PROTEIN EXPRESSION AND PURIFICATION
·
2024
·
IF 1.4
Junzhang Chang,Yuxin Zhang,Zhiwei Li,Yunfeng Ma,Xueqin Hu,Jingwen Yang,Hongbin Zhang
Chiral amino acids and their deamination products, α-keto acids, have important applications in food, medicine, and fine chemicals. In this study, two l -amino acid deaminase genes from Proteus mirabilis , PM473 of type Ⅰ and PM471 of type Ⅱ were cloned and expressed in Escherichia coli respectively, expected to achieve the chiral separation of amino acids . Extensive substrate preference testing showed that both deaminases had catalytic effects on the d -amino acid component of the D, l -amino acids, and PM473 has a wider catalytic range for amino acids. When D, L-Cys was used as the substrate, all L-Cys components and 75.1 % of D-Cys were converted to mercapto pyruvate , and the remaining D-Cys was a single chiral enantiomer . Molecular docking analysis showed that the interaction between the substrate and the key residues affected the stereoselectivity of enzymes . The compatibility of hydrophobicity between the binding pocket and substrate may be the basic factor that affects the substrate selectivity. This work provides an alternative method for the production of α-keto acids and the resolution of chiral amino acids.
研究领域:
生物化学
·
生物催化
·
酶学
·
药物化学
✓ 已验证
DOI
PubMed
高影响因子
Frontiers of Chemical Science and Engineering
·
2011
·Vol. 5
·期 4
·409-415
·
IF 0
Cao Hongfei,Feng Yakai,Wang Heyun,Zhang Li,Khan Musammir,Guo Jintang
By using the corresponding L -amino acid sodium as initiator, ɛ -caprolactone-depsipeptides CL-Ala and CL-Leu were prepared by the reactions of ɛ -caprolactone (CL) with L -alanine and L -leucine, respectively, and p -dioxanone-depsipeptide (PDO-Leu) was prepared by the reaction of p -dioxanone (PDO) with L leucine. Two poly( ɛ -caprolactone) oligomers (PCL-Ala and PCL-Leu) of different molecular weights with depsipeptide unit were synthesized by controlling the feed ratio of L -amino acid sodium and CL. The presence of the depsipeptide structure in these obtained products was confirmed by 1 H NMR spectra and the molecular weight of the poly( ɛ -caprolactone) oligomers was measured by gel permeation chromatography (GPC). These products contain a hydroxyl group and a carboxyl group in one molecule, which means they could act as bifunctional monomers for further polymerization to prepare high molecular weight polymers. By this way, the depsipeptide unit could be introduced into the polymers and the biodegradation rates of the novel polymers could be well controlled in vivo by the tailored molecular structures.
研究领域:
有机化学
·
高分子化学
·
生物材料学
✓ 已验证
DOI
Analytical Methods
·
2017
·Vol. 9
·期 43
·6131-6138
·
IF 1.9
Xiangyu Wang,Huihui Wu,Rongying Luo,Donghai Xia,Zhengjin Jiang,Hai Han
D-amino acids are currently paid attention to as new physiologically active substances. Foodstuffs and beverages containing D-amino acids are a matter of interest. Until now, the profiles of D-amino acids have not been reported in natural and fermented teas except theanine. In this study, an off-line 2D-HPLC method combining a Gemini C18 column and a CHIRALPAK® IC-3 column or a self-prepared poly(MQD-co-HEMA-co-EDMA) monolithic capillary column was employed for the separation and detection of D-amino acids in tea samples with the co-existence of a large amount of L-amino acids. The free amino acid fractions in longjing, black, oolong, and pu-erh tea samples were separated and collected after pre-column derivatization using 9-fluorenylmethoxycarbonyl (FMOC) chloride in the reversed-phase mode, and then were concentrated and separated to D- and L-forms on a chiral column. Among them, the D-form of isoleucine (Ile) (1.0–1.6%), alanine (Ala) (0.1–0.8%), phenylalanine (Phe) (0.1–0.4%), valine (Val) (0.2–0.3%), serine (Ser) (0.1–0.2%), aspartic acid (Asp) (0.2%), and proline (Pro) (0.1%) were detected in longjing and oolong teas. Differences between natural tea and fermented tea in the profiles of D-amino acids as well as the total amino acids were also observed. The results provided useful information for the bio-function research of D-amino acids in tea.
研究领域:
食品科学
·
分析化学
·
生物化学
✓ 已验证
产品关联
DOI
高影响因子
Analytical Methods
·
2016
·Vol. 8
·期 17
·3481-3487
·
IF 1.915
Lili Wang,Wencheng Gong,Fang Wang,Zhenya Yu,Zilin Chen
An efficient bienzyme nanocomposite film-based biosensor for L-amino acid recognition has been developed and characterized. The biosensor was fabricated by immobilizing horseradish peroxidase (HRP) and L-amino acid oxidase (L-AAODx) in poly(toluidine blue) film (polyTB) at the surface of a multi-walled carbon nanotube (CNTs) modified glassy carbon electrode (GCE). Only L-amino acids can be electrochemically oxidized by L-AAODx in the presence of oxygen (O2) to produce hydrogen peroxide (H2O2), which can be further reduced to H2O by HRP at a low potential of −0.1 V. The concentration of L-amino acids was estimated by the reduction current of H2O2. The polyTB film, acting as the enzyme-immobilization carrier and redox mediator, accelerated electron transfer between analytes and the electrode. At the same time, CNTs with excellent conductivity and strong adsorption ability enhanced the utilization of the enzymes. The performance of this biosensor was evaluated by electrochemical techniques and it showed high selectivity and fast response for the chiral recognition of L-amino acids, including L-tryptophan (L-Trp), L-phenylalanine (L-Phe) and L-tyrosine (L-Tyr). The low potential reduced the interference of some coexisting biomolecules for the determination of the L-amino acids. The recovery of the three amino acids in milk indicated the feasibility of this biosensor for use in complex samples.
研究领域:
生物化学
·
分析化学
✓ 已验证
产品关联
DOI
高影响因子
Scientific Reports
·
2017
·Vol. 7
·期 1
·1-9
·
IF 4.259
Liu Qingju,Chen Li,Zhang Zhikun,Du Bibai,Xiao Yating,Yang Kunhao,Gong Lingling,Wu Li,Li Xiangjun,He Yujian
D-amino acid oxidases (DAAO) are stereospecific enzymes which are generally almost inactive towards L-enantiomer in neutral solution when L-, D-amino acids are supplied as substrates. In this paper, the D-amino acid oxidase can catalytic oxidize L-amino acids by modulating pH of aqueous solution. With L-Pro as substrate, the catalytic rate ( k
cat ) and the affinity ( K
m ) of DAAO were 6.71 s −1 and 33 mM at pH 8.0, respectively, suggesting that optimal pH condition enhanced the activity of DAAO towards L-Pro. Similar results were obtained when L-Ala (pH 9.8), L-Arg (pH 6.5), L-Phe (pH 9.0), L-Thr (pH 9.4), and L-Val (pH 8.5) were catalyzed by DAAO at various pH values. The racemization of the L-amino acids was not found by capillary electrophoresis analysis during oxidation, and quantification analysis of L-amino acids before and after catalytic reaction was performed, which confirmed that the modulation of enantioselectivity of DAAO resulted from the oxidation of L-amino acids rather than D-amino acids by changing pH. A mechanistic model was proposed to explain enhanced activity of DAAO towards L-amino acids under optimal pH condition.
研究领域:
生物化学
·
酶学
✓ 已验证
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Yancheng Xie,Xue Feng,Jiaqi Tao,Qianhui Gao,Yue Li,Xinyi Liu,Mingyu Xia,Dong Wang
5-Hydroxytryptamine (5-HT) and its derivative bufotenine, which possess important physiological functions, are the primary active components in the secretions of toad parotid and skin gland. However, the biosynthetic pathway of these substances remains unclear in toads. To characterize toad's Aromatic-L-amino-acid decarboxylase (AADC), the key enzyme in the predicted 5-HT derivatives biosynthetic pathway, the full-length cDNA of AADC from Bufo bufo gargarizans ( Bbg AADC) was cloned from the parotoid gland of B. bufo gargarizans . The recombinant Bbg AADC exhibited optimal expression in E. coli BL21 (DE3) containing pCold- Bbg AADC after induction for 16 h at 15 °C with 0.3 mM IPTG, resulting in substantial yields of soluble proteins . The enzymological properties of Bbg AADC were assessed, and it was determined that the optimal reaction temperature was 37 °C, the optimal pH was 8.6, and the optimum molar ratio of pyridoxal-5′-phosphate (PLP) to Bbg AADC was found to be 3.6:1. Additionally, high substrate specificity was observed, as Bbg AADC could catalyze the production of 5-HT from 5-hydroxytryptophan (5-HTP) but not dopamine or tryptamine from levodopa or tryptophan , respectively. The Km of the recombinant protein Bbg AADC was 0.2918 mM and the maximum reaction rate (Vmax) was 1.182 μM·min −1 when 5-HTP was used as substrate. The Kcat was 0.0545 min −1 , and Kcat/Km was 0.1868 mM −1 ·min −1 . To elucidate the mechanism of Bbg AADC, molecular docking was performed with PLP and 5-HTP, or the external aldimine formed by 5-HTP and PLP. The results indicated that the active sites for Bbg AADC to bind with PLP were K303, H192, N300, A148, F309, T246, A273, and T147. W71, Y79, F80, P81, T82, H192, T246, N300, H302, F309, and R477 served as catalytically active sites for the binding of Bbg AADC to 5-HTP. Furthermore, R447, W71, S149, N300, A148, and T147 of Bbg AADC were involved in the decarboxylation reaction of the aldimine formed by PLP and 5-HTP.
研究领域:
生物化学
·
分子生物学
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Journal of Magnetic Resonance Open
·
2025
·
IF 2
Bowen Han,Jing He,Shaohua Huang
Chiral amino acids play an indispensable role in living organisms. Diffusion-ordered NMR spectroscopy is an effective NMR tool and a noninvasive analytical method for the analyses of mixture without the need for physical separation of the analytes. However, conventional diffusion-ordered NMR spectroscopy method usually fails to resolve the mixtures of chiral amino acids because of their same molecular masses, sizes, and shapes. Microcrystalline cellulose has been gradually gained more and more interest owing to its wide compatibility, surface area, excellent separation efficiency, non-toxicity, cost-effective, and mechanical stability in many fields. Herein we provide a fast, simple, specific and sensitive method to resolve some D and L-amino acids mixtures by using microcrystalline cellulose as a matrix. This work recognized some D and L- proteinogenic amino acid enantiomers with diffusion-ordered NMR for the first time.
研究领域:
分析化学
·
生物化学
✓ 已验证
产品关联
DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2024
·
IF 3.4
Yuguang Wang,Mengjing Wu,Huifang Zheng,Dongmei Wu,Panpan Yao,Wenjing Li,Kexin Jin,Xinjun Yu
A strain high-performance of esterase producing bacteria was screened from soil, which could selectively hydrolyze D -homoserine lactone from its racemate to achieve the resolution of L - homoserine lactone with more than 99% e.e. in 48% yield. L -homoserine lactone building block was then converted to L -α-amino-γ-bromobutyronic acid chiral blocks, which reacted with various nucleophilic reagent modules could to be applied to prepare L -γ- substituted α-amino acids such as L -selenomethionine, L -methionine, L -glufosinate and L -selenocystine. Its advantages included high selectivity of biocatalytic resolution reactions, high optical purity of products, racemic recycle of D -substrates and modular reaction, which simplified the production process of these products and highlighted the power of biological manufacturing.
研究领域:
生物化学
·
生物制造
·
酶工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF THE ELECTROCHEMICAL SOCIETY
·
2020
·Vol. 167
·期 6
·067517
·
IF 3.721
Lijun Zhang,Qianwen Li,Zhongbo Li,Zhaofang Du,Xia Hong,Longzhen Qiu
The chiral recognition of α-amino acids has attracted considerable interest because different enantiomeric amino acids show differences in their biological behavior and pharmacological activity. An organic electrochemical transistor (OECT)-based biosensor modified with L-amino acid oxidases (L-AAODX) was fabricated and successfully used as a biosensor for enantioselectively discriminating α-amino acids, such as D/L-tryptophan (D/L-Trp), D/L-tyrosine (D/L-Tyr) and D/L-phenylalanine (L-Phe) with high performance because L-AAODX selectively catalyzed the oxidation of L-Trp, L-Tyr, and L-Phe along with the amplification of OECT. The biosensor showed a sensitivity of 19.67 μA/μM with a LOD of 10 nM (S/N > 3) over the range of 10 nM to 30 μM for L-Trp, 16.71 μA/μM with a LOD of 2 nM (S/N >3) over the range of 2 nM to 30 μM for L-Tyr and 15.51 μA/μM with a LOD of 10 nM (S/N > 3) over the range of 30 nM to 30 μM for L-Phe. Furthermore, the biosensor can both be used for the chiral recognition of Trp, Tyr and Phe, and distinguish L-amino acids from its interferences (e.g., Glu, UA, L-Asp, L-Lys and L-Glu) and common metal ions (K+, Na+ and Fe3+).
研究领域:
生物传感
·
分析化学
·
生物医学工程
✓ 已验证
产品关联
DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2025
·
IF 8.5
Lijun Yang,Tiantian Zhou,Zhiyi Zhang,Wei Qi,Mengfan Wang
The enzymatic oxidation of amino acids using amino acid oxidases (AAOs) as biocatalysts for α-keto acid synthesis has garnered increasing attention due to its environmental friendliness and exceptional reaction efficiency. However, the by-product H 2 O 2 inevitably causes the decarboxylation of α-keto acids and subsequently deactivates the enzyme. Herein, we used a simple in-situ biomineralization method to immobilize Pt NPs with catalase-like activity and L-amino acid oxidase (LAAO) in to a hydrogen-bonded organic framework ( TW HOF) under a mild condition, and the synthesized LAAO-Pt@ TW HOF cascade catalytic system was applied to synthesis of α-keto acid. Compared with free LAAO, the LAAO-Pt@ TW HOF exhibited a 3.3-fold activity, well reusability and complete conversion (100 %) of L-tryptophan to indole-3-pyruvic acid was achieved. This work offers a potential application of HOF materials in constructing high-efficiency enzyme-metal coupled catalyst for cascade reactions.
研究领域:
生物化学
·
材料科学
·
催化反应
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Biomedicines
·
2021
·Vol. 9
·期 10
·1320
·
IF 6.081
Xiaoke Zheng,Hanyu Yang,Lan Qin,Siqian Wang,Lei Xie,Lu Yang,Weimin Kong,Liang Zhu,Li Liu,Xiaodong Liu
Liver failure is associated with increased levels of brain aromatic amino acids (AAAs), whose transport across the blood–brain barrier (BBB) is mainly mediated by L-amino acid transporter 1 (LAT1). We aimed to investigate whether liver failure induced by bile duct ligation (BDL) increases levels of brain AAAs by affecting the expression and function of LAT1. The LAT1 function was assessed using the brain distribution of gabapentin. It was found that BDL significantly increased levels of gabapentin, phenylalanine, and tryptophan in the cortex, hippocampus, and striatum of rats, and upregulated the expression of total LAT1 protein in hippocampus and striatum as well as cortex membrane LAT1 protein. HCMEC/D3 served as in vitro BBB model, and the data showed that both the serum of BDL rats and bilirubin induced LAT1 expression and function, while bilirubin oxidase almost abolished the upregulation of LAT1 protein by bilirubin and the serum of BDL rats. The enhanced function and expression of LAT1 were also observed in the hippocampus and striatum of hyperbilirubinemia rats. Both aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone and AhR silencing obviously attenuated the upregulation of LAT1 protein by bilirubin or omeprazole. This study provides the first evidence that BDL upregulates LAT1 at the rat BBB, attributed to the activation of AhR by the increased plasma bilirubin. The results highlight the mechanisms causing BDL-increased levels of brain AAAs and their physiological significance.Keywords:L-amino acid transporter;blood–brain barrier;liver failure;unconjugated bilirubin;aryl hydrocarbon receptor
研究领域:
神经科学
·
药理学
·
肝病学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
RSC Advances
·
2016
·Vol. 6
·期 56
·50837-50845
·
IF 3.289
Bin Liu,Ji-Mei Ma,Hang-Wei Chen,Zi-Long Li,Lin-Hao Sun,Zhen Zeng,Hong Jiang
α-Glucosidase inhibitors can effectively control postprandial hyperglycemia. In this study, a series of phenolic acids with the L-amino acid moiety were synthesized and their inhibitory activities against α-glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20) were evaluated. The results suggested that all these compounds showed strong α-glucosidase inhibitory activities. In particular, N-(4-hydroxyl-phenylpropenoyl)-L-alanine (c2) and N-(4-hydroxyl-phenylpropenoyl)-L-methionine (c8) exhibited much higher potency (IC50 values 0.04 mM) than the positive control, acarbose (IC50 values 1.70 mM). Three-dimensional quantitative structure–activity relationship (3D-QSAR) model for comparative molecular field analysis (CoMFA) was generated and the result showed that bulk groups and high electron density groups on the amino acid residues were a benefit to their activities. Moreover, the substituents with low electron density and little steric hindrance on the para position of the benzene ring were helpful in improving the activities. Kinetic analysis indicated that compound (c2) acted as a mixed-type inhibitor with a Ki value of 0.0124 mM. Docking analysis showed that they could bind to α-glucosidase at the catalytic site via hydrogen bonds and a π–π stacking.
研究领域:
药理学
·
生物化学
·
计算化学
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）