PROCESS BIOCHEMISTRY
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2017
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IF 2.497
Jinsha Huang,Wei Zhuang,Ce Wei,Liwen Mu,Jiahua Zhu,Yudan Zhu,Jinglan Wu,Yong Chen,Hanjie Ying
Orientation immobilization of enzymes has attracted intensive interest owing to the retainable specific activity and stability. Specially, glycoprotein immobilized onto Concanavalin A (Con A) modified carriers induces the orientation of the enzyme. However, the effects of the interface properties of carriers and enzymes are still not well understood yet. In this study, we synthesized the activated porous poly (styrene- divinylbenzene) resin carriers with 30 nm pore sizes and 72 m 2 g −1 specific surface areas and decorated with Con A. The resultant loading capacity of NP 1 on Con A modified carriers was as high as 4.02 mg g −1 wet support as a result of strong affinity between the enzyme and Con A decorated on carriers. It was found that the acid resistance, thermal stability, reusability and degradation efficiency of the immobilized enzyme on Con A modified porous carriers were significantly improved. The reduction of K m from 18.40 ± 0.55 mg mL −1 to 17.19 ± 0.51 mg mL −1 illustrated the improved substrate affinity of HA-GA-ConA-NP 1 . Moreover, Con A-affinity NP 1 exhibited the best operational stability that only 7% of its initial activity was lost even after 9 batches repeated reaction. This work demonstrates that surface property manipulation of porous carriers and its derivatives has great potential in efficient biocatalytic systems.
研究领域:
生物化学
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酶工程
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DOI
高影响因子
TALANTA
·
2019
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IF 4.916
Zuhai Bai,Yan Chen,Fei Li,Yunlei Zhou,Huanshun Yin,Shiyun Ai
Herein, a simple and selective electrochemical method was developed for sulfadimethoxine detection based on the triggered cleavage activity of nuclease P1 by the formation of aptamer and sulfadimethoxine conjugate. After probe DNA was immobilized on gold electrode surface, aptamer DNA labeled with biotin at its 5′-terminal was then captured on electrode surface through the hybridization reaction between probe DNA and aptamer DNA. The formed double-stranded DNA (dsDNA) can block the digestion activity of Nuclease P1 towards the single-stranded probe DNA. Then, the anti -dsDNA antibody was further modified on electrode surface based on the specific interaction between dsDNA and antibody. Due to the electrostatic repulsion effect and steric-hindrance effect, a weak electrochemical signal was obtained at this electrode. However, in the presence of sulfadimethoxine, it can interact with aptamer DNA, and then the formation of dsDNA can be blocked. As a result, the probe DNA at its single-strand state can be digested by Nuclease P1, which leads to the failure of the immobilization of anti -dsDNA antibody. At this state, a strong electrochemical signal was obtained. Based on the change of the electrochemical signal, sulfadimethoxine can be detected with linear range of 0.1–500 nmol/L. The detection limit was 0.038 nmol/L. The developed method possesses high detection selectivity and sensitivity. The applicability of this method was also proved by detecting sulfadimethoxine in veterinary drug and milk with satisfactory results.
研究领域:
生物化学
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分析化学
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兽医学
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DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2021
·Vol. 193
·期 11
·3753-3764
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IF 2.926
Chen Xiaochun,Huang Xiaoquan,Tang Yiwen,Zhang Lei
Nuclease P1 (NP1) can hydrolyze nucleic acids into four 5′-mononucleotides, which are widely used in the pharmaceutical and food industries. In this paper, an aqueous two-phase system (ATPS) was developed to purify NP1 from Penicillium citrinum . Polyethylene glycol (PEG) and nucleotides salts were studied to form ATPSs, among which PEG3000/disodium guanosine monophosphate (GMPNa 2 ) was researched, including the phase composition and pH. Using 14% (w/w) PEG3000 and 20% (w/w) GMPNa 2 ATPS at pH 5.0, the best recovery and purification factor, 82.4% and 3.59, were obtained. The recovery of NP1 was 98.3% by the separation of ultrafiltration from the PEG-rich phase. The recycling use of GMPNa 2 was also studied, and 95.1% of GMPNa 2 in the salt-rich phase was obtained with the addition of ethanol as the solvent. These results showed that the ATPS was effective for purification of NP1.
研究领域:
生物化学
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工业微生物学
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DOI
PubMed
高影响因子
COLLOIDS AND SURFACES B-BIOINTERFACES
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2016
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IF 3.902
Wei Zhuang,Linjiao He,Jiahua Zhu,Jianwei Zheng,Xiaojing Liu,Yihui Dong,Jinglan Wu,Jingwei Zhou,Yong Chen,Hanjie Ying
The use of graphene oxide (GO) nanosheets for functional enzyme support has attracted intensive interest owing to their unique planar structure and intriguing physical and chemical properties. However, the detailed effects of the interface properties of GO and its functionalized derivatives on active biomolecules are not well understood. We immobilize nuclease P 1 , a common industrial nucleic acid production enzyme, on pristine and amino poly(ethylene glycol) (PEG-NH 2 ) modified GO nanosheets with interface property heterogeneity using two approaches, physical adsorption and chemical crosslinking. It is demonstrated that nuclease P 1 could be stable immobilized on the surface of pristine GO by physical adsorption and on the edge of modified GO nanosheets by chemical crosslinking. The resultant loading capacity of nuclease P 1 on pristine GO is as high as 6.45 mg/mg as a consequence of strong electrostatic and hydrophobic interactions between the enzyme and carrier. However, it is determined that the acid resistance, thermal stability, reusability and degradation efficiency of the immobilized enzyme on PEG-NH 2 -modified GO are obviously improved compared to those of the enzyme immobilized on pristine GO. The enhanced catalytic behavior demonstrates that GO and its derivatives have great potential in efficient biocatalytic systems.
研究领域:
生物化学
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纳米生物学
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酶工程
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
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2014
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IF 2.745
Bingbing Li,Yong Chen,Zhi Cao,Huanqing Niu,Dong Liu,Ying He,Xiaochun Chen,Jinglan Wu,Jingjing Xie,Wei Zhuang,Hanjie Ying
The reversible immobilization of crude nuclease P 1 fermented by Penicllium citrinum and used without further purification was performed via adsorption on a weak base anion resin activated by polyethylenimine (PEI). The immobilization conditions, including PEI concentration, the amount of support, the immobilization time, and the reusability of the PEI-activated resin were investigated. The results have shown that the PEI-activated resin has the capability of selectively adsorbing nuclease P 1 . Subsequently, the functional properties of the immobilized nuclease P 1 were studied and compared to those of the free enzyme. The apparent K m value for immobilized nuclease P 1 on the activated resin (15.31 g L −1 ) was about 4.41-fold higher than that of the free enzyme (3.47 g L −1 ), and the apparent V max value of the immobilized enzyme (530 U g −1 ) was about 3.9-fold less than that of the free enzyme (2082 U mL −1 ). The optimum temperature was observed to be 70 °C, 15 °C higher than that of the free enzyme. The optimum pH was the same for both free and immobilized nuclease P 1 (pH 5.0). The apparent activation energies ( E a ) of the free and immobilized nuclease P 1 were 163.09 kJ mol −1 and 156.32 kJ mol −1 , respectively, implying that the catalytic efficiency of the immobilized enzyme was restricted by mass-transfer rather than kinetic limitations.
研究领域:
生物化学
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酶工程
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）