Improving the specific activity and thermo-stability of alkaline pectate lyase from Bacillus subtilis 168 for bioscouring

Wang, Xiaowen; Lu, Zhenghui; Xu, Ting; Selvaraj, Jonathan Nimal; Li, Yang; Zhang, Guimin

doi:10.1016/j.bej.2017.11.001

Cited by 27 publications

(23 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Escherichia coli Rosetta (DE3)/pET28a-pel3 for the expression of pectate lyase PEL3 was constructed in our previous work (Wang et al, 2018). Glycine with purity 98.5% and polygalacturonic acid (PGA) with purity 90% were purchased from Sigma-Aldrich (St. Louis, MO, United States).…”

Section: Methodsmentioning

confidence: 99%

“…For the synthesis of the protein-inorganic hybrid nanoflowers with improved catalytic performance, five different metal ions including Cu 2+ , Ca 2+ , Mn 2+ , Co 2+ , and Zn 2+ were screened. First, we expressed and purified PEL3 according to our previously published method (Wang et al, 2018). Then, the purified protein PEL3 was diluted in PBS (pH 7.4) to get the protein solution of 0.02 mg/mL.…”

Section: Preparation Of Pel3-inorganic Hybrid Nanoflowersmentioning

confidence: 99%

“…Due to its industrial applications, isolation and purification of Pels from wide-type strains such as Bacillus (Klug-Santner et al, 2006), Erwinia (Lojkowska et al, 1995), and Aspergillus (Dinu et al, 2007) were widely reported. Also, gene cloning and heterologous expression, molecular modification, and construction of high yield strain, etc., were studied and reported (Zhang et al, 2013;Kohli and Gupta, 2015;Liang et al, 2015;Wang et al, 2018).…”

mentioning

confidence: 99%

“…Therefore, under the industrial processing conditions, Pels with higher activity and stability are still in demand. At present, directed evolution and rational design were the most successful strategies applied for engineering natural enzymes, and some successful examples of modified Pels have been reported to be compatible with the target industrial process (Liang et al, 2015;Wang et al, 2018). However, lacking of long-term operational stability and difficulty in reusability of Pels still hamper their use in industrial process.…”

mentioning

confidence: 99%

“…In our previous studies, a pectate lyase PEL168 from Bacillus subtilis 168 was cloned, characterized, and molecular modified (Zhang et al, 2013;Wang et al, 2018). A triple-site mutagenesis mutant K47D/V132F/R272W (PEL3) was obtained with specific activity of 5610 U/mg at 1 mM Ca 2+ , which showed the highest activity of reported alkaline Pels, and an extended T 50 to 330 min at 50 • C (Wang et al, 2018). In order to further improve the thermostability and reusability of PEL3, a facile and rapid method of preparing immobilized enzyme PEL3-inorganic hybrid nanoflowers was developed in this study.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Improving the Catalytic Performance of Pectate Lyase Through Pectate Lyase/Cu3(PO4)2 Hybrid Nanoflowers as an Immobilized Enzyme

Luo

et al. 2020

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

Pectate lyases (Pels) can be used in the textile industrial process for cotton scouring and ramie degumming, and its hydrolyzed products oligo galacturonic acid, are high-value added agricultural and health products. In our previous studies, an alkaline pectate lyase PEL168 mutant, PEL3, was obtained with improved specific activity and thermostability. Here, a facile and rapid method for preparing an immobilized PEL3-inorganic hybrid nanoflower was developed, as it could improve its biocatalytic performance. With 0.02 mg/mL (112.2 U/mL) PEL3 in PBS buffer, five different divalent ions, including Mn 2+ , Ca 2+ , Co 2+ , Zn 2+ , and Cu 2+ , were used as inorganic component. The results showed that PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers presented the highest relative activity with 2.5-fold increase, compared to the free PEL3. X-ray diffraction analysis confirmed that the composition of PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers were pectate lyase PEL3 and Cu 3 (PO 4) 2 •5H 2 O. The optimum temperature and pH of PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers were ascertained to be 55 • C and pH 9.0, respectively, exhibiting subtle difference from the free PEL3. However, the PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers maintained 33% residual activity after 24 h incubation at 55 • C, while the free PEL3 completely lost its activity after 18 h incubation at 55 • C. Furthermore, over 50% residual activity of the PEL3/Cu 3 (PO 4) 2 hybrid nanoflowers was remained, even after four times of repetitive utilization, demonstrating its promising stability for practical application.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Preparation Of Pel3-inorganic Hybrid Nanoflowersmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Improving the Catalytic Performance of Pectate Lyase Through Pectate Lyase/Cu3(PO4)2 Hybrid Nanoflowers as an Immobilized Enzyme

Luo

et al. 2020

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

show abstract

Enhancing thermal stability and lytic activity of phage lysin PlyAB1 from Acinetobacter baumannii

Yuan

Zhang

et al. 2022

Biotech & Bioengineering

View full text Add to dashboard Cite

With the increasingly serious drug resistance of Acinetobacter baumannii, there is an increasingly urgent need for new antibacterial drugs. Phage lysin PlyAB1 has a bactericidal effect on drug‐resistant A. baumannii, which has the potential to replace antibiotics to fight infection caused by A. baumannii. However, its application is limited by its thermal stability and lytic activity. To solve these problems, molecular dynamics (MD) simulations combined with Hotspot wizard 3.0 were used to identify key residue sites affecting thermal stability, and evolutionary analysis combined with multiple sequence alignment was used to identify key residue sites affecting lytic activity. Four single‐point variants with significantly increased thermal stability and four single‐point variants with significantly lytic activity were obtained, respectively. Furthermore, by superimposing mutations, we obtained three double‐point variants, G100Q/K69R, G100R/K69R, and G100K/K69R, with significantly improved thermal stability and improved lytic activity. At 45°C, the lytic activity and half‐life of the optimal variant G100Q/K69R were 1.51‐ and 24‐fold higher than those of the wild PlyAB1, respectively. These results deepen our understanding of the structure and function of phage lysin and contribute to the application of phage lysin in antibiotic substitution.

show abstract

Identification of a Hotspot Residue for Improving the Thermostability of a Flavin‐Dependent Monooxygenase

et al. 2019

View full text Add to dashboard Cite

HadA is a flavin‐dependent monooxygenase that can catalyze the denitration and dehalogenation of a wide variety of toxicants such as pesticides. Although these enzymatic reactions are useful for bioremediation or biocatalysis, the application of HadA for these purposes is not yet possible because of its low thermostability. In this work we have engineered HadA to be more thermostable through the use of structural, in silico, and rational approaches. The X‐ray structure of HadA was solved to obtain a reliable three‐dimensional protein model for further prediction of thermostable variants. In silico analysis by using two bioinformatic tools—FireProt and Disulfide by Design—suggested 102 variants that we then further refined by applying rational criteria including the location of a particular residue and its nearby interactions, as well as other biophysical parameters to narrow down the list to six candidates. The G513Y variant was found to be an optimal engineered candidate because it has significantly improved stability relative to the wild‐type enzyme and equivalent activity. G513Y has an activity half‐life 72 (50 °C) and 160 times (45 °C) longer than that of the wild‐type enzyme. Coupled together with thermostable reactions of reduced flavin and NADH‐regenerating systems, the G513Y variant can be used to catalyze denitration of 4nitrophenol at 45 °C. Structure/sequence alignments of HadA and its homologues indicate that several flavin‐dependent monooxygenases also contain amino acid residues homologous to the G513 of HadA, hence opening up the possibility of applying this engineering approach to improving their thermostabilities as well. Molecular dynamics (MD) simulations confirmed that the improved thermostability of the G513Y variant was due to aromatic hydrocarbon interactions between Y513 and N359, L347, G348, and F349.

show abstract

Improving the specific activity and thermo-stability of alkaline pectate lyase from Bacillus subtilis 168 for bioscouring

Cited by 27 publications

References 59 publications

Improving the Catalytic Performance of Pectate Lyase Through Pectate Lyase/Cu3(PO4)2 Hybrid Nanoflowers as an Immobilized Enzyme

Improving the Catalytic Performance of Pectate Lyase Through Pectate Lyase/Cu3(PO4)2 Hybrid Nanoflowers as an Immobilized Enzyme

Enhancing thermal stability and lytic activity of phage lysin PlyAB1 from Acinetobacter baumannii

Identification of a Hotspot Residue for Improving the Thermostability of a Flavin‐Dependent Monooxygenase

Contact Info

Product

Resources

About