric assay was established and validated using conventional HPLC analysis (<10% divergence). This method was then used to characterize an ω-TA from thermophilic Geobacillus thermoleovorans and an ω-TA obtained from the metagenome of a soda lake. The results demonstrated that ω-transaminase enzymatic properties could be characterized simply, rapidly, and at low cost, using this newly established visible spectrophotometric assay method.
研究领域:
生物化学
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工业酶学
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DOI
PubMed
高影响因子
MOLECULES
·
2023
·Vol. 28
·期 9
·3895
·
IF 4.6
Hongpeng Wang,Mercy Vimbai Masuku,Yachen Tao,Jiayao Yang,Yi Kuang,Changjiang Lyu,Jun Huang,Shengxiang Yang
The efficient biosynthesis of chiral amines at an industrial scale to meet the high demand from industries that require chiral amines as precursors is challenging due to the poor stability and low catalytic efficiency of ω-transaminases (ω-TAs). Herein, this study adopted a green and efficient solvent engineering method to explore the effects of various aqueous solutions of deep eutectic solvents (DESs) as cosolvents on the catalytic efficiency and stability of ω-TA. Binary- and ternary-based DESs were used as cosolvents in enhancing the catalytic activity and stability of a ω-TA variant fromAspergillus terreus(E133A). The enzyme exhibited a higher catalytic activity in a ternary-based DES that was 2.4-fold higher than in conventional buffer. Moreover, the thermal stability was enhanced by a magnitude of 2.7, with an improvement in storage stability. Molecular docking studies illustrated that the most potent DES established strong hydrogen bond interactions with the enzyme’s amino acid, which enhanced the catalytic efficiency and improved the stability of the ω-TA. Molecular docking is essential in designing DESs for a specific enzyme.Keywords:catalytic efficiency;deep eutectic solvents;thermal stability;molecular docking
研究领域:
生物催化
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酶工程
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绿色化学
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PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2025
·
IF 6.2
Puhong Yi,Yue Xu,Yuhua Hao,Zhiqiang Liu,Yaping Xue,Liqun Jin,Yuguo Zheng
ω-Transaminases (ω-TAs) are attractive biocatalysts in the asymmetric synthesis of chiral amines. Recently, a promising ω-TA from Salmonella enterica (SeTA) has been used to produce the valuable herbicide l-phosphinothricin (l-PPT). To improve the catalytic activity of SeTA, a combination strategy involving structural analysis and mutability landscape construction was conducted. A flexible region (loop1) enhancing the catalytic activity of SeTA was identified, and a distal mutant I22D (M1) was successfully screened. M1 showed a 3.24-fold increase in specific activity, along with greater thermal stability. Its half-life (t1/2) was 30.95-fold (45 °C) and 17.53-fold (55 °C) longer than that of WT, and Tm increased by 7.6 °C. MD simulations revealed that the distal mutation induced long-range effects through allostery, resulting in the substrate tunnel being more spacious, reshaping the active pocket, and stabilizing the local conformation. These findings can provide insights into the distal sites and functional relationships of ω-TAs for improving their performance.
研究领域:
生物化学
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酶工程
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计算生物学
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PubMed
高影响因子
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
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2021
·Vol. 193
·期 11
·3624-3640
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IF 2.926
Jia Dong-Xu,Peng Chen,Li Jun-Liang,Wang Fan,Liu Zhi-Qiang,Zheng Yu-Guo
ω-Transaminase (ω-TA) is an attractive biocatalyst for stereospecific preparation of amino acids and derivatives, but low catalytic efficiency and unfavorable substrate specificity hamper their industrial application. In this work, to obtain applicable ( R )-ω-TA responsible for amination of α-keto acids substrates, the reactivities of eight previously synthesized ω-TAs toward pyruvate using ( R )-α-methylbenzylamine (( R )-α-MBA) as amine donor were investigated, and Gibberella zeae TA (GzTA) with the highest ( R )-TA activity and stereoselectivity was selected as starting scaffold for engineering. Site-directed mutagenesis around enzymatic active pocket and access tunnel identified three positive mutation sites, S214A, F113L, and V60A. Kinetic analysis synchronously with molecular docking revealed that these mutations afforded desirable alleviation of steric hindrance for pyruvate and α-MBA. Furthermore, the constructed single-, double-, and triple-mutant exhibited varying degrees of improved specificities toward bulkier α-keto acids. Using 2-oxo-2-phenylacetic acid ( 1d ) as substrate, the conversion rate of triple-mutant F113L/V60A/S214A increased by 3.8-fold relative to that of wide-type GzTA. This study provided a practical engineering strategy for improving catalytic efficiency and substrate specificity of ( R )-ω-TA. The obtained experience shed light on creating more industrial ω-TAs mutants that can accommodate structurally diverse substrates. Graphical abstract
研究领域:
生物化学
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酶工程
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合成生物学
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PubMed
高影响因子
ORGANIC PROCESS RESEARCH & DEVELOPMENT
·
2022
·
IF 3.858
Xiangxiang Wang,Youyu Xie,Zhicai Wang,Ke Zhang,Hualei Wang,Dongzhi Wei
We developed a continuous flow system using ω-transaminase covalently immobilized on an epoxy resin modified with ethylenediamine (EDA) to synthesize (S)-1-Boc-3-aminopiperidine, which is a key intermediate for several novel inhibitors. A second functional group (−NH3+) was introduced to rapidly adsorb ω-transaminase, which was further rigidified by multipoint covalent attachment to the modified resin with high binding efficiency (>95%) and activity recovery (75%). The resulting immobilized ω-transaminase was stable with 90% residual activity after 15 reuse cycles. We synthesized (S)-1-Boc-3-aminopiperidine using this continuous flow system, and 95% conversion was obtained within 10 min of residence. The system was continuously operated for 24 h and achieved a space–time yield of 930.73 g·L–1·day–1, indicating outstanding reaction efficiency. This efficient continuous bioprocess has potential for large-scale production of (S)-1-Boc-3-aminopiperidine.
研究领域:
生物化学
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制药工程
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酶工程
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DOI
高影响因子
GREEN CHEMISTRY
·
2023
·Vol. 25
·期 18
·7372-7380
·
IF 9.8
Ruke Wang,Xuan Tang,Xinchun Jv,Yaxu Liu,Bo Wang
α-Chiral amines are key intermediates for scalable preparation of bioactive compounds; herein we present a novel palladium-based nanocatalyst capable of selectively catalyzing the reductive amination of carbonyl compounds, which enables the in situ regeneration of amino donors from wasteful co-products in a one-enzyme cascade using ω-transaminase, without the requirement of the expensive coenzyme NAD(P)H. The cascade network combines a ω-transaminase-assisted transamination with a selective reductive amination reaction facilitated by a heterogeneous palladium-based nanocatalyst. Nitrogen is sourced from hydroxylamine ions to convert generated co-products back into amino donors, yielding chiral amines with exceptional yields of up to 99% and excellent enantioselectivity. This chemoenzymatic one-enzyme transamination-reductive amination cascade network is highly atom-efficient and generates H2O as its sole by-product, demonstrating its potential impact in synthetic chemistry and beyond.
研究领域:
合成化学
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生物催化
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有机化学
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DOI
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2022
·
IF 4.446
Shan Yuan,Miaomiao Jin,Chao Xu,Wencheng Yin,Mingsha Zhao,Mo Xian,Wei Liu
3-(Aminomethyl) pyridine (3-AMP) is a versatile compound for the synthesis of pharmaceuticals, agricultural chemicals and numerous polymers. Efficient and mild biocatalytic synthesis is often an excellent supplement to traditional chemical synthesis methods. However, there has been no report of biocatalytic 3-AMP synthesis. In this paper, 3-AMP was synthesized for the first time by using biocatalysts co-expressing carboxylic acid reductase from Neurospora crassa (ncCAR) and ω-transaminase from Ochrobactrum anthropi (OATA) in one pot. In addition, the metabolic pathway was optimized by knocking out the transcriptional dual regulator arcA , which resulted in a 90% increase in the yield of 3-AMP. Furthermore, efficient self-sufficient whole-cell bioconversion was established by optimization, achieving 93% yield and a 40.6 mM (4.4 g/L) titer of 3-AMP without additional carbon sources. This whole-cell bioconversion system was also suitable for the high-efficiency biocatalytic synthesis of other aromatic or heterocyclic primary amines, such as benzylamine, 2-furfurylamine, 4-fluorobenzylamine, 2,4-difluorobenzylamine and 2-(aminomethyl) pyridine. Being entirely biocatalytic, our one-pot procedure provides considerable advantages in terms of economic, environmental and safety impacts over reported chemical methods.
研究领域:
生物化学
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合成生物学
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生物技术
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DOI
高影响因子
危险品化学品经营许可证（带存储）