Improvement of catalytic efficiency and thermal stability of l-asparaginase from Bacillus subtilis 168 through reducing the flexibility of the highly flexible loop at N-terminus

Feng, Yue; Liu, Song; Pang, Cuiping; Gao, Hui; Wang, Miao; Du, Guocheng

doi:10.1016/j.procbio.2019.01.001

Cited by 14 publications

(7 citation statements)

References 31 publications

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“…69 Reducing the flexibility of the highly flexible Nterminal loop has been reported to increase L-ASNase activity. 70 RMSF analysis of mutant D172W/E207A (Figure S3) indicates that the mutation leads to a change in the Nterminal loop, which affects the activity of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…A conserved highly flexible loop exists in L-asparaginases of different sources, which regulates the catalytic function of the enzyme . Reducing the flexibility of the highly flexible N-terminal loop has been reported to increase l -ASNase activity . RMSF analysis of mutant D172W/E207A (Figure S3) indicates that the mutation leads to a change in the N-terminal loop, which affects the activity of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

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Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

Chi

Wang

Xia

et al. 2022

J. Agric. Food Chem.

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L-Asparaginase has gained much attention for effectively treating acute lymphoblastic leukemia (ALL) and mitigating carcinogenic acrylamide in fried foods. Due to high-dose dependence for clinical treatment and low mitigation efficiency for thermal food processes caused by poor thermal stability, a method to achieve thermostable L-asparaginase has become a critical bottleneck. In this study, a rational design including free energy combined with structural and conservative analyses was applied to engineer the thermostability of L-asparaginase from Bacillus licheniformis (BlAsnase). Two enhanced thermostability mutants D172W and E207A were screened out by site-directed saturation mutagenesis. The double mutant D172W/E207A exhibited highly remarkable thermostability with a 65.8-fold longer half-life at 55 °C and 5 °C higher optimum reaction temperature and melting temperature (T m ) than those of wild-type BlAsnase. Further, secondary structure, sequence, molecular dynamics (MD), and 3D-structure analysis revealed that the excellent thermostability of the mutant D172W/E207A was on account of increased hydrophobicity and decreased flexibility, highly rigid structure, hydrophobic interactions, and favorable electrostatic potential. As the first report of rationally designing L-asparaginase with improved thermostability from B. licheniformis, this study offers a facile and efficient process to improve the thermostability of L-asparaginase for industrial applications.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

Chi

Wang

Xia

et al. 2022

J. Agric. Food Chem.

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“…Consequently, the k cat / K m of mutant ILRAC increased 6.32-fold more than that of wild-type. The k cat / K m value of the mutant ILRAC was higher than some other ASNases from different source, such as Bacillus megaterium H-1 (3.39 min −1 -mM −1 ) and Bacillus subtilis (397.8 min −1 -mM −1 ) [ 32 , 33 ]. Although the recorded K m value of mutant ILRAC was still higher than clinical ASNase from E. coli (10 μM) and E. chrysanthemi (12 μM), it was much lower than other types II ASNases from multiple sources such as B. subtilis (5.29 mM), Bacillus licheniformis (49.995 mM), Pseudomonas aeruginosa (10.904 mM), and Streptomyces fradiae (10.07 mM) [ 34 , 35 , 36 , 37 ].…”

Section: Resultsmentioning

confidence: 99%

Enhancing the Catalytic Activity of Type II L-Asparaginase from Bacillus licheniformis through Semi-Rational Design

Zhou

Jiao

Shen

et al. 2022

IJMS

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Low catalytic activity is a key factor limiting the widespread application of type II L-asparaginase (ASNase) in the food and pharmaceutical industries. In this study, smart libraries were constructed by semi-rational design to improve the catalytic activity of type II ASNase from Bacillus licheniformis. Mutants with greatly enhanced catalytic efficiency were screened by saturation mutations and combinatorial mutations. A quintuple mutant ILRAC was ultimately obtained with specific activity of 841.62 IU/mg and kcat/Km of 537.15 min−1·mM−1, which were 4.24-fold and 6.32-fold more than those of wild-type ASNase. The highest specific activity and kcat/Km were firstly reported in type II ASNase from Bacillus licheniformis. Additionally, enhanced pH stability and superior thermostability were both achieved in mutant ILRAC. Meanwhile, structural alignment and molecular dynamic simulation demonstrated that high structure stability and strong substrate binding were beneficial for the improved thermal stability and enzymatic activity of mutant ILRAC. This is the first time that enzymatic activity of type II ASNase from Bacillus licheniformis has been enhanced by the semi-rational approach, and results provide new insights into enzymatic modification of L-asparaginase for industrial applications.

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“…The consensus design paradigm reasons that common, naturally occurring residues at a given position in related proteins are more likely to be active and/or stabilizing than a random mutation because they are frequently found in functional homologs and are likely present in the ancestral protein. For example, sampling a contiguous stretch of residues from homologs within flexible regions of l ‐arginase, an enzyme with applications in cancer treatment, was recently shown to greatly stabilize and increase enzyme activity . This interesting study—which demonstrates that both activity and stability can be simultaneously increased if mutations are judiciously selected—involved optimizing l ‐arginase through exchanging homologous sequences in a flexible loop adjacent to the enzyme active site.…”

Section: Design Methods For Improving Directed Evolution Of Protein Amentioning

confidence: 99%

Directed evolution methods for overcoming trade‐offs between protein activity and stability

2019

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Engineered proteins are being widely developed and employed in applications ranging from enzyme catalysts to therapeutic antibodies. Directed evolution, an iterative experimental process composed of mutagenesis and library screening, is a powerful technique for enhancing existing protein activities and generating entirely new ones not observed in nature. However, the process of accumulating mutations for enhanced protein activity requires chemical and structural changes that are often destabilizing, and low protein stability is a significant barrier to achieving large enhancements in activity during multiple rounds of directed evolution. Here we highlight advances in understanding the origins of protein activity/stability trade-offs for two important classes of proteins (enzymes and antibodies) as well as innovative experimental and computational methods for overcoming such trade-offs. These advances hold great potential for improving the generation of highly active and stable proteins that are needed to address key challenges related to human health, energy and the environment. K E Y W O R D Saffinity, antibody, catalysis, enzyme, protein design, protein engineering

show abstract

Improvement of catalytic efficiency and thermal stability of l-asparaginase from Bacillus subtilis 168 through reducing the flexibility of the highly flexible loop at N-terminus

Cited by 14 publications

References 31 publications

Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

Enhancing the Catalytic Activity of Type II L-Asparaginase from Bacillus licheniformis through Semi-Rational Design

Directed evolution methods for overcoming trade‐offs between protein activity and stability

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