BIOTECHNOLOGY LETTERS
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2021
·Vol. 43
·期 8
·1617-1624
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IF 2.461
Wang Liuzhu,Jiang Shuiqin,Sun Yangyang,Yang Zeyu,Chen Zhi,Wang Hualei,Wei Dongzhi
Objectives Catalytic promiscuity, or the ability to catalyze a secondary reaction, provides new opportunities for industrial biocatalysis by expanding the range of biocatalytic reactions. Some nitrilases converting nitriles to amides, referred to as the secondary activity, show great potential for amides production. And our goal was exploiting the amide-forming potential of nitrilases. Results In this study, we characterized and altered the secondary activity of nitrilase from Acidovorax facilis 72 W (Nit72W) towards different substrates. We increased the secondary activity of Nit72W towards 2-cyanopyridine by 196-fold and created activity toward benzonitrile and p-nitrophenylacetonitrile by modifying the active pocket. Surprisingly, the best mutant, W188M, completely converted 250 mM 2-cyanopyridine to more than 98% 2-picolinamide in 12 h with a specific activity of 90 U/mg and showed potential for industrial applications. Conclusions Nit72W was modified to increase its secondary activity for the amides production, especially 2-picolinamide.
研究领域:
生物化学
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工业生物技术
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DOI
PubMed
高影响因子
LETTERS IN APPLIED MICROBIOLOGY
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2022
·Vol. 76
·期 2
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IF 2.813
Wang Li,Huang Jiao,Cui Baocheng,Mu Er,Liang Changhai
Our previous study identified a novel nitrile hydratase (NHase) with remarkable biotransformation activity toward adipamide during the production of 5-cyanovaleramide (5-CVAM), an important intermediate of herbicide and chemical raw material. Nevertheless, free NHase will face harsh conditions if they are applied directly in industrial processes. In this study, we, therefore, prepared Fe3(PO4)2 hybrid nanoflowers for NHase immobilization based on the protein–inorganic hybrid self-assembly by establishing a novel and facile method. The results showed that the NHase@Fe3(PO4)2 nanoflowers had significantly enhanced tolerance to the temperature ranging from 40°C to 60°C when compared with free NHase. The catalytic activity of NHase@Fe3(PO4)2 nanoflowers remained high in extreme pH environments such as weak acid (pH 5) and strong alkali (pH 10) environments. In addition, the storage stability and reusability of encapsulated NHase were also superior to that of free NHase. NHase@Fe3(PO4)2 nanoflowers had a notable feature of high substrate tolerance. We found NHase@Fe3(PO4)2 nanoflowers still had 65% activity as the adiponitrile concentration increased up to 200 mmol L−1, whereas free NHase almost lost their catalytic activity when the adiponitrile concentration was just 100 mmol L−1. All of these results clearly demonstrated that ferrous phosphate nanocrystals might offer a novel strategy for 5-CVAM production with nanobiocatalytic 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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DOI
PubMed
高影响因子
BIOORGANIC CHEMISTRY
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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.
研究领域:
生物化学
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分子生物学
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酶工程
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DOI
PubMed
高影响因子
Catalysis Science & Technology
·
2023
·Vol. 13
·期 20
·5880-5891
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IF 5
Jinling Xu,Haisheng Zhou,Jiaqi Xu,Ziyuan Wang,Zhonglang Yu,Zhe Wang,Hongyu Zhang,Haoran Yu,Jianping Wu,Lirong Yang
Nitrile hydratase (NHase), an excellent biocatalyst, has been widely used for the production of amides, but the exothermic hydration reaction leads to its rapid inactivation, hindering its industrial applications, which requires the thermostability of NHase to be enhanced. In this study, employing NHase from Bordetella petrii DSM 12804 (NHAB) as the object, a computational strategy using synergetic energy and correlated configuration for redesigning enzymes (SECURE) was proposed to prune the reasonable mutant library and assemble effective single mutations, thus maximizing the thermostability of NHAB. Among the mutants, the best variant, A6M/B4M, combined six mutations in its α-subunit (S30T, A71D, A74D, A78R, S81T, and A133P) and four mutations in its β-subunit (L25F, G27Y, N59P, and A173N), showing an increase in Tm by 13.2 °C, an 866.0-fold prolonged half-life at 50 °C, and an 11.2% increase in activity. Then, the catalytic efficiency dramatically increased to 249.5 g L−1 acrylamide in 5 batches compared with that of 166.5 g L−1 by the wild type in 3 batches. Finally, the synergistic effect on the mutant represented by an overall change in structure and newly formed intermolecular interactions through dynamic simulations accounted for the enhanced thermostability. Thus, SECURE was demonstrated to be practical for the redesign of multimeric NHase, which also provided guidance for computational thermostability engineering of other industrial biocatalysts.
研究领域:
生物化学
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生物技术
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计算生物学
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酶学
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DOI
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
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2016
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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.
研究领域:
生物化学
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材料科学
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酶工程
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产品关联
DOI
高影响因子
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