Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

Tu, Tao; Luo, Huiying; Meng, Kun; Cheng, Yanli; Ma, Rui; Shi, Pengjun; Huang, Huoqing; Bai, Yingguo; Wang, Yaru; Zhang, Lujia; Yao, Bin

doi:10.1128/aem.01363-15

Cited by 45 publications

(25 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4A), all enzymes displayed an equilibrium state within the last 12.5 ns. Generally, lowering the overall RMSD is one way to modify the enzyme structure to make it more rigid and consequently enhance its stability (19, 20). The variants A10C fdh and A10C/I239C fdh had lower RMSD values than wild-type Cbo FDH, suggesting that their structures were more stable.…”

Section: Resultsmentioning

confidence: 99%

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Zheng

Yang

Zhou

et al. 2017

Appl Environ Microbiol

View full text Add to dashboard Cite

NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10Cfdh) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10Cfdh, which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances.IMPORTANCE FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.

show abstract

Section: Resultsmentioning

confidence: 99%

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Zheng

Yang

Zhou

et al. 2017

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Several polygalacturonases from Aspergillus carbonarius and Achaetomium sp. Xz8 have been reported to have the capacities to improve the juice yield and clarity (Nakkeeran et al 2011) and reduce the viscosity of papaya juice (Tu et al 2015). Polygalacturonase produced by A. niger using banana peel as a substrate has been used for clarification of banana juice (Barman et al 2015).…”

Section: Application Of Purified Pg In Fruit Juice Clarificationmentioning

confidence: 99%

Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice

Anand

Yadav

2017

3 Biotech

View full text Add to dashboard Cite

Polygalacturonases (PG) represent an important member of pectinases group of enzymes with immense industrial applications. A fungal strain Aspergillus niger MTCC478 was used for the production of polygalacturonase both under submerged and solid-state fermentation condition. Further its production was optimized under solid-state fermentation condition with media comprising of wheat bran and tea extract. Purification of an exo-PG was achieved by acetone precipitation (60-90%) and CMcellulose column chromatography revealing 15.28-fold purification with a specific activity of 33.47 U/mg protein and 1.2% yield. A relative molecular mass of purified PG was approximately 124.0 kDa. The pH and temperature optimum was found to be 4 and 50°C, respectively. The k cat and K m value for degradation of PGA by the purified enzyme was found to be 194 s -1 and 2.3 mg/mL, respectively. Cu 2? was found to enhance the PG activity while Ag ? completely inhibited the enzyme activity. The application of the purified PG in orange juice clarification was elucidated.

show abstract

“…The thermodynamic stabilities ( T m , melting temperature) of wild-type PG63 and its mutants were determined on a MicroCal™ VP-Capillary DSC (GE Healthcare) as described previously13. Aliquots of proteins (0.35 mg) were dissolved in 1 mL of 20-mM McIlvaine buffer (pH 4.0) and loaded onto the capillary automatically.…”

Section: Methodsmentioning

confidence: 99%

“…The best binding conformation was selected by the criteria of interacting energy combined with the geometrical matching quality and the binding affinity (ΔG, kcal/mol) of the AutoDock Vina software. After that, energy minimization was used to relax atomic clashes for the complex using YASARA software313. This process ultimately obtained a reliable model for the PG63-GalpA6 complex.…”

Section: Methodsmentioning

confidence: 99%

“…Based on the action modes, PGs are grouped into endo-PGs (EC 3.2.1.15) and exo-PGs (EC 3.2.1.67, EC 3.2.1.82)12. Endo-PGs are the most well-characterized and studied pectinases that catalyze the hydrolysis of the α-1,4-glycosidic bonds between the polygalacturonic acid (PGA) at the smooth pectin regions, and thus are often preferred for technical applications, such as in fruit juice, feed, paper, and textile industries, as well as for the extraction of oils13. Therefore, a full understanding of the catalytic mechanism of endo-PGs is a key towards the engineering of new endo-PGs for various applications.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Probing the role of cation-π interaction in the thermotolerance and catalytic performance of endo-polygalacturonases

et al. 2016

Sci Rep

Self Cite

View full text Add to dashboard Cite

Understanding the dynamics of the key pectinase, polygalacturonase, and improving its thermotolerance and catalytic efficiency are of importance for the cost-competitive bioconversion of pectic materials. By combining structure analysis and molecular dynamics (MD) simulations, eight mutagenesis sites having the potential to form cation-π interactions were identified in the widely used fungal endo-polygalacturonase PG63. In comparison to the wild-type, three single mutants H58Y, T71Y and T304Y showed improved thermostability (the apparent Tms increased by 0.6−3.9 °C) and catalytic efficiency (by up to 32-fold). Chromatogram analysis of the hydrolysis products indicated that a larger amount of shorter sugars were released from the polygalacturonic acid by these three mutants than by the wild-type. MD analysis of the enzyme-substrate complexes illustrated that the mutants with introduced cation-π interaction have modified conformations of catalytic crevice, which provide an enviable environment for the catalytic process. Moreover, the lower plasticity of T3 loop 2 at the edge of the subsite tunnel appears to recruit the reducing ends of oligogalacturonide into the active site tunnel and initiates new hydrolysis reactions. This study demonstrates the importance of cation-π interaction in protein conformation and provides a realistic strategy to enhance the thermotolerance and catalytic performance of endo-polygalacturonases.

show abstract

Improvement in Thermostability of an Achaetomium sp. Strain Xz8 Endopolygalacturonase via the Optimization of Charge-Charge Interactions

Cited by 45 publications

References 37 publications

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Production, purification and biochemical characterization of an exo-polygalacturonase from Aspergillus niger MTCC 478 suitable for clarification of orange juice

Probing the role of cation-π interaction in the thermotolerance and catalytic performance of endo-polygalacturonases

Contact Info

Product

Resources

About