SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
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2025
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IF 4.6
Yingxin Wu,Kainan Hong,Juzhen Yi,Juncheng Shen,Liqun Yang
Surface-enhanced Raman spectroscopy (SERS) shows unique advantages for the study of protein structure in water environment, because it can provide plenty of structural information of proteins in addition to very weak Raman scattering signal of water. To achieve the Raman-enhanced effect of proteins, the challenge is to prompt protein molecules to approach the surface of noble metal (such as Ag and Au). Based on the enzyme–substrate specificity, the Ag nanoparticles-encapsulated amylopectin (Amyp/AgNPs) colloid was prepared as an aqueous SERS substrate to obtain the Raman-enhanced effect of amylase. Dynamic light scattering analysis showed that the hydrodynamic diameter of the Amyp/AgNPs colloid was in the range of 40–700 nm. UV–vis spectroscopy and energy dispersive spectroscopy confirmed the formation of Ag nanoparticles in the Amyp/AgNPs colloid. The Ag nanoparticles in the Amyp/AgNPs colloid were observed as globular homogeneous particles in the sizes ranging from 10 to 20 nm using transmission electron microscopy (TEM). The crystalline Ag nanoparticles were further observed in the high-resolution TEM image. The obviously enhanced Raman bands assigned to the amino acid residues and the secondary structures of α -amylase and β -amylase appeared in their SERS spectra, which were further analyzed following the principal component analysis (PCA) process. The Raman-enhanced mechanism is considered as that the specificity of two amylases with amylopectin in the Amyp/AgNPs colloid may prompt them to approach the surface of Ag nanoparticles and then produce the significant SERS effect. The SERS strategy based on enzyme–substrate specificity is thus anticipated to apply for structural analysis of enzyme.
研究领域:
生物化学
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分析化学
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纳米技术
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产品关联
DOI
PubMed
高影响因子
BIOORGANIC & MEDICINAL CHEMISTRY
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2012
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IF 2.921
Hui-juan Jin,Jing Lu,Xue Wu
A new self-immolative spacer conjugate based on a chemical adaptor unit aiming at controlled releasing drugs was designed and synthesized. It releases a fluorophore which was used as a model drug via a spontaneous cyclization mechanism after cleavage of an enzyme substrate. This system was proved to be stable under physiological conditions and only decomposed triggered by enzyme. It provides a generic linkage allowing connection of a variety of drugs and targeted devices to the chemical adaptor.
研究领域:
药用化学
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药物递送系统
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酶学
✓ 已验证
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2021
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IF 3.493
Xinqi Xu,Xiangzhen Deng,Juan Lin,Jie Yang
Arylsulfatase is useful in industrial agar processing by removing sulfate groups. A full-length arylsulfatase gene, designated ArySMA1, was obtained from marine bacteria Serratia sp. SM1. The ArySMA1 gene encoded a novel serine-type arylsulfatase and the enzymatic properties were characterized. The enzyme presented notable capacity of removing sulfate groups from natural algae substrates. Kinetic study suggested that the microscopic thermal inactivation rate of ArySMA1 in free form was smaller than that of the enzyme-substrate complex. The presence of substrate could unexpectedly accelerate ArySMA1 to deactivate at high temperature. Such phenomenon was opposite to many findings that substrate could stabilize enzymes against heat. Molecular dynamics simulation and ANS fluorescent assay indicated the substrate led the hydrophobic regions of the active site more flexible and the sulfate group of the substrate could retard the processivity of ArySMA1 catalysis. This study provides guidance for agar desulfation and down-stream processing industry.
研究领域:
酶学
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海洋生物技术
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工业生物技术
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产品关联
DOI
PubMed
高影响因子
CARBOHYDRATE POLYMERS
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2024
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IF 10.7
Jun Yang,Shuzhen Ma,Sihong Tao,Muci Wu,Qian Wu,Jingyi Wang
β-Galactosidase (lactase) plays a crucial role as a dietary supplement in managing lactose intolerance. Here, the catalytic activity of lactase was successfully activated for the first time through complexation with water-extractable β-glucans from highland barley (WHBG). Under simulated gastric/small intestinal digestive conditions, WHBG and lactase spontaneously formed complexes, resulting in a remarkable increase in catalytic activity up to 172.6 %. Structural analyses revealed that the incorporation of WHBG caused partial unfolding of lactase, thereby exposing its hydrophobic regions with active sites, and the electrostatic and hydrophobic interactions between the two played pivotal roles. Meanwhile, according to microstructure and particle size analyses, the dissociation of aggregates and the re-distribution of lactase molecules were also observed. Consequently, the enzyme-substrate contact was promoted, and the hydrolysis efficiency of complexed lactase in the digestion of lactose in milk was superior to that of native lactase. Notably, among WHBG30/50/70 obtained by continuous fractionation of WHBG with 30 %/50 %/70 % ethanol, WHBG70 exhibited the lowest molecular weights and size, and the highest negative ζ-potential, potentially contributing to its superior activation abilities on lactase. These findings challenge the traditional view of polysaccharides as enzyme inhibitors and highlight their potential for diverse applications.
研究领域:
食品科学
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酶学
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营养学
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生物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
iScience
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2024
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IF 4.6
Jiawei Wang,Xingyu Ouyang,Shiyu Meng,Jiayi Li,Liangxu Liu,Chaofeng Li,Hengrun Li,Haotian Zheng,Chao Liao,Yi-Lei Zhao,Jun Ni
Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of Th Ado, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes.
研究领域:
生物化学
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生物技术
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酶学
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可持续化学
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
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2016
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IF 7.476
Xiaonan Wang,Meiwen Wang,Yuanyuan Zhang,Xiaocao Miao,Yuanyuan Huang,Juan Zhang,Lizhou Sun
A new strategy to fabricate electrochemical biosensor is reported based on the linkage of enzyme substrate , thereby an electrochemical method to detect aldolase activity is established using pectin-thionine complex (PTC) as recognization element and signal probe . The linkage effect of fructose-1,6-bisphosphate (FBP), the substrate of aldolase, can be achieved via its strong binding to magnetic nanoparticles (MNPs)/aminophenylboronic acid (APBA) and the formation of phosphoramidate bond derived from its reaction with p -phenylenediamine (PDA) on the surface of electrode. Aldolase can reversibly catalyze the substrates into the products which have no binding capacity with MNPs/APBA, resulting in the exposure of the corresponding binding sites and its subsequent recognization on signal probe. Meanwhile, signal amplification can be accomplished by using the firstly prepared PTC which can bind with MNPs/APBA, and accuracy can be strengthened through magnetic separation. With good precision and accuracy, the established sensor may be extended to other proteins with reversible catalyzed ability.
研究领域:
分析化学
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生物化学
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生物传感器
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电化学
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纳米技术
✓ 已验证
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2023
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IF 6.1
Jian Ming,Yueru Sun,Yuanyuan Chen,Qiming Wang,Jinlong Li
According to the “interfacial activation” mechanism, constructing a sufficient interface is the key strategy for lipase-catalytic system designing. Based on the “infinite interface in finite three-dimensional space” logic, in the current study, poly(N,N-dimethylacrylamide) (PDMA)-polybutyl methacrylate (PBMA) hybrid gels were prepared by a two-step crosslinking strategy, subsequently constructed as lipase-interfacial catalytic systems. The results confirm that the PDMA-PBMA hybrid gels with “networks in pores” structures could swell both the aqueous phase and organic phase. The balance between water swelling and isooctane swelling, hybrid gel space (height control), and the lipase entry manner significantly affect the interface construction and consequently the catalytic efficiency. The enzyme–substrate contact rate affected by swelling leads to three catalytic stages. Considering the spatial barrier and distribution of lipases, a potential high-performance lipase reactor can be assembled from small-size, lamellar-like, and porous hybrid gels. The reactors also show good time storage and low temperature tolerance.
研究领域:
生物技术
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化学工程
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材料科学
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生物化学
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催化
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Nature Protocols
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2024
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IF 13.1
Luo Juan,Yu Yao,Wang Ke,He Sizhe,Wang Longjie,Liang Fangfang,Chin Jason W.,Tang Shan
Catalytic mechanism-based, light-activated traps have recently been developed to identify the substrates of cysteine or serine hydrolases. These traps are hydrolase mutants whose catalytic cysteine or serine are replaced with genetically encoded 2,3-diaminopropionic acid (DAP). DAP-containing hydrolases specifically capture the transient thioester- or ester-linked acyl–enzyme intermediates resulting from the first step of the proteolytic reaction as their stable amide analogs. The trapped substrate fragments allow the downstream identification of hydrolase substrates by mass spectrometry and immunoblotting. In this protocol, we provide a detailed step-by-step guide for substrate capture and identification of the peptidase domain of the large tegument protein deneddylase (UL36 USP ) from human herpesvirus 1, both in mammalian cell lysate and live mammalian cells. Four procedures are included: Procedure 1, DAP-mediated substrate trapping in mammalian cell lysate (~8 d); Procedure 2, DAP-mediated substrate trapping in adherent mammalian cells (~6 d); Procedure 3, DAP-mediated substrate trapping in suspension mammalian cells (~5 d); and Procedure 4, substrate identification and validation (~12–13 d). Basic skills to perform protein expression in bacteria or mammalian cells, affinity enrichment and proteomic analysis are required to implement the protocol. This protocol will be a practical guide for identifying substrates of serine or cysteine hydrolases either in a complex mixture, where genetic manipulation is challenging, or in live cells such as bacteria, yeasts and mammalian cells.
研究领域:
生物化学
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分子生物学
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细胞生物学
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病毒学
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蛋白质组学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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