WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2024
·Vol. 41
·期 1
·1-12
·
IF 4
Li Yang-Nan,Zhu Chen-Yuan,Xu Chen-Lu,Yu Shen,Huan Tong,Zhang Ye-Wang
A heparinase III ( Ns Hep-III) from Niabella sp . was identified, cloned, and expressed as soluble form in E. coli BL21 (DE3). With heparin as substrate, the maximum activity of Ns Hep-III of 90.0 U·mg − 1 was achieved at pH 7.1 Tris-HCl (containing 15 mM Mg 2+ ) and 30 o C. The half-life of Ns Hep-III was determined to be 5 h at 30 o C. The interactions between Ns Hep-III and substrates were studied by molecular docking. The combination of Ns Hep-III and a heparinase I from Bacteroides eggthii ( Be Hep-I) was employed to cleave heparin. Analysis of the enzymatic products of Ns Hep-III by SAX-HPLC showed seven different modified disaccharides, indicating that Ns Hep-III has a wide range of substrate specificity. The results of GPC analysis demonstrated that the average molecular weight of the product of heparin cleavage by the combination of Ns Hep-III/ Be Hep-I was reduced to 3969 Da, which accounted for 90% of all the components, and complied with the requirements of the European Pharmacopoeia. Ns Hep-III has notable activity and efficiency in cleaving heparin, which is potentially useful for the industrial production of low molecular weight heparin.
研究领域:
生物化学
·
药学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2016
·
IF 4.758
Dan Yan,Yu He,Yili Ge,Gongwu Song
A novel fluorescence “turn on-off” method for sensitive and selective detection of heparin and heparinase I was developed based on near infrared Polyethyleneimine Capped Ag 2 S Quantum Dots (PEI-Ag 2 S QDs). The fluorescence “turn-on” arose from aggregation-induced emission enhancement (AIEE) characteristics of PEI-Ag 2 SQDs induced by electrostatic interaction between PEI-Ag 2 SQDs and heparin. Heparinase I, which can specifically hydrolyze heparin into short fragments would de-aggregate PEI-Ag 2 S QDs/heparin system and lead to fluorescence “turn-off”. Under optimal conditions described, the fluorescence enhancement at 685 nm was linearly related to the concentration of heparin and heparinase I in the range of 0.069–2.275 μM and 0–103 ng mL −1 with the relative correlation coefficient of R 2 = 0.997 and 0.99 and detection limit as low as 6 nM and 0.5 ng mL −1 , respectively. The activity of the heparinase I was further investigated using steady-state kinetic for the reaction. In ddition, the proposed method was successfully utilized in serum samples, which presaged more opportunities for application in related biochemical and biomedical researches.
研究领域:
生物化学
·
医学检测
·
纳米技术
✓ 已验证
产品关联
DOI
高影响因子
CARBOHYDRATE POLYMERS
·
2019
·
IF 6.044
Xiqi Ma,Xiaojuan Wang,Qi Chen,Hua He,Yixin Sun,Hui Liu,Yanan Wang,Jianbo Qu,Fang Huang
A novel fluorescent heparanase assay based on hybrid nano-assembly of gold nanocluster and glycosaminoglycan is developed. The nanoparticle probes are fabricated through the co-assembly of positively charged gold nanoclusters with negatively charged heparin molecules, which is accompanied by a dramatic size change and a 2.5-fold fluorescence enhancement. It is demonstrated that the fluorescence enhancement is due to denser aggregation of Au-thiolate complexes in the hybrid nanoparticle and the fluctuation of the fluorescence intensity is an indicator of the variation in assembly efficiency. Experiments in solution and in cell lysis media showed that the heparanase could turn-off the fluorescence with a high selectivity, which could be utilized for the assessment of heparanase activity and the metastatic potentials of different tumour cells. This assay technique is low cost, easy to prepare, and showing good performance. The co-assembly strategy has potential to be transferable to construct other functional nanomaterial.
研究领域:
生物化学
·
纳米医学
·
癌症研究
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2019
·
IF 4.784
Rui Jiang,Song Zhao,Langkun Chen,Maoyu Zhao,Wenjing Qi,Wensheng Fu,Lianzhe Hu,Yan Zhang
In this research, a new DSA (Distyryl-anthracene) derivative with four carboxyl groups was designed and synthesized. This molecule exhibits aggregation-induced emission property (AIE). With the AIE character, a convenient and sensitive fluorescent probe for the detection of protamine , heparin and heparinase has been developed. The protamine can directly induce the aggregation of probe, which caused by electrostatic attraction. In this way, the turn-on detection of protamine is achieved, and the detection limit is as low as 30 ng mL −1 . When heparin appears, the probes will be redisperse in solution, which causes a decrease in fluorescence intensity. Besides, this method also shows good selectivity and sensitivity, and the linear range of heparin is from 0.08 to 8 μg mL −1 with detection limit of 37 ng mL −1 . After hydrolyzing heparin by heparinase, the probes rebind with protamine and the fluorescence enhance. The fluorescence enhancement was linearly related to the concentration of heparinase in the range of 0.02–2.0 μg mL −1 and detection limit as low as 143.7 ng mL −1 . In addition, the results exhibited that the recovery percentage of heparinase in bovine samples reached to 96–101%.
研究领域:
生物化学
·
分析化学
✓ 已验证
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2019
·
IF 6.35
Shao-Bin He,Quan-Quan Zhuang,Liu Yang,Mei-Ying Lin,Ye Kuang,Hua-Ping Peng,Hao-Hua Deng,Xing-Hua Xia,Wei Chen
A visual assay for the detection of heparinase was developed on the basis of a ternary system of Hg2+–heparin–osmium nanoparticles (OsNPs). First, heparin-capped OsNPs (heparin–OsNPs) were synthesized by a facile reduction method using heparin as the protecting/stabilizing agent. The oxidase-like activity of heparin–OsNPs, however, turned out to be low, which somewhat limits their application. We discovered that Hg2+ can significantly/specifically boost the oxidase-like activity of heparin–OsNPs via electrostatic interaction. The oxidase-like activity of heparin–OsNPs toward the oxidation of the substrate, 3,3′,5,5′-tetramethylbenzidine, by dissolved O2 was found to increase by 76-fold in the presence of Hg2+. More significantly, heparin in heparin–OsNPs could be specifically hydrolyzed into small fragments in the presence of heparinase, which resulted in the weakening of the oxidase-like activity of Hg2+/heparin–OsNPs. On the basis of these findings, a linear response of the sensor for heparinase was obtained in the range 20–1000 μg/L with a low detection limit (15 μg/L), which is comparable to those of other reported sensors. Further, the colorimetric sensor was employed for the detection of heparinase in human serum samples with satisfactory results. We speculate that combining such surface modification of the osmium nanozyme with a sensing element could be an interesting direction for promoting nanozyme research in medical diagnosis.
研究领域:
生物化学
·
医学诊断
·
纳米生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Marine Drugs
·
2021
·Vol. 19
·期 3
·162
·
IF 5.118
Xiang Shi,Ruowei Guan,Lutan Zhou,Zhichuang Zuo,Xuelin Tao,Pin Wang,Yanrong Zhou,Ronghua Yin,Longyan Zhao,Na Gao,Jinhua Zhao
Unique fucosylated glycosaminoglycans (FG) have attracted increasing attention for various bioactivities. However, the precise structures of FGs usually vary in a species-specific manner. In this study, HfFG was isolated fromHolothuria floridanaand purified by anion exchange chromatography with the yield of ~0.9%. HfFG was composed of GlcA, GalNAc and Fuc, its molecular weight was 47.3 kDa, and the -OSO3−/-COO−molar ratio was 3.756. HfFG was depolymerized by a partial deacetylation–deaminative cleavage method to obtain the low-molecular-weight HfFG (dHfFG). Three oligosaccharide fragments (Fr-1, Fr-2, Fr-3) with different molecular weights were isolated from the dHfFG, and their structures were revealed by 1D and 2D NMR spectroscopy. HfFG should be composed of repeating trisaccharide units -{(L-FucS-α1,3-)d-GlcA-β1,3-d-GalNAc4S6S-β1,4-}-, in which sulfated fucose (FucS) includes Fuc2S4S, Fuc3S4Sand Fuc4Sresidues linked to O-3 of GlcA in a ratio of 45:35:20. Furthermore, the heparanase inhibitory activities of native HfFG and oligosaccharide fragments (Fr-1, Fr-2, Fr-3) were evaluated. The native HfFG and its oligosaccharides exhibited heparanase inhibitory activities, and the activities increased with the increase of molecular weight. Additionally, structural characteristics such as sulfation patterns, the terminal structure of oligosaccharides and the presence of fucosyl branches may be important factors affecting heparanase inhibiting activity.Keywords:Holothuria floridana;fucosylated glycosaminoglycans;oligosaccharide;chemical structure;heparanase
研究领域:
海洋生物学
·
生物化学
·
药理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
·
2023
·
IF 2.9
Hua-Ping Zhou,Ding-Ran Wang,Chen-Lu Xu,Ye-Wang Zhang
Heparinase I (EC 4.2.2.7), is an enzyme that cleaves heparin, showing great potential for eco-friendly production of low molecular weight heparin (LMWH). However, owing to its poor catalytic activity and thermal stability, the industrial application of heparinase I has been severely hindered. To improve the catalytic activity, we proposed to engineer both the substrate and Ca2+ binding domains of heparinase I. Several heparinases I from different organisms were selected for multiple sequence alignment and molecular docking to screen the key residues in the binding domain. Nine single-point mutations were selected to enhance the catalytic activity of heparinase I. Among them, T250D was the most highly active one, whereas mutations around Ca2+ binding domain yielded two active mutants. Mutant D152S/R244K/T250D with significantly increased catalytic activity was obtained by combined mutation. The catalytic efficiency of the mutant was 118,875.8 min−1·µM−1, which was improved 5.26 times. Molecular modeling revealed that the improved activity and stability of the mutants were probably attributed to the formation of new hydrogen bonds. The highly active mutant had great potential applications in industry and the strategy could be used to improve the performance of other enzymes.
研究领域:
生物化学
·
酶工程
✓ 已验证
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2017
·
IF 3.671
Shuqin Xu,Meiling Qiu,Xuanyue Zhang,Jinghua Chen
Heparinase I (Hep I) can efficiently depolymerize heparin and heparin sulfate to oligosaccharides or unsaturated disaccharides, which resulted in loss of physiological function such as blood coagulation. In order to realize the immobilization of Hep I on chitin carriers, we cloned Hep I with the chitin binding domain (ChBD) as a chitin-affinity tag, and the Small Ubiquitin-like MOdifier (SUMO) linker as a solvation enhancer in different fusion sequence. DNA and protein gels suggested that 4 kinds of recombinants were successfully constructed and expressed in Escherichia coli ( E. coli ). And the triple functional heparinases isolated from cell lysate could be efficiently purified by chitin beads. After optimizing fermentation conditions, it gave the specific enzyme activities of 1.88 ± 0.11, 3.69 ± 0.45, 3.44 ± 0.38, and 2.73 ± 0.29 IU/mg total proteins for ChBD-Hep I, ChBD-SUMO-Hep I, SUMO-ChBD-Hep I, and ChBD-Hep I-SUMO, respectively, with unfractionated heparin as substrate. The optimal reaction temperature and pH were determined to be 30 °C and 7.0 for all the fusion enzymes. ChBD-SUMO-Hep I exhibited the maximum half-life (48 min) at 30 °C and best thermo-stability under 15–50 °C. All the fusion enzymes showed broad pH-stability in the range of 5.4–9.0.
研究领域:
生物化学
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Biomaterials Science & Engineering
·
2020
·
IF 4.152
Zhihua Wang,Yingxin Xu,Guangyu Wu,Tiantian Zuo,Jun Zhang,Jie Yang,Yifan Yang,Tianxu Fang,Qi Shen
Tumor microenvironment (TME), with complex composition, plays a vital role in the occurrence, development, and metastasis of tumors. TME becomes an important obstacle to the accessibility of nanotherapy, thus indicating the need to improve the functional design to overcome this challenge. In this study, we generate an intelligent nano-drug-delivery system (DOX@PssP-Hh NPs) with dual environmental response, which involves heparanase (HPSE) in TME and glutathione (GSH) in tumor cells. The nanosystem consists of a nanoskeleton formed by self-assembly of mPEG-ss-PEI and α-CD (PssP), chemotherapy drug doxorubicin (DOX) for enhancing antitumor efficacy, together with hyaluronidase (HAase), which is designed to degrade extracellular matrix to increase drug penetration, and an outer shell of heparin. Through the process of “responsive disintegration–remodeling tumor microenvironment–enhancing drug penetration–inducing oxidative stress”, the semi-rotaxaneself-assembled nanomicelles were constructed to achieve the progressive function. DOX@PssP-Hh NPs with the size of 81.85 ± 1.85 nm exhibited satisfactory cytotoxicity (IC50 = 0.80 ± 0.33 μg/mL). With the disulfide bond-mediated GSH depletion and DOX-mediated reactive oxygen species (ROS) production, treatment with DOX@PssP-Hh NPs prominently reduced glutathione peroxidase 4 (GPX4) level and would lead to enhanced oxidative stresses. Hyaluronic acid (HA), collagen I, and α-smooth muscle actin (α-SMA) were significantly reduced for TME remodulation. Moreover, the antitumor effect in vivo implied that DOX@PssP-Hh NPs could inhibit tumor growth effectively and reduce tumor interstitial fluid pressure (IFP) evidently. In conclusion, DOX@PssP-Hh NPs improved the penetration of drugs and exhibited enhanced antitumor efficacy.
研究领域:
药剂学
·
肿瘤学
·
生物材料学
✓ 已验证
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2024
·
IF 5.7
Chen-Lu Xu,Chen-Yuan Zhu,Yang-Nan Li,Jian Gao,Ye-Wang Zhang
A novel heparinase III from Pedobacter schmidteae (PsHep-III) with high activity and good stability was successfully cloned, expressed, and characterized. PsHep-III displayed the highest specific activity ever reported of 192.8 U mg–1 using heparin as the substrate. It was stable at 25 °C with a half-life of 323 h in an aqueous solution. PsHep-III was employed for the depolymerization of heparin, and the enzymatic hydrolyzed products were analyzed with gel permeation chromatography and high-performance liquid chromatography. PsHep-III can break glycosidic bonds in heparin like →4]GlcNAc/GlcNAc6S/GlcNS/GlcNS6S/GlcN/GlcN6S(1 → 4)ΔUA/ΔUA2S[1 → and efficiently digest heparin into seven disaccharides including N-acetylated, N-sulfated, and N-unsubstituted modification, with molecular masses of 503, 605, 563, 563, 665, 360, and 563 Da, respectively. These results indicated that PsHep-III with broad substrate specificity could be combined with heparinase I to overcome the low selectivity at the N-acetylated modification binding sites of heparinase I. This work will contribute to the application of PsHep-III for characterizing heparin and producing low-molecular-weight heparin effectively.
研究领域:
生物化学
·
分子生物学
·
制药工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
PLoS One
·
2020
·Vol. 15
·期 10
·e0240920
·
IF 2.74
Li-Wei Gao,Hong-Tao Zhu,Cai-Yun Liu,Zhi-Xiang Lv,Xiao-Man Fan,Ye-Wang Zhang
As one of the most extensively studied glycosaminoglycan lyases, heparinase I has been used in producing low or ultra-low molecular weight heparin. Its’ important applications are to neutralize the heparin in human blood and analyze heparin structure in the clinic. However, the low productivity and activity of the enzyme have greatly hindered its applications. In this study, a novel Hep-I from Bacteroides cellulosilyticus (BcHep-I) was successfully cloned and heterologously expressed in E . coli BL21 (DE3) as a soluble protein. The molecular mass and isoelectric point (pI) of the enzyme are 44.42 kDa and 9.02, respectively. And the characterization of BcHep-I after purified with Ni-NTA affinity chromatography suggested that it is a mesophilic enzyme. BcHep-I can be activated by 1 mM Ca 2+ , Mg 2+ , and Mn 2+ , while severely inhibited by Zn 2+ , Co 2+ , and EDTA. The specific activity of the enzyme was 738.3 U·mg -1 which is the highest activity ever reported. The K m and V max were calculated as 0.17 mg·mL -1 and 740.58 U·mg -1 , respectively. Besides, the half-life of 300 min at 30°C showed BcHep-I has practical applications. Homology modeling and substrate docking revealed that Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 were mainly involved in the substrate binding of Hep-I, and 11 hydrogen bonds were formed between heparin and the enzyme. These results indicated that BcHep-I with high activity has great potential applications in the industrial production of heparin, especially in the clinic to neutralize heparin.
研究领域:
生物化学
·
分子生物学
·
药理学
✓ 已验证
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2015
·
IF 6.409
Meiding Yang,Jian Chen,Huipeng Zhou,Wenying Li,Yan Wang,Juanmin Li,Cuiyun Zhang,Chuibei Zhou,Cong Yu
A benzoperylene probe excimer emission in an aqueous buffer solution is observed for the first time, and a novel ratiometric fluorescence method based on the probe excimer emission for the sensitive detection of heparin and heparinase is demonstrated. A negatively charged benzoperylene derivative, 6-(benzo[ghi]perylene-1,2-dicarboxylic imide-yl)hexanoic acid (BPDI), was employed. A polycation , poly(diallyldimethylammonium) chloride (poly-DDA), could induce aggregation of BPDI through noncovalent interactions. A decrease of BPDI monomer emission and a simultaneous increase of BPDI excimer emission were observed. Upon the addition of heparin, the strong binding between heparin and poly-DDA caused release of BPDI monomer molecules, and an excimer–monomer emission signal transition was detected. However, after the enzymatic hydrolysis of heparin by heparinase, heparin was hydrolyzed into small fragments, which weakened the competitive binding of heparin to poly-DDA. Poly-DDA induced aggregation of BPDI, and a monomer–excimer emission signal transition was detected. Our assay is simple, rapid, inexpensive, sensitive and selective, which could facilitate the heparin and heparinase related biochemical and biomedical research.
研究领域:
分析化学
·
生物化学
·
生物医学工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）