ANALYTICA CHIMICA ACTA
·
2018
·
IF 5.123
Lele Zhou,Qi Kang,Oiongzheng Hu,Li Yu
Glutathione (GSH) is of significance in human pathology due to its many cellular functions. γ-Glutamyl transpeptidase (γ-GT) plays a key role in the balance of GSH by breaking down extracellular GSH. In this study, we proposed a novel strategy for sensitive and selective detection of GSH based on LC sensing platform coupled with its inhibition of γ-GT. The γ-GT cleavable surfactant, dodecyl N -ethanamide N, N, N -trimethylammonium bromide (DAA), was synthesized and utilized to decorate the aqueous/LC interface. A black optical image of LCs was observed due to formation of the self-assembled DAA monolayer at the interface. Subsequently, the optical response of LCs returned to bright appearance after transferring γ-GT to the DAA-laden interface because DAA could be enzymatically hydrolyzed by γ-GT and the monolayer was disrupted. Interestingly, LCs remained dark images after the DAA-decorated interface in contact with the pre-incubated GSH and γ-GT. The detection limit of GSH reached 1 pg/mL, which demonstrates high competitiveness with the current assays. The detection mechanism of GSH was explained by Isothermal titration calorimetry (ITC) measurements and Fourier transform infrared (FTIR) spectroscopy. This work presents an appealing route to achieve ultrasensitive detection of GSH according to its hydrolysis by γ-GT and the as-prepared simple and robust LC-based sensing platform has potentials in the diagnosis of GSH-related diseases (e.g. cancer, liver damage, and malignant neoplasms).
研究领域:
生物化学
·
医学诊断
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产品关联
DOI
PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2024
·Vol. 108
·期 1
·1-12
·
IF 3.9
Chen Qianlin,Wang Bin,Pan Li
Abstract In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis , Bacillus amyloliquefaciens , and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. Key points
• Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide.
• Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system.
• The process of immobilizing BlGGT with epoxy resin was optimized.
研究领域:
微生物学
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食品科学
·
分子生物学
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DOI
PubMed
高影响因子
JOURNAL OF FOOD SCIENCE AND TECHNOLOGY-MYSORE
·
2013
·Vol. 52
·期 3
·1724-1729
·
IF 1.123
Xu Xiaobian,Miao Yelian,Chen Jie Yu,Zhang Qimei,Wang Jining
S-allyl-L-cysteine (SAC) is a bioactive compound in garlic ( Allium sativum ). A novel process including soaking and homogeneous reaction was applied for the effective production of SAC with endogenous γ-glutamyltranspeptidase (γ-GTP, EC 2.3.2.2) in garlic. The effects of temperature and CaCl 2 concentration on γ-GTP activity in soaking, and the relationship of SAC production with γ-GTP activity in homogeneous reaction were investigated, using fresh garlic as raw material. The experimental results showed that the γ-GTP in fresh garlic was activated by soaking. The yield rate and the final content of SAC increased linearly with increasing initial γ-GTP activity in the homogeneous reaction at 37 °C. The final SAC content reached 606.3 μg/g (i.e. 32 times higher than that in fresh garlic) after soaking for 72 h in a 10-mM CaCl 2 solution at 10 °C, and the homogeneous reaction for 8 h at 37 °C. SAC was produced effectively through the homogeneous reaction with activated endogenous γ-GTP in garlic.
研究领域:
植物学
·
生物化学
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DOI
高影响因子
RSC Advances
·
2021
·Vol. 11
·期 31
·18738-18747
·
IF 3.361
Dingyao Gao,Yinxing Miao,Siqin Ye,Chunmei Lu,Gaochao Lv,Ke Li,Chunjing Yu,Jianguo Lin,Ling Qiu
The expression level of γ-glutamyltranspeptidase (GGT) in some malignant tumors is often abnormally high, while its expression is low in normal tissues. Therefore, GGT is considered as a key biomarker for cancer diagnosis. Several GGT-targeting fluorescence probes have been designed and prepared, but their clinical applications are limited due to their shallow tissue penetration. Considering the advantages of positron emission tomography (PET) such as high sensitivity and deep tissue penetration, we designed a novel PET imaging probe for targeted monitoring of the expression of GGT in living subjects, ([18F]γ-Glu-Cys-PPG(CBT)-AmBF3)2, hereinafter referred to as ([18F]GCPA)2. The non-radioactive probe (GCPA)2 was synthesized successfully and [18F]fluorinated rapidly via the isotope exchange method. The radiotracer ([18F]GCPA)2 could be obtained within 0.5 h with the radiochemical purity over 98% and the molar activity of 10.64 ± 0.89 GBq μmol−1. It showed significant difference in cellular uptake between GGT-positive HCT116 cells and GGT-negative L929 cells (2.90 ± 0.12% vs. 1.44 ± 0.15% at 4 h, respectively). In vivo PET imaging showed that ([18F]GCPA)2 could quickly reach the maximum uptake in tumor (4.66 ± 0.79% ID g−1) within 5 min and the tumor-to-muscle uptake ratio was higher than 2.25 ± 0.08 within 30 min. Moreover, the maximum tumor uptake of the control group co-injected with the non-radioactive probe (GCPA)2 or pre-treated with the inhibitor GGsTop decreased to 3.29 ± 0.24% ID g−1 and 2.78 ± 0.32% ID g−1 at 10 min, respectively. In vitro and in vivo results demonstrate that ([18F]GCPA)2 is a potential PET probe for sensitively and specifically detecting the expression level of GGT.
研究领域:
医学影像学
·
肿瘤学
·
分子生物学
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DOI
PubMed
高影响因子
Theranostics
·
2021
·
IF 11.556
Fangyuan Zhou,Shikui Yang,Chao Zhao,Wangwang Liu,Xufeng Yao,Hui Yu,Xiaolian Sun,Yi Liu
Rationale: Precise treatment of tumors is attracting increasing attention. Molecular probes simultaneously demonstrating the diagnostic signal and pharmacological effect in response to tumor microenvironment are highly desired. γ-glutamyl transpeptidase (GGT) is a biomarker with significantly up-regulated expression in the tumor area. We developed a GGT responsive near-infrared (NIR) nanoassembly for tumor-specific fluorescence imaging-guided photothermal therapy. Methods: The GGT responsive NIR probe was constructed by conjugating GGT-specific substrate γ-glutamic acid (γ-Glu) with cyanine fluorophore (NRh-NH 2 ) via amide reaction. The resulting NRh-G spontaneously assembled into nanoparticles (NRh-G-NPs) around 50 nm. The NPs were characterized and the properties evaluated in the presence or absence of GGT. Subsequently, we studied fluorescence imaging and photothermal therapy of NRh-G-NPs in vitro and in vivo . Results: NRh-G-NPs, upon specific reaction with GGT, turned into NRh-NH 2 -NPs, showing a ~180-fold fluorescence enhancement and excellent photothermal effect recovery. NRh-G-NPs could selectively light up U87MG tumor cells while their fluorescence was weak in L02 human normal liver cells. The NPs also showed excellent tumor cell ablation upon laser irradiation. After intravenous injection into tumor-bearing mice, NRh-G-NPs could arrive in the tumor area and specifically light up the tumor. Following laser irradiation, the tumor could be completely erased with no tumor reoccurrence for up to 40 days. Conclusions: NRh-G-NPs were specifically responsive to GGT overexpressed in U87MG tumor cells and selectively lit up the tumor for imaging-guided therapy. Besides, the recovery of photothermal property in the tumor area could improve cancer therapy precision and decreased side effects in normal tissues.
研究领域:
医学影像学
·
肿瘤学
·
纳米医学
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产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2025
·
IF 3.8
Wenyu Gao,Guanying Shi,Lihua Zhang,Zhaogai Wang,Wei Zong
γ-glutamyl transpeptidase (GGT, E.C.2.3.2.2) has great applications in the food industry, but there is no commercially available bacterial GGT on the market. In this paper, response surface methodology was used to optimize the yield of Bacillus licheniformis GGT ( Bl GGT). The enzyme was extracted and used to catalyze the synthesis of volatile sulfur compounds (VSCs) in Toona sinensis (TS). The results showed that the optimal conditions for fermentation of GGT production were: lactose 8 g/L, tryptone: soybean meal (1:1) 15 g/L, MgSO 4 ·7 H 2 O 0.5 g/L, K 2 HPO 4 1 g/L, inoculum volume of 5 % v/v, initial pH 8.0, loading volume of 50 mL, and fermentation temperature of 37.0 ℃. Under this condition, the enzyme activity of GGT reached 918.38 ± 0.012 U/L, which was 19 times higher than the activity under the initial medium conditions. In addition, when the GGT was added to the precursor extract from TS, it was noteworthy that VSCs in TS were regained, with the total amount reaching 30.38 μg/mL. The optimization of the fermentation medium to increase the yield of Bl GGT may also provide valuable data for the large-scale production of GGT. The results of this study provide an effective pathway for the synthesis of unique VSCs in TS.
研究领域:
食品科学
·
微生物学
·
生物化学
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产品关联
DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2024
·
IF 5.7
Guanying Shi,Wenyu Gao,Zuobing Xiao,Le Zhang,Pengfei Jiang,Lili Zhao,Zhaogai Wang,Zhimin Xu
γ-Glutamyl transpeptidase (GGT) in Toona sinensis shoot (TS) was extracted and purified through salt precipitation, ion-exchange column, and gel filtration chromatography methods. The GGT had two peptides with M.W. 35 and 50 kDa. Its function in the formation of TS sulfur-containing volatiles (SCVs) was evaluated. The highest GGT hydrolytic activity was at pH 7.0 and 50 °C. Its activity was enhanced by Ca2+, Mg2+, K+, Na+, and Ba2+ and reduced by Cu2+, Fe3+, Mn2+, and Zn2+. The SCVs reached the highest level at GGT to TS sulfur-containing precursors extract 1:20. The aroma produced by GGT from the extract was similar to that of raw TS, especially for the cooked onion-like/TS-like flavor. These results revealed the characteristics and function of GGT in TS on sulfurous aroma formation. It could be applied to retrieve the production of sulfur-containing aroma from thermally preserved TS or similar vegetables, which lost GGT activity irreversibly during heat drying.
研究领域:
植物生物化学
·
食品科学
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2020
·
IF 4.192
Ya Gao,Haipu Li,Yang Song,Fenglin Zhang,Yi Lu,Fangyuan Peng,Zhaoguang Yang
In plants, glutathione (GSH) is crucial for the detoxification and tolerance of heavy metals. However, the change characteristics and decisive enzymes involved in GSH metabolism under heavy metal exposure are still unclear. Based on long-term exposure cultivation of spinach and monitoring of the change trends of enzyme activity and GSH contents in response to cadmium (Cd) stress, these issues were clarified. Spinach goes through three statuses in sequence in response to Cd stress, that is, perception status (PS), response status (RS), and new stable status. With the increase in the Cd concentration, the durations of the PS and RS and the time to reach the peaks in the roots were shorter. However, the durations of the PS and the time to reach the peaks in the leaves were longer. The enzyme activities changed significantly in response to diverse Cd stress in RS. γ-glutamyl transpeptidase was vital to the GSH content in roots. Glutathione synthase was important for the GSH content in leaves. The results of this study provide valuable information to find an efficient way to perform GSH adjustments to fulfill the goal of ensuring food safety.
研究领域:
植物生理学
·
环境毒理学
·
农业科学
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产品关联
DOI
PubMed
高影响因子
ACS Nano
·
2024
·
IF 15.8
Hanchen Shen,Lidong Du,Changhuo Xu,Bingzhe Wang,Qingqing Zhou,Ruquan Ye,Ryan T. K. Kwok,Jacky W. Y. Lam,Guichuan Xing,Jianwei Sun,Tzu-Ming Liu,Ben Zhong Tang
Activity-based detection of γ-Glutamyltranspeptidase (GGT) using near-infrared (NIR) fluorescent probes is a promising strategy for early cancer diagnosis. Although NIR pyridinium probes show high performance in biochemical analysis, the aggregation of both the probes and parental fluorochromes in biological environments is prone to result in a low signal-to-noise ratio (SBR), thus affecting their clinical applications. Here, we develop a GGT-activatable aggregate probe called OTBP-G for two-photon fluorescence imaging in various biological environments under 1040 nm excitation. By rationally tunning the hydrophilicity and donor–acceptor strength, we enable a synergistic effect between twisted intramolecular charge transfer and intersystem crossing processes and realize a perfect dark state for OTBP-G before activation. After the enzymatic reaction, the parental fluorochrome exhibits bright aggregation-induced emission peaking at 670 nm. The fluorochrome-to-probe transformation can induce 1000-fold fluorescence ON/OFF ratio, realizing in vitro GGT detection with an SBR > 900. Activation of OTBP-G occurs within 1 min in vivo, showing an SBR > 400 in mouse ear blood vessels. OTBP-G can further enable the early detection of pulmonary metastasis in breast cancer by topically spraying, outperforming the clinical standard hematoxylin and eosin staining. We anticipate that the in-depth study of OTBP-G can prompt the development of early cancer diagnosis and tumor-related physiological research. Moreover, this work highlights the crucial role of hydrophilicity and donor–acceptor strength in maximizing the ON/OFF ratio of the TICT probes and showcases the potential of OTBP as a versatile platform for activity-based sensing.
研究领域:
生物医学工程
·
肿瘤学
·
分子影像学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOCONJUGATE CHEMISTRY
·
2020
·
IF 4.031
Siqin Ye,Shijie Wang,Dingyao Gao,Ke Li,Qingzhu Liu,Bainian Feng,Ling Qiu,Jianguo Lin
γ-Glutamyltranspeptidase (GGT) is a cell -membrane-associated enzyme which has been recognized as a promising biomarker for the diagnosis of many malignant tumors. Herein, we rationally designed a fluorine-18 labeled small-molecule probe, [18F]γ-Glu-Cys(StBu)-PPG(CBT)-AmBF3 (18F-1G), by applying a biocompatible CBT-Cys condensation reaction and ingeniously decorating it with a GGT-recognizable substrate, γ-glutamate (γ-Glu), for enhancing PET imaging to detect GGT level of tumors in living nude mice. The probe had exceptional stability at physiological conditions, but could be efficiently cleaved by GGT, followed by a reduction-triggered self-assembly and formation of nanoparticles (NPs) progressively that could be directly observed by transmission electron microscopy (TEM). In in vitro cell experiments, 18F-1G showed GGT-targeted uptake contrast of 2.7-fold to that of 18F-1 for the detection of intracellular GGT level. Moreover, the higher uptake in GGT overexpressed HCT116 tumor cells (∼4-fold) compared to GGT-deficient L929 normal cells demonstrated that 18F-1G was also capable of distinguishing some tumor cells from normal cells. In vivo PET imaging revealed enhanced and durable radioactive signal in tumor regions after 18F-1G coinjecting with 1G, thus allowing real-time detection of endogenous GGT level with high sensitivity and noninvasive effect. We anticipated that our probe could serve as a new tool to investigate GGT-related diseases in the near future.
研究领域:
医学影像学
·
分子生物学
·
肿瘤学
✓ 已验证
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2016
·
IF 7.476
Xianfeng Hou,Fang Zeng,Shuizhu Wu
γ-Glutamyl transpeptidase (GGT) plays crucial roles in some physiological processes . Herein a turn-on fluorescent probe for γ-glutamyl transpeptidase (GGT) assay based on aggregation-induced-emission (AIE) effect and the enzyme-induced transformation of hydrophilicity to hydrophobicity has been developed by functionalizing tetraphenylethylene (TPE) derivative with two γ-glutamyl amide groups, which simultaneously work as recognition units and hydrophilic groups. When the γ-glutamyl amide groups are cleaved through GGT enzymatic reaction , the hydrophobic reaction product readily aggregate and correspondingly strong blue fluorescence can be observed, as a result of activated AIE process. By virtue of the probe's good solubility in totally aqueous solution, high sensitivity and excellent photostability, the probe can be employed to detect GGT level in human serum samples. Furthermore, the probe can be used for imaging endogenous GGT in living A2780 cells. Hence, the probe holds great promise for acting as a convenient one-step straightforward assay for GGT detection in diagnostic-related applications, and also it could provide a useful approach for conducting pathological analysis for diseases involving GGT.
研究领域:
生物化学
·
分析化学
·
分子生物学
·
医学诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
·
2020
·
IF 3.448
Zelong Li,Runtao Zhu,Yongqi Liu,Jiaqi Li,Haofeng Gao,Nan Hu
L-theanine, a unique amino acid in green tea with health benefits, can be enzymatically synthesized by γ-glutamyltranspeptidase (γ-GT; EC 2.3.2.2). Here, a salt-tolerant γ-glutamyltranspeptidase from a marine bacterium Bacillus amyloliquefaciens was expressed in Escherichia. coli BL21 (DE3) and was shown to be optimally active at 55 °C, pH 8.5 and alkali stable. A mutant, with higher transpeptidation activity, was obtained following two rounds of directed evolution using error-prone PCR and site-saturation mutagenesis. The mutation increased the ratio of transpeptidation to hydrolysis from 1.6 to 35.6. Additionally, Kinetic analysis exhibited 17.5% decrease of K m , 13.0-fold increase of K cat , and 16.3-fold increase of K cat / K m in mutant V319A/S437 G versus the wild-type. The 3-D modelling analysis revealed a tighter binding pocket in mutant V319A/S437 G. The frequency of hydrogen bond between donor substrate and two residues in the catalytic pocket (Gly437 and Thr375) was enhanced, which stabilized the ligand binding and thus improved the catalytic efficiency. The optimal conditions for the biocatalytic synthesis were determined as pH 10.0, 20 μg mL -1 Ba GT, 200 mM L-glutamine, 2 M ethylamine, and a reaction time of 5 h. The V319A/S437 G mutant was shown to increase the percentage yield of L-theanine from 58% to 83%. These results indicate the great potential of V319A/S437 G in L-theanine production after further study.
研究领域:
生物化学
·
生物技术
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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