Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design

Chang, Meihui; Chu, Xiaoyu; JinZhi, Lv; Li, Qingbin; Tian, Jian; Wu, Ningfeng

doi:10.1371/journal.pone.0165006

Cited by 25 publications

(14 citation statements)

References 38 publications

(41 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Hence, the ECSM approach compares very favorably with site‐directed mutagenesis and regional truncation methods, which increased the catalytic efficiency only about two folds (Table S7).…”

Section: Resultsmentioning

confidence: 99%

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

et al. 2019

View full text Add to dashboard Cite

Pullulanases are well‐known debranching enzymes hydrolyzing α‐1,6‐glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis‐involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the ‘evolutionary coupling saturation mutagenesis’ (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.

show abstract

Section: Resultsmentioning

confidence: 99%

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

et al. 2019

View full text Add to dashboard Cite

show abstract

“…There are several works focused on improvement of the themostability of pullulanase derived from bacteria (Chen et al, 2015;Li et al, 2015;Chang et al, 2016;Wang et al, 2016). However, it is still lacking the understanding on the factor affecting the themostability of HvLD at the atomic level.…”

Section: Introductionmentioning

confidence: 99%

Understanding Thermostability Factors of Barley Limit Dextrinase by Molecular Dynamics Simulations

Dong²,

et al. 2020

Front. Mol. Biosci.

View full text Add to dashboard Cite

Limit dextrinase (LD) is the only endogenous starch-debranching enzyme in barley (Hordeum vulgare, Hv), which is the key factor affecting the production of a high degree of fermentation. Free LD will lose its activity in the mashing process at high temperature in beer production. However, there remains a lack of understanding on the factor affecting the themostability of HvLD at the atomic level. In this work, the molecular dynamics simulations were carried out for HvLD to explore the key factors affecting the thermal stability of LD. The higher value of root mean square deviation (RMSD), radius of gyration (R g), and surface accessibility (SASA) suggests the instability of HvLD at high temperatures. Intra-protein hydrogen bonds and hydrogen bonds between protein and water decrease at high temperature. Long-lived hydrogen bonds, salt bridges, and hydrophobic contacts are lost at high temperature. The salt bridge interaction analysis suggests that these salt bridges are important for the thermostability of HvLD, including E568-R875, D317-R378, D803-R884, D457-R214, D468-R395, D456-R452, D399-R471, and D541-R542. Root mean square fluctuation (RMSF) analysis identified the thermal-sensitive regions of HvLD, which will facilitate enzyme engineering of HvLD for enhanced themostability.

show abstract

“…Naturally, the DSB formation between cysteine residues occurs during the folding of proteins in their secretory pathway catalyzed by members of the protein disulfide isomerase family [36]. The introduction of extra DSBs through protein engineering without affecting the catalytic efficacy of enzymes was considered to improve the thermostability of mesophilic enzymes [22,37]. Therefore, in this study, we used rational DSB engineering to improve the thermostability of the EGLII from P. verruculosum .…”

Section: Discussionmentioning

confidence: 99%

Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability

Bashirova

Pramanik

Volkov

et al. 2019

IJMS

View full text Add to dashboard Cite

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.

show abstract

Improving the Thermostability of Acidic Pullulanase from Bacillus naganoensis by Rational Design

Cited by 25 publications

References 38 publications

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

Evolutionary coupling saturation mutagenesis: Coevolution‐guided identification of distant sites influencing Bacillus naganoensis pullulanase activity

Understanding Thermostability Factors of Barley Limit Dextrinase by Molecular Dynamics Simulations

Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability

Contact Info

Product

Resources

About