Molecular Catalysis
·
2023
·
IF 4.6
Yangyang Sun,Zhuzhu Tang,Tingze Pan,Ke Zhang,Liuzhu Wang,Xingye Zhai,Yiran Jia,Tianqi Yuan,Yujia Qian,Hualei Wang,Dongzhi Wei,Shengli Yang
Catalytic promiscuity makes nitrilase-mediated unnatural reactivity a promising pathway in the production of primary amides . However, during the functional switch of nitrilase, a trade-off between the newly developed hydration activity and relative activity hinders the application of nitrilase in the production of amides. Our previous study showed that the mutant F193N (Mut0) of nitrilase NIT6803 from Synechocystis sp. PCC6803 improved the hydration activity as the amide ratio increased to 73.4%, whereas the relative activity was only 50.1% of that of the wild-type (WT). In this study, we succeeded in alleviating the trade-off of Mut0 through an evolutionary combination of the key identified sites 101, 192, and 201. Inside the active pocket, the co-evolution of residue Q192 was identified by hotspot prediction to correlate with N193, and Q192H exhibited an improved amide ratio of 76.9% with a relative activity of up to 72%. Outside the active pocket, the flexible loop surface residue G101 and homodimer “A” interface residue I201 were identified using sequence alignment-based mutations. Further evolutionary combination of the three key sites yielded the best triple mutant G101K/Q192H/I201M, which had the highest amide ratio of 98.5% and considerably recovered relative activity of 82.6% while retaining only 1.5% of the carboxylic acid ratio. The transition-state analogue sheds light on the basis of the almost identical switch of the reaction pathways, and our results expand the toolbox of nitrilase-catalysed promiscuity for amide formation.
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
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2025
·Vol. 41
·期 3
·1-11
·
IF 4.2
Liu Ming-Yu,Wang Yu-Yue,Li Han-Zhi,Luo Xue-Mei,Feng Jia-Xun,Zhao Shuai
Nitriles exhibit acute cytotoxicity to human and animal cells. Nitrilase is a green biocatalyst that can directly convert nitrile into nontoxic carboxylic acids and ammonia. However, the nitrilases capable of degrading 3-butenenitrile and 4-pentenenitrile derived from glucosinolate present in rapeseed meals are still limited. This study expressed the nit rilase 2 gene from bacterium Paraburkholderia g raminis ( PgNIT2 ) in Escherichia coli . The purified recombinant nitrilase rPgNIT2 showed specific activities of 5.94 U/mg and 6.66 U/mg when using 3-butenenitrile and 4-pentenenitrile as substrates at the optimal pH 7.0 and temperature 45 °C. The substrate binding pocket of PgNIT2 comprising 12 amino acids was required for rPgNIT2 activity. Remarkably, the specific activity of mutant enzyme A190I increased by 68.3% and 57.3%, respectively, against 3-butenenitrile and 4-pentenenitrile compared with the wild-type enzyme. Furthermore, the A190I showed improved thermostability and hydrolysis ability towards 3-butenenitrile and 4-pentenenitrile compared with rPgNIT2 but shared the same optimal pH and temperature. These results provided theoretical guidance for engineering of bacterial nitrilases used for rapeseed meal detoxification.
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
CHEMICAL PAPERS
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2017
·Vol. 71
·期 10
·1985-1992
·
IF 1.258
Fan Haiyang,Chen Lifeng,Sun Huihui,Wang Hualei,Liu Qinghai,Ren Yuhong,Wei Dongzhi
This study aimed at developing an efficient biotransformation process for phenylacetic acid production from phenylacetonitrile by using recombinant Escherichia coli M15 harboring a double mutant MG nitrilase (I113M/Y199G) from Burkholderia cenocepacia J2315. A yield of 2310 U/mL nitrilase was obtained by fermentation after the optimization of cultivation conditions, with a specific activity of 64 U/mg dcw. The MG nitrilase showed high substrate tolerance and completely hydrolyzed 100 mM phenylacetonitrile in 30 min under optimal conditions. To alleviate substrate inhibition, periodic or continuous batch-feeding of substrate was used during the biotransformation. Up to 164 g/L substrate was completely hydrolyzed in 9 h with continuous batch-feeding using resting cells, corresponding to 400 U/mL of nitrilase activity, and leading to production of 163.4 g/L phenylacetic acid. The hydrolysis process has potential application for phenylacetic acid production on a large scale.
研究领域:
生物化学
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生物工程
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酶学
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DOI
高影响因子
PROCESS BIOCHEMISTRY
·
2018
·
IF 2.616
Mujiao Zhang,Peiyuan Yao,Shanshan Yu,Tongcun Zhang,Qiaqing Wu,Dunming Zhu
An efficient method for the production of 4-cyanobenzoic acid from terephthalonitrile was developed by using a novel nitrilase from Pantoea sp. AS-PWVM4 (PaNIT). Under the optimized reaction conditions (pH 8.0 and 37 °C), up to 100 g/L substrate concentration was almost completely converted to 4-cyanobenzoic acid with wet whole cells loading of 5.0 wt% in 4 h and the isolated yield was 91%. The substrate concentration, yield and productivity (26.13 g/(L h)) are much higher than those in the previous studies. Furthermore, PaNIT has broad substrate specificity of catalyzing the hydrolysis of various aromatic nitriles and aliphatic nitriles. These results suggest that PaNIT is a valuable nitrilase with potential practical applications.
研究领域:
生物化学
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酶工程
·
有机合成
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DOI
高影响因子
Journal of Engineered Fibers and Fabrics
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2018
·
IF 0.678
Setara Begum,Jindan Wu,Carrington M. Takawira,Jiping Wang
Subtilisin treatment is feasible in benign conditions, and yields a clean, dynamic approach for Polyamide 6,6 (PA 6,6) modification. In this study, the water contact angle (WCA) method, capillary height and moisture regain were used measure hydrophilicity. To characterization the modification, the scanning electron microscope (SEM), amino group quantification and x-ray photoelectron spectrometry (XPS) were used. Fabric burst strength and hand properties were characterized. Optimum conditions for subtilisin treatment were found to be 55oC, neutral pH with 10% (owf) enzyme concentration for a fixed 90 min treatment period. X-ray photoelectron spectrometry (XPS) results showed that carboxyl groups were produced on the modified fiber surface. The modified fibers showed much improved hydrophilicity as indicated by reduced wicking time, improved capillary height and moisture regain percentage. The creation of carboxylic acid group sand increased number of amino groups detected on the fabric surface may positively influence hydrophilicity. In comparison to harsh alkali modification, the enzyme treatment led to smaller weight loss. In general, enzymatic treatments- a more eco-friendly alternative to alkaline treatments, resulted in a significant improvement in surface properties of PA 6,6 fabrics.
研究领域:
纺织科学
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生物化学
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DOI
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2017
·
IF 2.892
Ya-Ping Xue,Hu-Jun Zhong,Shu-Ping Zou,Yu-Guo Zheng
Gabapentin has been widely used for the treatment of epilepsy and neuropathic pain in the world. In this work, an efficient and greener chemoenzymatic manufacturing process for gabapentin has been developed. The whole cells expressing engineered nitrilase were immobilized in diatomite cross-linked with polyethyleneimine and glutaraldehyde to improve their reusability and stability for the synthesis of gabapentin intermediate 1-cyanocyclohexaneacetic acid from 1-cyanocyclohexaneacetonitrile. The initial enzyme activity in the bioreactors at each batch biotransformation was maintained at a constant value by introducing fresh immobilized cells into the system to keep the high conversion (100%) and the same operation time (8 h) in each batch. The resulting 1-cyanocyclohexaneacetic acid was then converted to gabapentin efficiently by simple chemical steps. An E factor (exclude water) of 11.5 was calculated for the chemoenzymatic route relative to 55.7 for the mentioned chemical process. This new route dramatically improved process efficiency compared to the chemical process.
研究领域:
生物技术
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制药工程
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酶学
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DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2021
·
IF 6.953
Neng Xiong,Yan Dong,Dong Xie,Zi-qi Li,Ya-Ping Xue,Yu-Guo Zheng
In the chemical-biological synthesis route of gabapentin, immobilized Escherichia coli cells harboring nitrilase are used to catalyze the biotransformation of intermediate 1-cyanocyclohexaneacetonitile to 1-cyanocyclohexaneacetic acid. Herein, we present a novel cell immobilization method, which is based on cell adsorption using 75 g/L Escherichia coli cells and 6 g/L zeolite, cell crosslinking using 3 g/L polyethylenemine and biomimetic silicification using 18 g/L hydrolyzed tetramethylorthosilicate. The constructed “hybrid biomimetic silica particles (HBSPs)” with core-shell structure showed a specific activity of 147.2 ± 2.3 U/g, 82.6 ± 2.8% recovery of nitrilase activity and a half-life of 19.1 ± 1.9 h at 55 °C. 1-Cyanocyclohexaneacetonitrile (1.0 M) could be completely hydrolyzed by 50 g/L of HBSPs at pH 7.5, 35 °C in 4 h, providing 92.1 ± 3.2% yield of 1-cyanocyclohexaneacetic acid. In batch reactions, the HBSPs could be reused for 13 cycles and maintained 79.9 ± 4.1% residual activity after the 10th batch, providing an average product yield of 92.6% in the first 10 batches with a productivity of 619.3 g/L/day. In addition, multi-layer structures consisting of silica coating and polyethylenemine/glutaraldehyde crosslinking were constructed to enhance the mechanical strength of immobilized cells, and the effects of coating layers on the catalytic properties of immobilized cells was discussed.
研究领域:
生物化学
·
酶工程
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产品关联
DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
·
2023
·
IF 4.4
Neng Xiong,Zi-Qi Li,Rui Xu,Kai-Qi Guo,Yu-Xiao Liu,Ya-Ping Xue,Yu-Guo Zheng
Surface-crosslinked hydrogel particles encapsulating E. coli cells harboring nitrilase were constructed and used as a durable biocatalyst in the synthesis of gabapentin intermediate. E. coli cells (50 g/L) expressing nitrilase were encapsulated in 2% (w/v) alginate hydrogel, 0.5%(w/v) glutaraldehyde and 1%(w/v) polyethyleneimine were sequentially used as crosslinker. The constructed alginate particles with GA&PEI composite crosslinking had a similar catalytic capacity compared to equivalent free cells, which could completely hydrolyze 1.0 M substrate 1-cyanocyclohexaneacetonitrile within 4 h at pH 7.5 and 35 °C, and have a specific activity of 134.6 U/g, an optimum temperature at 55 °C and a corresponding half-life up to 19.1 ± 1.9 h. The particles showed excellent storage stability, reusability and mechanical strength, which retained 90.3% activity after 2-month storage at 4 °C, and 83.2% activity after 14-day storage at 35 °C. In batch reactions, 50 g/L particles could be reused for maximum 32 cycles, maintained 63.5% residual activity and 50.7% particle integrity, providing an average product yield of 94.6% in the first 30 batches and a productivity of 1467.03 g/L/day. Besides, the stability and catalytic activity of the particles were almost unaffected in several organic co-solvents and in purified water, showing their good industrial potential.
研究领域:
生物化学
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工业微生物学
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DOI
高影响因子
Green Synthesis and Catalysis
·
2024
·
IF 8.2
Wenlong Zheng,Liyuan Yang,Yan Meng,Lirong Yang,Jianping Wu
Nitrilases are enzymes capable of converting nitriles into carboxylic acids under mild conditions. However, wild type nitrilases typically exhibit poor catalytic efficiency towards sterically hindered substrates. This study investigates the role of distal amino acid residues in enhancing the catalytic efficiency of nitrilase for hydrolyzing sterically hindered substrates. Initially, consensus sequence analysis and structure-guided loop scanning strategies were employed to identify key distal residues. Subsequently, site-directed saturation mutagenesis and combinatorial mutations led to the generation of a mutant T171P/T173E/H181F/T225A/N146D, which exhibited a 9.5-fold increase in enzyme activity compared to the wild type when p -methoxybenzonitrile was used as a substrate. Moreover, the mutant also showed excellent catalytic performance with various sterically hindered substrates and in scaled-up reactions. Molecular dynamics simulations revealed that distal mutations can induce conformational changes through residue interaction networks, thereby optimizing the enzyme's substrate-binding pocket and enhancing its catalytic efficiency. This study offers a comprehensive investigation into the role of distal amino acid residues in modulating the catalytic efficiency of nitrilase towards sterically hindered substrates and provides insights into enzyme function modification via distal site mutations.
研究领域:
生物化学
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酶工程
·
计算生物学
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DOI
高影响因子
Journal of Computational and Theoretical Nanoscience
·
2016
·Vol. 13
·期 4
·2264-2268
·
IF 1.666
He Li,Chen Ying,Wu Xiaomei,Xu Yi
The magnetic Fe3O4/Alginate cell beads (FACB) were prepared by entrapping cells of E. coli BL21(DE3) pET21a-NIT (a recombinant nitrilase producing strain) and nano-Fe3O4 with alginate. The FACB can be applied as an immobilized biocatalyst
for the hydrolysis of 1-cyanocyclohexane acetonitrile to 1-cyanocyclohexane acetic acid, which is an important intermediate in the synthesis of Gabapentin. The optimal immobilization conditions were investigated in detail, including 3.0% (w/v) sodium alginate and 50 g/L Fe3O4.
The mechanical strength of FACB was improved significantly compared with the alginate cell beads without Fe3O4. Compared with free cell, FACB have better pH stability and thermo-stability. Moreover, FACB showed excellent operation stability could be reused for 35 batches
(420 h) and still remained 90% of its original activity.
研究领域:
生物化学
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制药工程
·
生物技术
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DOI
高影响因子
PROCESS BIOCHEMISTRY
·
2017
·
IF 2.497
Shu-Ping Zou,Ji-Wei Huang,Ya-Ping Xue,Yu-Guo Zheng
1-Cyanocyclohexaneacetic acid (1-CHAA) is an important intermediate in the preparation of gabapentin. Regioselective biotransformation of 1-cyanocyclohexaneacetonitile (1-CHAN) into 1-CHAA, mediated by the immobilization of recombinant microbial nitrilase, is a novel and promising strategy for its production. In this study, a recombinant E. coli expressing Acidovorax facilis nitrilase was flocculated with polyethylenemine, followed by cross-linking with glutaraldehyde to obtain cross-linked cell aggregates (CLCAs). Under the optimum preparation conditions, the activity recovery of CLCAs reached 68%. Characterization of the CLCAs showed that the optimum pH was not affected by cross-linking, but cross-linking resulted in slightly different optimum temperature. The half-life of CLCAs at 45 °C was 266.5 h, which is 11.6-times higher than that of the free cells. Preparative-scale biotransformation of 1-CHAN to 1-CHAA at high substrate loading (up to 1.0 M) was performed using CLCAs as the biocatalyst in a 2L stirred reactor, resulting in an conversion of 91%, with a productivity of 244.5 g L −1 d −1 . The CLCAs also exhibited a high operational stability and even after 30 cycles of reaction, it retained >80% of the initial conversion.
研究领域:
生物化学
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微生物学
·
制药工程
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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）