Effect and mechanism analysis of different linkers on efficient catalysis of subunit-fused nitrile hydratase

Guo, Junling; Cheng, Zhongyi; Berdychowska, Julia; Zhu, Xiaonan; Wang, Lingling; Pepłowski, Łukasz; Zhou, Zhemin

doi:10.1016/j.ijbiomac.2021.03.103

Cited by 18 publications

(9 citation statements)

References 50 publications

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“…Such creation of hydrophobic cores was observed during analysis of mesophilic and thermophilic proteins sequences (Haney et al 1999;Saelensminde et al 2009). During the process of MD simulations, when residue is mutated into hydrophobic amino acid, the hydrophobic core can be formed, such as in neurotensin receptor (Lee et al 2015), enzymes like trehalose synthase (Chen et al 2021), and nitrile hydratase (Cheng et al 2020;Guo et al 2021). The semi-rational design approach established in the present study sheds light on the future engineering of other industrial attractive SPases, such as the one from Bifidobacterium adolescentis.…”

Section: Discussionmentioning

confidence: 93%

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

Xia

Yang

et al. 2021

Appl Microbiol Biotechnol

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Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure–function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. Key points • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants

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Section: Discussionmentioning

confidence: 93%

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

Xia

Yang

et al. 2021

Appl Microbiol Biotechnol

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“…The cultivation of E. coli BL21 (DE3) cells and the process of NHase expression have been described in our previous study [16] . AKTA purifier (GE Healthcare UK Ltd., Amersham, UK) was used for enzyme purification.…”

Section: Methodsmentioning

confidence: 99%

“…The reduction of 3cyanopyridine in each reaction mixture was measured and the reduction ratio was calculated. [16]…”

Section: Analysis Of Thermal Stability and Product Tolerancementioning

confidence: 99%

Tailoring the Hinge Residue at the Substrate Access Tunnel Entrance Improves the Catalytic Performance of Industrialized Nitrile Hydratase Toward 3‐Cyanopyridine

et al. 2022

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Nicotinamide is a high-value chemical that has been widely applied in various aspects of daily life. Nitrile hydratase (NHase) is capable of carrying out biotransformation of nitriles to amides and has shown great potential in the green synthesis of nicotinamide. The high-molecular mass NHase (H-NHase) from Rhodococcus rhodochrous J1 has been applied in the industrialscale production of acrylamide; however, its activity is not ideal towards aromatic 3-cyanopyridines, resulting in its substrate preference towards aliphatic nitriles. In this study, a hinge residue at the entrance of the substrate access tunnel of H-NHase was identified through substrate access tunnel calculations, site-saturation mutagenesis, molecular docking and molecular dynamics simulations. The corresponding mutant βW48Y showed a 5.9-fold higher specific activity towards 3cyanopyridine than that of its parental enzyme, with better thermostability, proving itself to be a competitive candidate for the industrial production of nicotinamide.

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“…Subunit fusion of NHase via a linker had an important influence on NHase stability. Guo et al used three different types of linker to construct subunit-fused NHase variants and all of the variants exhibited higher stability than the wild-type, especially that were made using an α-helical linker …”

Section: Nhase Biochemical Propertiesmentioning

confidence: 99%

“…Guo et al used three different types of linker to construct subunit-fused NHase variants and all of the variants exhibited higher stability than the wild-type, especially that were made using an α-helical linker. 95 The activity of purified NHase is susceptible to the toxicity of substrate and product, and therefore, bacterial cells containing NHase are often selected to be used in the industrial amides production. Rhodococcus are the most popular industrial strains source for amides synthesis, because of their higher NHase activity and superior product tolerance.…”

Section: ■ Nhase Expressionmentioning

confidence: 99%

Nitrile Hydratases: From Industrial Application to Acetamiprid and Thiacloprid Degradation

Sun

Zhou

Jiang

et al. 2021

J. Agric. Food Chem.

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The widespread application of neonicotinoid insecticides (NEOs) in agriculture causes a series of environmental and ecological problems. Microbial remediation is a popular approach to relieve these negative impacts, but the associated molecular mechanisms are rarely explored. Nitrile hydratase (NHase), an enzyme commonly used in industry for amide production, was discovered to be responsible for the degradation of acetamiprid (ACE) and thiacloprid (THI) by microbes. Since then, research into NHases in NEO degradation has attracted increasing attention. In this review, microbial degradation of ACE and THI is briefly described. We then focus on NHase evolution, gene composition, maturation mechanisms, expression, and biochemical properties with regard to application of NHases in NEO degradation for bioremediation.

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Effect and mechanism analysis of different linkers on efficient catalysis of subunit-fused nitrile hydratase

Cited by 18 publications

References 50 publications

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

Tailoring the Hinge Residue at the Substrate Access Tunnel Entrance Improves the Catalytic Performance of Industrialized Nitrile Hydratase Toward 3‐Cyanopyridine

Nitrile Hydratases: From Industrial Application to Acetamiprid and Thiacloprid Degradation

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