B-factor-saturation mutagenesis as a strategy to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus

Ge, Lin; Li, Dongdong; Wu, Tao; Zhao, Linguo; Ding, Gang; Wang, Zhenzhong; Xiao, Wei

doi:10.1016/j.jbiotec.2018.03.013

Cited by 25 publications

(5 citation statements)

References 34 publications

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“…As BlaR-CTD protein did not contain free cysteine and disulfide bonds, disulfide bond were introduced in the flexible region of the protein to improve the stability by using the software Disulfide by Design 2 (http://cptweb.cpt.wayne.edu/DbD2/). The sites where disulfide bonds could be inserted were based on the predicted energy and B-Factors [11]. The ΣB-factor, which is related to the protein stability, indicates the smearing of atomic electron densities regarding to their equilibrium positions on account of thermal motion and positional disorder [24].…”

Section: Methodsmentioning

confidence: 99%

Analysis of the stability and affinity of BlaR-CTD protein to β-lactam antibiotics based on docking and mutagenesis studies

et al. 2019

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Owing to the thermal instability and low affinity of BlaR-CTD to some β-lactams, the receptor assay based on BlaR-CTD is limited in the detection of abundant variety of drugs and the result is often unstable. In this study, the three-dimensional structure of BlaR-CTD from Bacillus licheniformis ATCC14580 was constructed by homologous modeling based on the crystal structure of BlaR-CTD from B. licheniformis 749/I, and the binding sites of this protein to 40 β-lactams were also obtained by molecular docking. To improve the stability and affinity of the protein, 23 mutant proteins were designed based on docking and homologous alignment results as well as by inserting disulfide bond and building the salt bridge. The mutation was rationality evaluated by SIFT and PloyPhen2 software. The heterologous expressed and purified mutant proteins were then subjected to the activity and stability assay. It was shown that among all mutant proteins, I188K/S19C/G24C, A138E/R50C/Q147C and S190Y/E183C/I188K respectively exhibited a higher affinity to 33, 22 and 21 β-lactams than the wild-type protein, while I188K/S19C/G24C exhibited the best stability. This may due to that the conformation of the active site in mutant protein I188K/S19C/G24C changed, and the random coli in the surface of protein activity increased. Our study suggests a possible structure-function relationship on the stability and affinity of BlaR-CTD, which provides new insights into protein rational design study and lays a solid foundation for establishing the receptor-based screening assay for the detection of β-lactam residues. Electronic supplementary material The online version of this article (10.1186/s13036-019-0157-4) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Analysis of the stability and affinity of BlaR-CTD protein to β-lactam antibiotics based on docking and mutagenesis studies

et al. 2019

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“…Recently, multiple strategies have been explored to improve the stability of proteins, including rigidification of flexible sites, the introduction of disulfide bonds or salt bridges, consensus mutation, and so forth (Yu and Huang, 2014 ). Particularly, the B-factor values assisting engineering strategies have been developed (Parthasarathy and Murthy 2000 , Reetz et al., 2006 , Sun et al., 2019 ) and applied to increase the half-life of many enzymes such as 5-hydroxymethylfurfural oxidase (Wu et al., 2021 ), α- l -rhamnosidase (Ge et al., 2018 ), and arginine deiminase (Cai et al., 2018 ). Through a combination of protein truncation guided by the B-factor analysis and site-directed mutagenesis, the specific activity and half-life at 37°C of chondroitinase ABC I were significantly improved by 2.3-fold and 247-fold, respectively.…”

Section: Introductionmentioning

confidence: 99%

Improvement of the stability and catalytic efficiency of heparan sulfate N-sulfotransferase for preparing N-sulfated heparosan

Huang

et al. 2023

Journal of Industrial Microbiology and Biotechnology

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The chemo-enzymatic and enzymatic synthesis of heparan sulfate and heparin are considered as an attractive alternative to the extraction of heparin from animal tissues. Sulfation of the hydroxyl group at the position 2 of the deacetylated glucosamine is a prerequisite for subsequent enzymatic modifications. In this study, multiple strategies including truncation mutagenesis based on B-factor values, site-directed mutagenesis guided by multiple sequence alignment and structural analysis were performed to improve the stability and activity of human N-sulfotransferase. Eventually, a combined variant Mut02 (MBP-hNST-NΔ599-602/S637P/S741P/E839P/L842P/K779N/R782V) was successfully constructed, whose half-life at 37 °C and catalytic activity were increased by 105-fold and 1.35-fold, respectively. After efficient overexpression using the Escherichia coli expression system, the variant Mut02 was applied to N-sulfation of the chemically deacetylated heparosan. The N-sulfation content reached around 82.87% which was nearly 1.88-fold higher than that of the wild-type. The variant Mut02 with high stability and catalytic efficiency has great potential for heparin biomanufacturing.

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“…For example, α-L-rhamnosidase from Bifidobacterium breve can increase the yield of ginsenoside Rg1 in Panax ginseng [ 5 ], and also promote the biotransformation of rutin to isoquercitrin [ 6 ]. α-L-Rhamnosidases from Aspergillus terreus and Pichia pastoris Mut(S) strain, the current most preferred microorganisms for recombinant enzyme production owing to their efficient expression systems, can transform rutin into isoquercitrin [ 7 , 8 ]. There are many reports on α-L-rhamnosidase, but few about thermostable α-L-rhamnosidase.…”

Section: Introductionmentioning

confidence: 99%

Immobilization of GH78 α-L-Rhamnosidase from Thermotoga petrophilea with High-Temperature-Resistant Magnetic Particles Fe₃O₄-SiO₂-NH₂-Cellu-ZIF8 and Its Application in the Production of Prunin Form Naringin

Shi

Zhang

et al. 2021

J. Microbiol. Biotechnol.

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To efficiently recycle GH78 thermostable rhamnosidase (TpeRha) and easily separate it from the reaction mixture and furtherly improve the enzyme properties, the magnetic particle Fe 3 O 4 -SiO 2 -NH 2 -Cellu-ZIF8 (FSNcZ8) was prepared by modifying Fe 3 O 4 -NH 2 with tetraethyl silicate (TEOS), microcrystalline cellulose and zinc nitrate hexahydrate. FSNcZ8 displayed better magnetic stability and higher-temperature stability than unmodified Fe 3 O 4 -NH 2 (FN), and it was used to adsorb and immobilize TpeRha from Thermotoga petrophilea 13995. As for properties, FSNcZ8-TpeRha showed optimal reaction temperature and pH of 90°C and 5.0, while its highest activity approached 714 U/g. In addition, FSNcZ8-TpeRha had better higher-temperature stability than FN. After incubation at 80°C for 3 h, the residual enzyme activities of FSNcZ8-TpeRha, FN-TpeRha and free enzyme were 93.5%, 63.32%, and 62.77%, respectively. The organic solvent tolerance and the monosaccharides tolerance of FSNcZ8-TpeRha, compared with free TpeRha, were greatly improved. Using naringin (1 mmol/l) as the substrate, the optimal conversion conditions were as follows: FSNcZ8-TpeRha concentration was 6 U/ml; induction temperature was 80°C; the pH was 5.5; induction time was 30 min, and the yield of products was the same as free enzyme. After repeating the reaction 10 times, the conversion of naringin remained above 80%, showing great improvement of the catalytic efficiency and repeated utilization of the immobilized α-L-rhamnosidase.

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B-factor-saturation mutagenesis as a strategy to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus

Cited by 25 publications

References 34 publications

Analysis of the stability and affinity of BlaR-CTD protein to β-lactam antibiotics based on docking and mutagenesis studies

Analysis of the stability and affinity of BlaR-CTD protein to β-lactam antibiotics based on docking and mutagenesis studies

Improvement of the stability and catalytic efficiency of heparan sulfate N-sulfotransferase for preparing N-sulfated heparosan

Immobilization of GH78 α-L-Rhamnosidase from Thermotoga petrophilea with High-Temperature-Resistant Magnetic Particles Fe₃O₄-SiO₂-NH₂-Cellu-ZIF8 and Its Application in the Production of Prunin Form Naringin

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B-factor-saturation mutagenesis as a strategy to increase the thermostability of α-L-rhamnosidase from Aspergillus terreus

Cited by 25 publications

References 34 publications

Analysis of the stability and affinity of BlaR-CTD protein to β-lactam antibiotics based on docking and mutagenesis studies

Analysis of the stability and affinity of BlaR-CTD protein to β-lactam antibiotics based on docking and mutagenesis studies

Improvement of the stability and catalytic efficiency of heparan sulfate N-sulfotransferase for preparing N-sulfated heparosan

Immobilization of GH78 α-L-Rhamnosidase from Thermotoga petrophilea with High-Temperature-Resistant Magnetic Particles Fe3O4-SiO2-NH2-Cellu-ZIF8 and Its Application in the Production of Prunin Form Naringin

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Immobilization of GH78 α-L-Rhamnosidase from Thermotoga petrophilea with High-Temperature-Resistant Magnetic Particles Fe₃O₄-SiO₂-NH₂-Cellu-ZIF8 and Its Application in the Production of Prunin Form Naringin