Amino acid residues adjacent to the catalytic cavity of tetramer l-asparaginase II contribute significantly to its catalytic efficiency and thermostability

Shuiqing, Long; Zhang, Xian; Rao, Zhiming; Chen, Kaiyue; Yang, Taowei; Yang, Shang‐Tian

doi:10.1016/j.enzmictec.2015.08.009

Cited by 38 publications

(26 citation statements)

References 41 publications

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“…The L-asparaginase activity assay was conducted at 95 °C as described by Long and Zuo et al . 13 , 19 . The reaction mixture (1 mL) containing L-asparagine (25 mM) and Tris-HCl (50 mM, pH 8.0) was preheated at 95 °C.…”

Section: Methodsmentioning

confidence: 99%

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Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Zhang

et al. 2018

Sci Rep

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L-asparaginase, which catalyses the hydrolysis of L-asparagine to L-aspartate, has attracted the attention of researchers due to its expanded applications in medicine and the food industry. In this study, a novel thermostable L-asparaginase from Pyrococcus yayanosii CH1 was cloned and over-expressed in Bacillus subtilis 168. To obtain thermostable L-asparaginase mutants with higher activity, a robust high-throughput screening process was developed specifically for thermophilic enzymes. In this process, cell disruption and enzyme activity assays are simultaneously performed in 96-deep well plates. By combining error-prone PCR and screening, six brilliant positive variants and four key amino acid residue mutations were identified. Combined mutation of the four residues showed relatively high specific activity (3108 U/mg) that was 2.1 times greater than that of the wild-type enzyme. Fermentation with the mutant strain in a 5-L fermenter yielded L-asparaginase activity of 2168 U/mL.

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Section: Methodsmentioning

confidence: 99%

“…Long et al . identified some key amino acid residues adjacent to the catalytic cavity of L-asparaginase and improved its thermostability and catalytic efficiency via mutagenesis of these residues 13 . By employing a genetic algorithm in tandem with flexibility studies using molecular dynamics, Offman et al .…”

Section: Introductionmentioning

confidence: 99%

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Zhang

et al. 2018

Sci Rep

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“…l ‐asparaginase (EC 3.5.1.1) catalyzes the hydrolysis of l ‐asparagine to l ‐aspartate and ammonia . l ‐asparaginase from guinea pig serum was the first to exhibit antilymphoma effects, leading to interest in l ‐asparaginase research .…”

Section: Introductionmentioning

confidence: 99%

Design of a high‐efficiency synthetic system for l‐asparaginase production in Bacillus subtilis

Zhang

et al. 2019

Engineering in Life Sciences

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l‐asparaginase has high application value in medicine and food industry, but the low yield limits its application. In this study, we designed a synthetic system in Bacillus subtilis to produce l‐asparaginase by improving gene expression and optimizing the fermentation agitation speed. Gene expression was improved by respectively increasing transcription levels and translation speeds through screening promoters and RBS sequences. With the optimal promoter, P43, and the synthetic RBS sequence, the yield obtained in a shake flask was 371.87 U/mL, which was 2.09 times that with the original strain. To further enhance production in a 5‐L fermenter, a multistage agitation speed control strategy was adopted, involving agitation at 600 rpm for the first 12 h, followed by a gradual increase in speed to 900 rpm, which resulted in the highest yield of l‐asparaginase, 5321 U/mL, after 42 h of fermentation.

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“…The acidity constant K a of ionizables residues in proteins is a physico‐chemical property of great relevance to biological systems . Changes in the protonation states of acidic or basic residues alter the active conformations of proteins, their stability and solubility, and is directly related to the catalytic efficiency of enzymes . Prediction of the acidity constant—or its negative logarithm pK a —for ionizable side chains in proteins, therefore, is currently a challenge and has been addressed in the past with a variety of computational methods …”

Section: Introductionmentioning

confidence: 99%

“…1,2 Changes in the protonation states of acidic or basic residues alter the active conformations of proteins, 3 their stability and solubility, 1,2,4 and is directly related to the catalytic efficiency of enzymes. 5 Prediction of the acidity constant-or its negative logarithm pK a -for ionizable side chains in proteins, therefore, is currently a challenge and has been addressed in the past with a variety of computational methods. 1,2,6,7 Computational methods used to calculate the pK a are based on the Gibbs free energy change between the protonated and deprotontaed state of a residue in a protein.…”

Section: Introductionmentioning

confidence: 99%

Conformational sampling and polarization of Asp26 in pK_a calculations of thioredoxin

Gomez

Vöhringer‐Martinez

2019

Proteins

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Thioredoxin is a protein that has been used as model system by various computational methods to predict the pKa of aspartate residue Asp26 which is 3.5 units higher than a solvent exposed one (eg, Asp20). Here, we use extensive atomistic molecular dynamics simulations of two different protonation states of Asp26 in combination with conformational analysis based on RMSD clustering and principle component analysis to identify representative conformations of the protein in solution. For each conformation, the Gibbs free energy of proton transfer between Asp26 and Asp20, which is fully solvated in a loop region of the protein, is calculated with the Amber99sb force field in alchemical transformations. The varying polarization of the two residues in different molecular environments and protonation states is described by Hirshfeld‐I (HI) atomic charges obtained from the averaged polarized electron density. Our results show that the Gibbs free energy of proton transfer is dependent on the protein conformation, the proper sampling of the neighboring Lys57 residue orientations and on water molecules entering the hydrophobic cavity upon deprotonating Asp26. The inclusion of the polarization of both aspartate residues in the free energy cycle by HI atomic charges corrects the results from the non‐polarizable force field and reproduces the experimental ΔpKa value of Asp26.

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Amino acid residues adjacent to the catalytic cavity of tetramer l-asparaginase II contribute significantly to its catalytic efficiency and thermostability

Cited by 38 publications

References 41 publications

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Design of a high‐efficiency synthetic system for l‐asparaginase production in Bacillus subtilis

Conformational sampling and polarization of Asp26 in pK_a calculations of thioredoxin

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Amino acid residues adjacent to the catalytic cavity of tetramer l-asparaginase II contribute significantly to its catalytic efficiency and thermostability

Cited by 38 publications

References 41 publications

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Simultaneous cell disruption and semi-quantitative activity assays for high-throughput screening of thermostable L-asparaginases

Design of a high‐efficiency synthetic system for l‐asparaginase production in Bacillus subtilis

Conformational sampling and polarization of Asp26 in pKa calculations of thioredoxin

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Conformational sampling and polarization of Asp26 in pK_a calculations of thioredoxin