WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
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2024
·Vol. 41
·期 1
·1-12
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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
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药学
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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
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医学检测
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纳米技术
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DOI
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
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酶工程
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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
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分子生物学
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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
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分子生物学
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药理学
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DOI
PubMed
高影响因子
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
·
2020
·
IF 1.415
Cai-Yun Liu,Wen-Bin Su,Li-Bin Guo,Ye-Wang Zhang
Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg−1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL−1 and 647.93 U·mg−1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K−1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability.
研究领域:
生物化学
·
酶学
·
药学
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产品关联
DOI
PubMed
高影响因子
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