Engineering of a nitrilase through consensus sequence analysis and conserved site substitution to improve its thermostability and activity

Xiong, Neng; Lv, Pei-jin; Song, Ji-Wei; Shen, Qi; Xue, Ya‐Ping; Zheng, Yu‐Guo

doi:10.1016/j.bej.2022.108475

Cited by 8 publications

(4 citation statements)

References 40 publications

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“…utilized consensus‐guided mutagenesis to enhance the thermostability and activity of nitrilase. [ 18 ]…”

Section: Introductionmentioning

confidence: 99%

“…[17] Xiong et al utilized consensus-guided mutagenesis to enhance the thermostability and activity of nitrilase. [18] Computer-aided design can increase the efficiency of creating stable proteins. [19] Recently, a computational tool called Protein Repair One-Stop Shop (PROSS) has displayed its ability to improve the stability of proteins, which incorporates the multiple sequence alignment (MSA) of homologous sequences to locate potential amino acid substitutions and Rosetta suite to calculate the energy difference introduced by the point mutations.…”

Section: Introductionmentioning

confidence: 99%

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Computer‐aided design to enhance the stability of aldo‐keto reductase KdAKR

Dai,

Tian,

Chen

et al. 2024

Biotechnology Journal

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The aldo‐keto reductase (AKR) KdAKR from Kluyvermyces dobzhanskii can reduce t‐butyl 6‐chloro‐(5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) to t‐butyl 6‐chloro‐(3R,5S)‐dihydroxyhexanoate ((3R,5S)‐CDHH), which is the key chiral intermediate of rosuvastatin. Herein, a computer‐aided design that combined the use of PROSS platform and consensus design was employed to improve the stability of a previously constructed mutant KdAKRM6. Experimental verification revealed that S196C, T232A, V264I and V45L produced improved thermostability and activity. The “best” mutant KdAKRM10 (KdAKRM6‐S196C/T232A/V264I/V45L) was constructed by combining the four beneficial mutations, which displayed enhanced thermostability. Its T5015 and Tm values were increased by 10.2 and 10.0°C, respectively, and half‐life (t1/2) at 40°C was increased by 17.6 h. Additionally, KdAKRM10 demonstrated improved resistance to organic solvents compared to that of KdAKRM6. Structural analysis revealed that the increased number of hydrogen bonds and stabilized hydrophobic core contributed to the rigidity of KdAKRM10, thus improving its stability. The results validated the feasibility of the computer‐aided design strategy in improving the stability of AKRs.

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“…utilized consensus‐guided mutagenesis to enhance the thermostability and activity of nitrilase. [ 18 ]…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Computer‐aided design to enhance the stability of aldo‐keto reductase KdAKR

Dai,

Tian,

Chen

et al. 2024

Biotechnology Journal

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“…By calculating the frequency of amino acid occurrences at each site, evolutionarily conserved consensus sequences are obtained. Amino acids that occur with higher frequencies are considered relatively stable throughout evolution [34][35][36]. Conserved amino acid residues within enzyme structures play a crucial role in proper folding, catalytic activity, and stability [37].…”

Section: Introductionmentioning

confidence: 99%

Thermal Stability Enhancement of L-Asparaginase from Corynebacterium glutamicum Based on a Semi-Rational Design and Its Effect on Acrylamide Mitigation Capacity in Biscuits

Chi,

Jiang,

Feng

et al. 2023

Foods

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Acrylamide is present in thermally processed foods, and it possesses toxic and carcinogenic properties. L-asparaginases could effectively regulate the formation of acrylamide at the source. However, current L-asparaginases have drawbacks such as poor thermal stability, low catalytic activity, and poor substrate specificity, thereby restricting their utility in the food industry. To address this issue, this study employed consensus design to predict the crucial residues influencing the thermal stability of Corynebacterium glutamicum L-asparaginase (CgASNase). Subsequently, a combination of site-point saturating mutation and combinatorial mutation techniques was applied to generate the double-mutant enzyme L42T/S213N. Remarkably, L42T/S213N displayed significantly enhanced thermal stability without a substantial impact on its enzymatic activity. Notably, its half-life at 40 °C reached an impressive 13.29 ± 0.91 min, surpassing that of CgASNase (3.24 ± 0.23 min). Moreover, the enhanced thermal stability of L42T/S213N can be attributed to an increased positive surface charge and a more symmetrical positive potential, as revealed by three-dimensional structural simulations and structure comparison analyses. To assess the impact of L42T/S213N on acrylamide removal in biscuits, the optimal treatment conditions for acrylamide removal were determined through a combination of one-way and orthogonal tests, with an enzyme dosage of 300 IU/kg flour, an enzyme reaction temperature of 40 °C, and an enzyme reaction time of 30 min. Under these conditions, compared to the control (464.74 ± 6.68 µg/kg), the acrylamide reduction in double-mutant-enzyme-treated biscuits was 85.31%, while the reduction in wild-type-treated biscuits was 68.78%. These results suggest that L42T/S213N is a promising candidate for industrial applications of L-asparaginase.

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“…Liang et al (2021) improved catalytic efficiency and thermostability of the β-mannanase using the consensus sequence design strategy. The thermostability and activity of the nitrilase were also improved through the consensus-guided mutagenesis (Xiong et al, 2022).…”

mentioning

confidence: 99%

Structural‐guided design to improve the catalytic performance of aldo‐keto reductase KdAKR

Dai,

Cao,

Tian

et al. 2023

Biotech & Bioengineering

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Aldo‐keto reductases (AKRs) are important biocatalysts that can be used to synthesize chiral pharmaceutical alcohols. In this study, the catalytic activity and stereoselectivity of a NADPH‐dependent AKR from Kluyveromyces dobzhanskii (KdAKR) toward t‐butyl 6‐chloro (5S)‐hydroxy‐3‐oxohexanoate ((5S)‐CHOH) were improved by mutating its residues in the loop regions around the substrate‐binding pocket. And the thermostability of KdAKR was improved by a consensus sequence method targeted on the flexible regions. The best mutant M6 (Y28A/L58I/I63L/G223P/Y296W/W297H) exhibited a 67‐fold higher catalytic efficiency compared to the wild‐type (WT) KdAKR, and improved R‐selectivity toward (5S)‐CHOH (dep value from 47.6% to >99.5%). Moreover, M6 exhibited a 6.3‐fold increase in half‐life (t1/2) at 40°C compared to WT. Under the optimal conditions, M6 completely converted 200 g/L (5S)‐CHOH to diastereomeric pure t‐butyl 6‐chloro‐(3R, 5S)‐dihydroxyhexanoate ((3R, 5S)‐CDHH) within 8.0 h, with a space‐time yield of 300.7 g/L/day. Our results deepen the understandings of the structure−function relationship of AKRs, providing a certain guidance for the modification of other AKRs.

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Engineering of a nitrilase through consensus sequence analysis and conserved site substitution to improve its thermostability and activity

Cited by 8 publications

References 40 publications

Computer‐aided design to enhance the stability of aldo‐keto reductase KdAKR

Computer‐aided design to enhance the stability of aldo‐keto reductase KdAKR

Thermal Stability Enhancement of L-Asparaginase from Corynebacterium glutamicum Based on a Semi-Rational Design and Its Effect on Acrylamide Mitigation Capacity in Biscuits

Structural‐guided design to improve the catalytic performance of aldo‐keto reductase KdAKR

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