Catalysts
·
2023
·Vol. 13
·期 2
·371
·
IF 3.9
Xue-Jiao Liu,Bao-Di Ma,Xiao-Mei Wu,Yi Xu
A recombinantE. coli, expressing nitrilase fromAcidovorax facilis72W with dual-site expression plasmid pRSFduet (E. colipRSF-AfNit2), was constructed. It showed higher soluble expression of nitrilase than that in the pET21a plasmid. The recombinant nitrilase can efficiently catalyze the hydrolysis of 3-cyanopyridine to nicotinic acid. The whole cells ofE. colipRSF-AfNit2 were immobilized by using sodium alginate/glutaraldehyde/polyethylene imine as the best immobilized reagents. The immobilized cells showed 95% activity recovery and excellent mechanical strength, with improved thermal stability and pH stability. They also retained 82% of initial activity after nearly two months of storage at 4 °C. A semi-continuous packed-bed bioreactor (sPBR) filled with the immobilized cells was studied for efficient production of nicotinic acid. After optimization, the highest space–time yield of 1576 g/(L·d) was obtained on 0.8 M substrate concentration at 2 mL/min of flow rate. The sPBR was repeatedly operated for 41 batches, keeping 100% conversion in the presence of 30 mM CaCl2. Finally, 95 g of nicotinic acid were obtained at 90% yield after separation and purification. The developed technology has potential application value.Keywords:nitrilase;cell immobilization;nicotinic acid;reaction optimization;semi-continuous packed-bed bioreactor
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Coatings
·
2021
·Vol. 11
·期 11
·1396
·
IF 2.881
Min Zhang,Dong Peng,Feifei Peng,Anwei Huang,Kaiqiang Song,Qingbing He,Changqing Yin,Jinsong Rao,Yuxin Zhang,Haitao Chen,Dalong Cong,Zhongsheng Li
The Al coatings were electrodeposited on the Cu substrate from AlCl3-EMIC ionic liquid (AlCl3:EMIC = 2:1 molar ratio) containing three cyanopyridine additives with different positions of the substituent group on the pyridine ring, which were 2-cyanopyridine, 3-cyanopyridine, and 4-cyanopyridine. The effects of cyanopyridine additives on the deposition potential, morphology, brightness, and corrosion properties of Al coatings were investigated. It was considered that the deposition potential of Al shifted to more negative overpotentials, the quality of Al coatings was promoted, and the corrosion property was improved by the cyanopyridine additives to a varying degree. Especially in the presence of 4-cyanopyridine, the flattest mirror bright Al coating was obtained, which had the smallest homogeneous nanocrystal grain size and strongest Al (200) crystallographic orientation. The average roughness Ra value was as low as 31 nm compared to that in the absence of cyanopyridine additives, which was 417 nm. Furthermore, the corrosion current density of the bright Al coating was three orders of magnitude lower than the rough Al coating, which resulted from the dense nanocrystal structure.Keywords:bright Al;electrodeposition;ionic liquids;cyanopyridine;additives
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BIOORGANIC CHEMISTRY
·
2023
·
IF 5.1
Yangyang Sun,Zhuzhu Tang,Yanjun Li,Yongkang Song,Hualei Wang,Dongzhi Wei,Shengli Yang
Hydration, a secondary activity mediated by nitrilase, is a promising new pathway for amide production. However, low hydration activity of nitrilase or trade-off between hydration and catalytic activity hinders its application in the production of amides. Here, natural C-terminal-truncated wild-type nitrilase, mined from a public database, obtained a high-hydration activity nitrilase as a novel evolutionary starting point for further protein engineering. The nitrilase Nit-74 from Spirosoma linguale DSM 74 was successfully obtained and exhibited the highest hydration activity level, performing 50.7 % nicotinamide formation and 87.6 % conversion to 2 mM substrate 3-cyanopyridine. Steric hindrance of the catalytic activity center and the N -terminus of the catalytic cysteine residue helped us identify three key residues: I166, W168, and T191. Saturation mutations resulted in three single mutants that further improved the hydration activity of N -heterocyclic nitriles. Among them, the mutant T191S performed 72.7 % nicotinamide formation, which was much higher than the previously reported highest level of 18.7 %. Additionally, mutants I166N and W168Y exhibited a 97.5 % 2-picolinamide ratio and 97.7 % isonicotinamide ratio without any loss of catalytic activity, which did not indicate a trade-off effect. Our results expand the screening and evolution library of promiscuous nitrilases with high hydration activity for amide formation.
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Molecular Catalysis
·
2021
·
IF 5.062
Yu-Ting Shang,Jiufu Qin,Jin-Song Gong,Zi-Kai Wang,Heng Li,Hui Li,Jin-Song Shi,Zheng-Hong Xu
A nicotinate dehydrogenase producing strain was isolated and identified to be Pseudomonas putida , which could bio-transform nicotinic acid into 6-hydroxynicotinic acid. The activity of nicotinate dehydrogenase towards nicotinic acid could reach up to 1.11 U/mL and the yield of 6-hydroxynicotinic acid accumulated to 176 g/L within 30 h with a space time yield of 1.36 g/(g•h). In order to further improve its catalytic potential, a mutant library was constructed through atmospheric and room temperature plasma (ARTP) and rapidly screened by a high-throughput screening strategy, which obtained a mutant strain mut-5 with activity of 2.68 U/mL. Via developing a nitrilase-nicotinate dehydrogenase enzymatic cascade system, 54.5 g/L 6-hydroxynicotinic acid could be obtained within 585 min with 3-cyanopyridine as the substrate, which would further reduce the production costs. The results indicated that the nicotinate dehydrogenase (separately or combining with nitrilase) could serve as a superior catalyst for 6-hydroxynicotinic acid production. To our knowledge, the nicotinate dehydrogenase activity reported in this study was the highest level in literature to date.
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ACS Applied Materials & Interfaces
·
2015
·
IF 6.723
Guojian Chen,Xiaochen Wang,Jing Li,Wei Hou,Yu Zhou,Jun Wang
A family of nitrogen-enriched ultramicroporous carbon materials was prepared by direct carbonization of task-specifically designed molecular carbon precursors of cyanopyridinium-based crystalline dicationic salts (CISs). Varying the molecular structure of CISs, large surface area (918 m2 g–1), high N content (20.10 wt %), and narrow distributed ultramicropores (0.59 nm) can be simultaneously achieved on the sample PCN-14 derived from methyl-linked 4-cyanopyridinium D[4-CNPyMe]Tf2N. It therefore exhibited exceptional performance in greenhouse gas CO2 capture, i.e., simultaneously possessing (1) high CO2 adsorption uptakes: 5.33 mmol g–1 at 273 K, and 3.68 mmol g–1 at 298 K (both at 1.0 bar); (2) unprecedented selectivity of CO2 versus N2: 156; and (3) a high adsorption ratio of CO2 to N2: 148 (at 1.0 bar). This is the first time such a high selectivity and adsorption ratio over carbon materials has been achieved, which is among the highest values over solid adsorbents.
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JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
·
2013
·
IF 2.823
Xiaolin Pei,Lirong Yang,Gang Xu,Qiuyan Wang,Jianping Wu
Microbial nitrile hydratases (NHases) are important industrial catalysts to produce valuable amides. However, only some NHase genes have been reported and studied at the molecular level. In this study, we developed a genome mining method to discover Fe-type NHases from GenBank. The putative NHase gene from Pseudomonas putida F1 was cloned and functionally expressed in Escherichia coli BL21 (DE3) by assisting of a putative activator gene adjacent to β-subunit region. Three recombinant plasmids containing NHase gene or the activator gene were designed and constructed. Maximal enzyme activity was obtained when the structural and activator genes were transcribed as one unit in plasmid pCDFDuet-1 at 18 °C. However, the expressed product did not show any NHase activity when the downstream activator gene was ignored, and the product completely existed in insoluble inclusion body. The activator gene might be involved in protein folding of the α- and β-subunits of NHase. In addition, the Fe-type NHase exhibited broad substrate specificity. The enzyme can efficiently hydrate aromatic nitriles, such as 3-cyanopyridine, 4-cyanopyridine, and benzonitrile, asides from aliphatic nitriles preferentially. Therefore, the recombinant NHase shows potential applications in some amides preparation.
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WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2013
·Vol. 30
·期 1
·245-252
·
IF 1.262
Wang Hualei,Li Guinan,Li Mingyang,Wei Dongzhi,Wang Xuedong
In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli . The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V
max and K
m toward 3-cyanopyridine were 77.5 μmol min −1 mg −1 and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.
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BIOCHEMICAL ENGINEERING JOURNAL
·
2016
·
IF 2.463
Jing Gao,Hao Yu,Liya Zhou,Ying He,Li Ma,Yanjun Jiang
Tannic-acid-templated magnetic mesoporous silica nanoparticles (TA-MMSNs) were synthesized for the first time. The TA-MMSNs were monodisperse spherical particles with a diameter of around 250 nm and a magnetization saturation value of 35.26 emu/g. The specific surface area of TA-MMSNs was 423.4 m 2 /g, and the diameter and cumulative volume of the pores were 9.349 nm and 1.071 cm 3 /g, respectively. The TA-MMSNs were used to prepare immobilized NHase (CLNHAs@TA-MMSNs) by forming cross-linked nitrile hydratase aggregates (CLNHAs) in pores of TA-MMSNs using glutaraldehyde as a cross-linker. CLNHAs@TA-MMSNs and free NHase had the same optimum pH (pH 7), and the optimum temperature of CLNHAs@TA-MMSNs (40 °C) was higher than that of free NHase (30 °C). Compared with free NHase, CLNHAs@TA-MMSNs exhibited improved thermal, pH, mechanical and storage stability. Furthermore, CLNHAs@TA-MMSNs was applied in production of nicotinamide, and yield of nicotinamide could reach more than 98%. The tolerance of CLNHAs@TA-MMSNs to high concentration of substrate was better than that of free NHase, and yield of nicotinamide could still reach 29.74% after seven cycles of reaction. The kinetic parameters were investigated and the results indicated a lower substrate affinity and catalytic efficiency of CLNHAs@TA-MMSNs in comparison with free NHase. This work demonstrated that TA-MMSNs could be efficiently employed as supports for enzyme immobilization.
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PROCESS BIOCHEMISTRY
·
2017
·
IF 2.497
Hui Zhang,Mengya Li,Jie Li,Guangli Wang,Feng Li,Minghua Xiong
Nitrile hydratase (NHase) has attracted substantial attention for industrial applications to produce large-scale amides. Several NHases have been investigated for functional expression in Escherichia coli ( E. coli ). A Fe-type NHase was obtained from an acetamiprid-degrading bacterium, Pseudoxanthomonas sp. AAP-7 and functionally expressed in E. coli BL21 (DE3). No significant NHase activity was detected from the E. coli expressing either the NHase gene alone or NHase and P46K genes transcribed as one unit. Purified recombinant NHase, co-expressed with P46K on two separate plasmids, exhibited the maximal enzyme activity. Furthermore, a GST tag attached to the N-terminus of α subunit resulted in a slight increase in the solubility and stability of NHase compared with a His tag at the C-terminus of β subunit. When co-expressed with the chaperones GroEL-GroES, the yield of the soluble recombinant NHase was improved substantially, while a small decrease in NHase activity was observed. The putative activator P46K was strictly required for production of the recombinant NHase for full enzyme activity, although the chaperones GroEL-GroES appeared to assist NHase to fold properly. This study of the expression of a fully active Fe-type NHase would provide another example to enhance our understanding of NHase biosynthesis.
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WATER RESEARCH
·
2025
·
IF 12.4
Yanping Wang,Longfang Ren,Xinqing Han,Huaqing Liu,Bing Shi,Taotao Qiang
The engineering design of uranium adsorbents encounters great challenge in terms of adsorption selectivity, adsorption capacity and antibacterial properties. Based on the designability of ionic liquids and good self-floating property of down fibers, we developed a novel cyanopyridine ionic liquid (IL-AN) and introduced it into the structure of down fibers by covalent binding (IDF-AN). Then the cyanogroup in IDF-AN was amidoximated to obtained IDF-AO with π-π conjugated structure, finally Ag NPs was in-situ loaded to construct an adsorbent IDF-AO/Ag with enhanced photothermal conversion and antibacterial properties. The rigid planar structure of pyridine ring and the flexible chelating arm of amidoxime group in IDF-AO/Ag make it exhibit distinguished selective adsorption for O = U = O . The K d (U/V) is as high as (2.97±0.13) × 10 5 mL/g, which is significantly higher than other adsorbents. Simultaneously, IDF-AO/Ag, just like camouflaged sargassum, can float on water surface to be exposed under solar irradiation for a long time. In this way, the adsorption capacity of IDF-AO/Ag under light condition (589.0 mg/g) is increased by 51.2 % than under dark conditions (389.78 mg/g) with the help of photothermal effects. Additionally, the water evaporation rate is improved to 2.78 kg m -2 h -1 under 1 solar irradiation. After 5 adsorption-desorption cycles, the adsorption capacity maintains 87.7 % of initial adsorption. A 30-day natural seawater experiment in Bohai Sea (121.11 °E, 40.83 °N) shows high adsorption capacity (8.45 mg/g) and excellent strength. The self-developed adsorbent has potential application in real seawater and this groundbreaking study provides a new concept for the engineering design of uranium adsorbents by synergistic strategy of bionic design and specific functional modification.
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