Characterization of a thermostable phytase from Bacillus licheniformis WHU and further stabilization of the enzyme through disulfide bond engineering

Zhang, Zhijie; Yang, Jian; Xie, Peijuan; Gao, Yanping; Bai, Jun; Zhang, Chun; Liu, Li; Wang, Qin; Gao, Xiaowei

doi:10.1016/j.enzmictec.2020.109679

Cited by 21 publications

(11 citation statements)

References 35 publications

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“…The number of hydrogen, ionic and disulfide bond interactions is believed to have a strong correlation with thermostability (Zhang 2007;Liao, et al, 2013;Fei et al, 2013;Hesampour et al, 2015;Kumar et al, 2015;Tan et al, 2016;Han et al, 2018;Li et al, 2019;Zhang et al, 2020). However, here we show that, for these enzymes, this is not true.…”

Section: Quantification and Analysis Of Interactions Of The Structura...contrasting

confidence: 51%

“…The association between the structure and the biochemical properties of enzymes can provide relevant information about factors that affect stability and catalytic activity. Several methods have been used for this purpose, including comparative sequence, structure analysis, molecular dynamics studies, or even the evaluation of random or rational mutations on enzyme activity (Zhang 2007;Liao, et al, 2013;Fei et al, 2013;Hesampour et al, 2015;Kumar et al, 2015;Tan et al, 2016;Han et al, 2018;Li et al, 2019;Zhang et al, 2020;Acquistapace et al, 2022). In this study, we report a comparative approach between sequences, threedimensional structures and biochemical characteristics of phytases already characterized and described in the literature, adopting factors such as: hydrogen, ionic and Van der Waals (VdW) interactions and disulfide bonds.…”

Section: Introductionmentioning

confidence: 99%

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Sequence diversity and catalytic properties of phytases

Pires

Polêto

Vidigal

et al. 2022

RSD

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Phytic acid is an antinutritional factor in cereal feeds, and the use of phytases increases the bioavailability of nutrients bound to this molecule. However, the application of these enzymes depends on their thermal stability and activity at acidic pH. Therefore, in this study we created a database composed of 59 phytase sequences and analyzed the interactions that stabilize their structures in order to understand whether they contribute to the biochemical properties observed. The sequences were aligned and grouped at 30 % similarity, generating 5 clusters, which highlights the high variability among them. A comparative structural analysis of the cluster 3 phytases revealed conserved catalytic domains, as well as eight cysteine residues along the primary sequence, forming disulfide bonds for stabilizing the three-dimensional structure. However, the number of Van der Waals, ionic, and hydrogen interactions, and disulfide bonds was not determinant for the biochemical characteristics presented by these enzymes. The phytase KM873028, from cluster 3, was selected for characterization studies, but its expression in Pichia pastoris generated a protein with properties distinct from those derived from the same sequence expressed in a prokaryotic system. It is likely that the differences observed are associated with the location of the interactions in the structures, non-conserved amino acid residues found around the catalytic site, and post-translational modifications inherent to the expression systems. These possibilities highlight the relevance of strategic choices related to enzyme expression aiming at its production and industrial feasibility.

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Section: Quantification and Analysis Of Interactions Of The Structura...contrasting

confidence: 51%

Section: Introductionmentioning

confidence: 99%

Sequence diversity and catalytic properties of phytases

Pires

Polêto

Vidigal

et al. 2022

RSD

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“…Analysis of the HypZn amino acid sequence shows that it contains only two cysteine residues, and PredictProtein online analysis ( https://www.predictprotein.org ) shows that the cause of the poor thermal stability of HypZn may be the protein structure, which lacks disulfide bonds. The disulfide bond between or within protein subunits was considered to be an important factor contributing to thermal stability [ 32 ].…”

Section: Resultsmentioning

confidence: 99%

Cloning, expression and characterization of metalloproteinase HypZn from Aspergillus niger

Song

Wang

et al. 2021

PLoS ONE

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A predicted metalloproteinase gene, HypZn, was cloned from Aspergillus niger CGMCC 3.7193 and expressed in Pichia pastoris GS115, and the physicochemical characteristics of recombinant HypZn were investigated after separation and purification. The results showed that the specific activity of the purified HypZn reached 1859.2 U/mg, and the optimum temperature and pH value of HypZn were 35°C and 7.0, respectively. HypZn remained stable both at 40°C and at pH values between 5.0 and 8.0. The preferred substrate of HypZn was soybean protein isolates, and the Km and Vmax values were 21.5 μmol/mL and 4926.6 μmol/(mL∙min), respectively. HypZn was activated by Co2+ and Zn2+ and inhibited by Cu2+ and Fe2+. The degree of soybean protein isolate hydrolysis reached 14.7%, and the hydrolysates were of uniform molecular weight. HypZn could tolerate 5000 mM NaCl and completely lost its activity after 30 min at 50°C. The enzymological characterizations indicated that HypZn has great application potential in the food industry, especially in fermented food processing.

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Section: ■ Introductionmentioning

confidence: 99%

“…Improving the thermostability of proteins through rational design has always been a hot topic in the field of computational biology and protein engineering. Engineering disulfide bonds into proteins is a feasible rational design strategy for modifying protein folding, which has been applied to improve the thermostabilities of various enzymes, such as α-amylase, cellulase, amadoriase, phytase, glucanase, and xylanase . Disulfide bonds can reduce the entropy of a protein in the unfolded state, thereby slowing the reversible unfolding process of the enzyme and stabilizing its structure. , In recent years, several software packages have been employed to analyze and predict potential residue pairs for constructing disulfide bonds, including Disulfide by Design 2.0 (DbD2) and the DiANNA webserver, which provided partial data support and improved screening efficiency.…”

Section: Introductionmentioning

confidence: 99%

Cysteine Engineering of an Endo-polygalacturonase from Talaromyces leycettanus JCM 12802 to Improve Its Thermostability

Wang

Meng

et al. 2021

J. Agric. Food Chem.

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Thermostable enzymes have many advantages for industrial applications. Therefore, in this study, computer-aided design technology was used to improve the thermostability of a highly active endo-polygalacturonase from Talaromyces leycettanus JCM12802 at an optimal temperature of 70 °C. The melting temperature and specific activity of the obtained mutant T316C/G344C were increased by 10 °C and 36.5%, respectively, compared with the wild-type enzyme. The crystal structure of the T316C/G344C mutant showed no formation of a disulfide bond between the introduced cysteines, indicating a different mechanism than the conventional mechanism underlying improved enzyme thermostability. The cysteine substitutions directly formed a new alkyl hydrophobic interaction and caused conformational changes in the side chains of the adjacent residues Asn315 and Thr343, which in turn caused a local reconstruction of hydrogen bonds. This method greatly improved the thermostability of the enzyme without affecting its activity; thus, our findings are of great significance for both theoretical research and practical applications.

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Characterization of a thermostable phytase from Bacillus licheniformis WHU and further stabilization of the enzyme through disulfide bond engineering

Cited by 21 publications

References 35 publications

Sequence diversity and catalytic properties of phytases

Sequence diversity and catalytic properties of phytases

Cloning, expression and characterization of metalloproteinase HypZn from Aspergillus niger

Cysteine Engineering of an Endo-polygalacturonase from Talaromyces leycettanus JCM 12802 to Improve Its Thermostability

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