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

Wang, Sheng; Meng, Kun; Su, Xiaoyun; Hakulinen, Nina; Wang, Yaru; Zhang, Jie; Luo, Huiying; Yao, Bin; Huang, Huoqing; Tu, Tao

doi:10.1021/acs.jafc.1c01618

Cited by 9 publications

(4 citation statements)

References 42 publications

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“…In rational design, the introduction of disulfide bonds has been successfully used in the engineering of various enzymes to improve their stability ( Navone et al, 2021 ; Wang et al, 2021 ). Tanghe et al (2017) obtained the stability improved ScLPMO10C, a cellulose-active LPMO from Streptomyces coelicolor , by introducing disulfide bonds into the protein, proving that this strategy is applicable to LPMOs.…”

Section: Discussionmentioning

confidence: 99%

Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

Zhou

et al. 2022

Front. Bioeng. Biotechnol.

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Lytic polysaccharide monooxygenases (LPMOs) oxidatively break down the glycosidic bonds of crystalline polysaccharides, significantly improving the saccharification efficiency of recalcitrant biomass, and have broad application prospects in industry. To meet the needs of industrial applications, enzyme engineering is needed to improve the catalytic performance of LPMOs such as enzyme activity and stability. In this study, we engineered the chitin-active CjLPMO10A from Cellvibrio japonicus through a rational disulfide bonds design. Compared with the wild-type, the variant M1 (N78C/H116C) exhibited a 3-fold increase in half-life at 60°C, a 3.5°C higher T5015, and a 7°C rise in the apparent Tm. Furthermore, the resistance of M1 to chemical denaturation was significantly improved. Most importantly, the introduction of the disulfide bond improved the thermal and chemical stability of the enzyme without causing damage to catalytic activity, and M1 showed 1.5 times the specific activity of the wild-type. Our study shows that the stability and activity of LPMOs could be improved simultaneously by selecting suitable engineering sites reasonably, thereby improving the industrial adaptability of the enzymes, which is of great significance for applications.

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Section: Discussionmentioning

confidence: 99%

Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

Zhou

et al. 2022

Front. Bioeng. Biotechnol.

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“…Hydrogen bonds and hydrophobic interactions play important roles in maintaining the stability of the enzyme . The number of hydrogen bonds in xylanases is positively correlated with the enzyme thermal stability. , Increasing the hydrophobic interactions formed by hydrophobic residues significantly reduces the conformational entropy of the protein, resulting in a more compact protein structure and thus improving the enzyme thermal stability . Compared with wild-type XynA, Mut1, and Mut2 showed lower RMSD, R g , and SASA values, indicating that the interactions introduced by the combinational mutations reduced the flexibility of amino acid residues in regions such as loop2, contributing to a more compressed xylanase structure and improving the conformational stability of the enzyme (Figure ).…”

Section: Discussionmentioning

confidence: 99%

Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from Streptomyces rameus L2001 by Rigidifying Flexible Regions in Loop Regions of the N-Terminus

Wu,

Zhang,

Dong

et al. 2023

J. Agric. Food Chem.

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The GH11 xylanase XynA from Streptomyces rameus L2001 has favorable hydrolytic properties. However, its poor thermal stability hinders its widespread application in industry. In this study, mutants Mut1 and Mut2 were constructed by rationally combining the mutations 11 YHDGYF 16 , 23 AP 24 / 23 SP 24 , and 32 GP 33 . The residual enzyme activity of these combinational mutants was more than 85% when incubated at 80 and 90 °C for 12 h, and thus are the most thermotolerant xylanases known to date. The reduced flexibility of the N-terminus, increased overall rigidity, as well as the surface net charge of Mut1 and Mut2 may be partially responsible for the improved thermal stability. In addition, the specific activity and catalytic efficiency of Mut1 and Mut2 were improved compared with those of wild-type XynA. The broader catalytic cleft and enhanced flexibility of the "thumb" of Mut1 and Mut2 may be partially responsible for the improved specific activity and catalytic efficiency.

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“…Furthermore, the co-evolutionary patterns of the residue pairs should also be favorable for the engineering of disulfide bonds. To verify this hypothesis, we used the thermostability modification data derived from Wang et al [81] as examples to test the potential relationship between the co-evolution and thermostability of disulfide residue pairs. A Python framework named EVcoulpings proposed by Thomas A Hopf et al [82] was employed.…”

Section: Discussionmentioning

confidence: 99%

“…Ev-coupling score and thermostability function of SS bond from Endo-polygalacturonase Talaromyces leycettanus JCM 12802[76].…”

mentioning

confidence: 99%

ThermoLink: Bridging Disulfide Bond and Enzyme Thermostability through Database Construction and Machine Learning Prediction

Xu,

Ye,

Xin

et al. 2023

Preprint

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Disulfide bonds (SS bonds), covalently formed by sulfur atoms in cysteine residues, play a crucial role in protein folding and structure stability. Due to their significance, artificial disulfide bonds are often introduced to enhance the thermostability of proteins. Although an increasing number of tools can assist with this task, significant amounts of time and resources were often wasted due to inadequate consideration. To enhance the accuracy and efficiency of designing disulfide bonds for protein thermostability improvement, we first collected disulfide-bond data with protein thermostability data from extensive literature sources. Then, various sequence- and structure-based features were extracted, and machine learning models were constructed to predict whether a disulfide bond could improve protein thermostability. Among all models, the neighborhood context model using the Adaboost-DT algorithm performs the best, and the AUC-ROC score and accuracy are 0.773 and 0.714, respectively. Alongside this, we also found that AlphaFold2 exhibits a high superiority in predicting disulfide bonds, and the coevolutionary relationship between residue pairs, to some extent, could also guide artificial disulfide-bond design. The SS-bond data has been integrated into an online server, named ThermoLink, available at guolab.mpu.edu.mo/thermoLink.

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

Cited by 9 publications

References 42 publications

Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from Streptomyces rameus L2001 by Rigidifying Flexible Regions in Loop Regions of the N-Terminus

ThermoLink: Bridging Disulfide Bond and Enzyme Thermostability through Database Construction and Machine Learning Prediction

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