JOURNAL OF CHROMATOGRAPHY A
·
2015
·
IF 4.169
Lulu Shangguan,Lingyi Zhang,Zhichao Xiong,Jun Ren,Runsheng Zhang,Fangyuan Gao,Weibing Zhang
The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bi-enzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic–inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24 h to 50 s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight.
研究领域:
生物化学
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蛋白质组学
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分析化学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY A
·
2016
·
IF 3.926
Bingbing Wang,Lulu Shangguan,Shulei Wang,Lingyi Zhang,Weibing Zhang,Fan Liu
The bottom up strategy has drawn much attention due to the high accuracy, reliability, and reproducibility in protein identification in which proteins are digested into peptides. However, conventional solution-based digestion and enzymatic reactor with one protease immobilized cannot satisfy high throughput proteolysis of complex samples. Application of consecutive hydrolysis by enzymatic reactor can be a new strategy for high throughput proteolysis of complex samples by adjusting immobilization amount of the enzymes, enzyme ratio, as well as hydrolysis order of two enzymes. In this work, we propose immobilized enzymatic reactor for consecutive digestion with two enzymes by combining two enzyme reactors with trypsin and chymotrypsin immobilized, respectively. Each reactor was prepared individually by immobilizing only one protease (trypsin or chymotrypsin) to hybrid monolith with SBA-15 particles embedded. Proteolysis conditions including hydrolysis order and trypsin to chymotrypsin ratio etc. were studied using standard proteins. Best digestion performance was obtained when the proteins were digested by trypsin first with trypsin to chymotrypsin ratio of 1:1. When applying them to digestion of rat liver proteins, total 1651 proteins and 11011 peptides were identified by combining four enzymolysis strategies with two enzymes including proteolytic digestion in two consecutive enzymatic reactors, synergy enzymolysis with two enzymes in one immobilized enzymatic reactor and consecutive hydrolysis with two enzymes in-solution digestion respectively, in which consecutive enzymolysis in enzymatic reactors gave the best results with 1091 proteins and 5071 peptides identified. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weights, and will play an important role in high efficient and high throughput proteomics research.
研究领域:
生物化学
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蛋白质组学
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DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2019
·
IF 8.456
Juan Qiao,Junfang Jiang,Lili Liu,Ji Shen,Li Qi
The challenge for polymeric enzyme reactors at present is to selectively control the enzymolysis rate in complex conditions. Additionally, the fabrication methodology is hindered by complex processes, especially for achieving diverse stimuli responsiveness and functions. Here, we reported a kind of pH-sensitive polymer, poly(styrene-co-maleic anhydride-acrylic acid) (PS-MAn-AA)-based hybrid enzyme reactor. It comprised magnetic nanoparticles and a pH-sensitive PS-MAn-AA porous polymer membrane made by breath figure method. The enzyme l-asparaginase (l-ASNase) could covalently bond on the surface of the pH-sensitive porous polymer membrane (pH-PPM), and the resultant enzyme reactor was characterized by Fourier transform infrared spectroscopy and vibrating sample magnetometer. The apparent Michaelis–Menten constants (Km and Vmax) of the l-ASNase enzyme reactor at different pH values were determined by a chiral ligand-exchange capillary electrophoresis method with l-asparagine as the substrate. The Vmax value of the l-ASNase enzyme reactor (0.67 mM/min) was almost 3-fold of that of the free l-ASNase (0.23 mM/min) at pH 8.2. Its ability to precisely control the enzymolysis rate in complex conditions is triggered primarily by the pH of the buffer solution, allowing controlled enzymatic reactions and displaying excellent stability and reusability of the proposed pH-PPM. This strategy for porous polymer membrane enzyme reactor fabrication has established a platform for enzyme efficiency adjusting. These valve-like distinguished features highlight the outstanding potential of stimuli-responsive enzyme reactor applied for enzyme immobilization and enzyme-related disease treatment.
研究领域:
生物化学
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材料科学
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酶工程
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF SEPARATION SCIENCE
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2014
·Vol. 37
·期 22
·3377-3383
·
IF 2.594
Tingting Hong,Cuijie Chi,Yibing Ji
Pepsin-modified affinity monolithic capillary electrochromatography, a novel microanalysis system, was developed by the covalent bonding of pepsin on silica monolith. The column was successfully applied in the chiral separation of (±)-nefopam. Furthermore, the electrochromatographic performance of the pepsin-functionalized monolith for enantiomeric analysis was evaluated in terms of protein content, pH of running buffer, sample volume, buffer concentration, applied voltage, and capillary temperature. The relative standard deviation (%RSD) values of retention time (intraday <0.53, n = 10; interday <0.53, n = 10; column-to-column <0.70, n = 20; and batch-to-batch <0.80, n = 20) indicated satisfactory stability of these columns. No appreciable change was observed in retention and resolution for chiral recognition of (±)-nefopam in 50 days with 100 injections. The proteolytic activity of this stationary phase was further characterized with bovine serum albumin as substrate for online protein digestion. As for monolithic immobilized enzyme reactor, successive protein injections confirmed both the operational stability and ability to reuse the bioreactor for at least 20 digestions. It implied that the affinity monolith used in this research opens a new path of exploring particularly versatile class of enzymes to develop enzyme-modified affinity capillary monolith for enantioseparation.
研究领域:
分析化学
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生物化学
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产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2025
·
IF 9.8
Shuo Liu,Hongli Chao,Meishan Guo,Ruifang Wang,Dongyu Gu,Yi Wang,Dajun He,Yi Yang
To achieve the biotransformation of cellulose into gluconic acid, a multi-enzyme reactor comprising cellulase, glucose oxidase, and catalase was constructed through artificial antibody-antigen-directed immobilization. Catechol was used as a template to prepare artificial antibodies, which were then used to modify the enzymes through a Schiff base reaction to form artificial antigens. The artificial antibody and antigen were subsequently assembled via affinity recognition to immobilize the enzymes. The immobilization yields for all three enzymes exceeded 85 %, with specific activities of 43.48 ± 1.54 U/mg, 0.88 ± 0.06 U/mg, and 18.47 ± 1.43 U/mg, respectively. The immobilized enzymes demonstrated excellent adaptability, storage stability, reusability, and regenerability. Through the modular assembly of artificial antigen-antibody, the multi-enzyme reactor facilitated the cascade conversion of cellulose to gluconic acid.
研究领域:
生物化学
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合成生物学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY A
·
2019
·
IF 3.858
Xue Zhao,Pei-Ru Fan,Chun-E Mo,Yan-Ping Huang,Zhao-Sheng Liu
In this study, a monolithic enzyme reactor based on a strategy of green synthesis was successfully prepared in a capillary with trypsin immobilized by “thiol-ene” click reaction. A polymer of poly(butyl methacrylate- co -α-methacrylic acid-co-ethylene glycol dimethacrylate) was prepared in a mixture of 1-butyl-3-methylimidazolium tetrafluoroborate and choline chloride/ethylene glycol as the support of enzyme reactor. After “thiol-ene” reaction was used for enzyme immobilization, the Michaelis constants and maximum reaction rate of the resulting immobilized enzyme reactors (IMER) were determined by capillary electrophoresis to be 2.1 mmol/L and 0.028 μmol/min, respectively. The enzymatic hydrolysis of the enzyme reactor under different experimental conditions were investigated. A on-line digestion of bovine serum albumin (BSA) on the new IMER can be achieved within 50 s, up to 864 times faster than in-solution digestion (12 h). BSA can be well digested and the numbers of identified peptides were 73 with the coverage rates of 82.7%. The IMER was further used for the analysis of protein extracts from rat liver, and 1034 protein groups were identified. All these results demonstrated that such a click reaction based IMER would be of great prospect in the high throughput analysis for proteome with high confidence.
研究领域:
生物化学
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分析化学
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蛋白质组学
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产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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2016
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IF 2.371
Guihua Ruan,Zhenwei Wu,Yipeng Huang,Meiping Wei,Rihui Su,Fuyou Du
A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N α -benzoyl- l -arginine ethyl ester to N α -benzoyl- l -arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas.
研究领域:
生物化学
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催化工程
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蛋白质组学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2018
·
IF 6.042
Jiqing Yang,Xiaotong Hu,Jia Xu,Xin Liu,Li Yang
A novel capillary electrophoresis-integrated immobilized enzyme reactor (CE-integrated IMER) is developed using single-step in situ acetylcholinesterase (AChE)-mediated alginate hydrogelation and enzyme encapsulation. Alginate hydrogelation with “egg-box” structure is triggered inside a capillary with releasing of Ca2+ by changing the pH of the sol solution, which is accomplished in situ by AChE-catalyzed hydrolysis reaction of acetylthiocholine to produce acetic acid. AChE and any other enzyme initially contained in the sol solution [e.g., xanthine oxidase (XO)] are efficiently encapsulated as the hydrogel network grows, forming CE-integrated IMERs without any additional manipulation process. The proposed method facilitates the analysis of different kinds of enzymes using the same IMER depending on the substrate injected for CE analysis. Approximately 68% of the original enzyme in the sol mixture can be encapsulated, indicating high loading capacity for the CE-integrated IMERs. The IMERs exhibit excellent intraday and interday stability and batch-to-batch reproducibility, and these characteristics imply the reliability of the proposed IMERs for accurate online enzyme assays. Enzymatic activities and inhibition of immobilized AChE and XO are analyzed, and the results are compared with those using free enzymes. The feasibility of the proposed method for potential application in real sample analysis is demonstrated by the successful application of the IMERs in detecting organophosphorus pesticides in apple juice samples using AChE-catalyzed reactions. The proposed method is a simple, efficient, and universal approach for online CE assays with immobilized enzymes, which can be widely applied in bioanalysis.
研究领域:
生物化学
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分析化学
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2015
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IF 4.513
Juan Qiao,Jin Yong Kim,Yuan Yuan Wang,Li Qi,Fu Yi Wang,Myeong Hee Moon
Fast and effective protein digestion is a vital process for mass spectrometry (MS) based protein analysis . This study introduces a porous polymer membrane enzyme reactor (PPMER) coupled to nanoflow liquid chromatography-tandem MS (nLC-ESI-MS/MS) for on-line digestion and analysis of proteins. Poly (styrene- co -maleic anhydride) (PS- co -MAn) was fabricated by the breath figure method to make a porous polymer membrane in which the MAn group was covalently bound to enzyme. Based on this strategy, microscale PPMER (μPPMER) was constructed for on-line connection with the nLC-ESI-MS/MS system. Its capability for enzymatic digestion with bovine serum albumin (BSA) was evaluated with varied digestion periods. The on-line proteolysis of BSA and subsequent analysis with μPPMER-nLC-ESI-MS/MS revealed that peptide sequence coverage increased from 10.3% (digestion time 10 min) to 89.1% (digestion time 30 min). μPPMER can efficiently digest proteins due to the microscopic confinement effect, showing its potential application in fast protein identification and protease immobilization. Applications of on-line digestion using μPPMER with human plasma and urinary proteome samples showed that the developed on-line method yielded equivalent or better performance in protein coverage and identified more membrane proteins than the in-solution method. This may be due to easy accommodation of hydrophobic membrane proteins within membrane pores .
研究领域:
生物化学
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分析化学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY A
·
2022
·
IF 4.601
Yang Wang,Xue Zhang,Ze-Hui Wei,Ya-Jie Jiao,Dong-Yu An,Yan-Ping Huang,Zhao-Sheng Liu,Chao Yan
A new dual-function enzyme reactor was prepared based on a dopamine/graphene oxide coated boron affinity monolithic column, which can be used for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment. Firstly, a boron affinity monolithic column was prepared as the carrier for enzyme reactor. Secondly, the monolithic column was coated with dopamine/graphene oxide to provide higher specific surface area for the increase in the amount of trypsin bound. Then, dopamine can self-polymerize under alkaline conditions to produce multiple reaction sites. By the Schiff base reaction and Michael addition reaction with amino, sulfhydryl groups to trypsin, enzyme were immobilized on the boron affinity monolithic carrier. The enzyme activity was characterized by kinetic parameters maximum rate (V max ) of the enzyme reaction and Michaelis constant (K m ). K m of the dual-function enzyme reactors doped with PDA/GO and without PDA/GO were 34.37 and 120.93 mM, Vmax were 1.35 and 3.35 mM/min, respectively. The performance of the dual-function enzyme reactor was evaluated by protein extraction of mouse liver. After digested by the dual-function enzyme reactor, the number of peptides was 4,801, which was 960 more than the number of peptides in the solution digestion. At the same time, the dual-function enzyme reactor displayed the ability to capture cis-dihydroxy compounds specificly. A total of 55 glycopeptides were enriched in the dual-functional enzyme reactor, corresponding to 33 glycoproteins. The dual-function enzyme reactor provided repeatable performance and robust with long-term storage.
研究领域:
生物化学
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蛋白质组学
·
分析化学
✓ 已验证
DOI
PubMed
高影响因子
Biosensors-Basel
·
2022
·Vol. 12
·期 11
·988
·
IF 5.743
Linmin Chen,Meihuang Zeng,Jingwen Jin,Qiuhong Yao,Tingxiu Ye,Longjie You,Xi Chen,Xiaomei Chen,Zhiyong Guo
Nanoenzyme reactors based on shell-isolated colloidal plasmonic nanomaterials are well-established and widely applied in catalysis and surface-enhanced Raman scattering (SERS) sensing. In this study, a “double wing with one body” strategy was developed to establish a reduced food antiseptic sensing method using shell-isolated colloidal plasmonic nanomaterials. Gold nano particles (Au NPs) were used to synthesize the colloidal plasmonic nanomaterials, which was achieved by attaching ferrous ions (Fe2+), ferric ions (Fe3+), nitroso (NO−) group, cyanogen (CN−) group, and dopamine (DA) via coordinative interactions. The oxidation-induced reaction was utilized to generate •OH following the Fe2+-mediated Fenton reaction with the shell-isolated colloidal plasmonic nanomaterials. The •OH generated in the cascade reactor had a high oxidative capacity toward acid preservatives. Importantly, with the introduction of the signal molecule DA, the cascade reactor exhibited also induced a Raman signal change by reaction with the oxidation product (malondialdehyde) which improved the sensitivity of the analysis. In addition, the stable shell-isolated structure was effective in realizing a reproducible and quantitative SERS analysis method, which overcomes previous limitations and could extend the use of nanoenzymes to various complex sensing applications.
研究领域:
分析化学
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纳米生物学
·
食品科学
✓ 已验证
DOI
高影响因子
ACS Applied Bio Materials
·
2020
·
IF 0
Ji Shen,Juan Qiao,Li Qi
Tuning the efficiency of enzymolysis in different environments is a challenge with important implications for the use of immobilized enzymes in biochemistry, pharmacology, and life science. Here, we demonstrate the use of a thermoresponsive porous polymer membrane for the construction of switchable enzyme reactors, which exhibited membrane surface property changes in diverse temperature environments. The enzyme reactor consisted of block copolymers, poly(styrene−maleic anhydride−oligo methacrylate−methoxyethoxy ethyl methacrylate) [P(St–MAn–OE–ME)], magnetic nanoparticles, and covalently bonded d-amino acid oxidase (DAAO). By varying the temperature between 25 and 39 °C, the self-assembly properties of the enzyme reactor could be controlled. Using d,l-Met as the substrate, the enzymolysis efficiency of the thermoresponsive porous polymer membrane enzyme reactor (t-PPMER) was evaluated by the chiral capillary electrophoresis technique. At higher temperatures, DAAO was embedded in the curled P(OE–ME) chains, resulting in improved enzymolysis efficiency owing to the confinement effect. At lower temperatures, inefficient enzymolysis occurred because of the stretching of the P(OE–ME) chains, which weakened the confinement effect. Remarkably, the enzymolysis rate of t-PPMER at 37 °C (3.1 mM·min–1) was 45 times higher than that at 25 °C (0.066 mM·min–1). The proposed protocol was successfully applied to the DAAO inhibitor screening. This approach capitalizes on the advantageous characteristics of the t-PPMER and provides inspirations for the construction of switchable enzyme reactors and the screening of enzyme inhibitors as potential drugs.
研究领域:
生物化学
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材料科学
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药物筛选
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产品关联
DOI
危险品化学品经营许可证（带存储）