Improved thermostability of D-allulose 3-epimerase from Clostridium bolteae ATCC BAA-613 by proline residue substitution

Wang, Huiyi; Chen, Jing; Zhao, Jingyi; Li, Hongwei; Wei, Xin; Liu, Jidong

doi:10.1016/j.pep.2022.106145

Cited by 9 publications

(5 citation statements)

References 37 publications

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“…Next, we attempted to explore the molecular mechanism underlying the thermostability of GeoCas9. The most well acknowledged protein thermostabilityassociated features include disulfide linkage and higher proportion of proline [35][36][37] . We found that GeoCas9, as well as all reported Cas9 structures, lack disulfide bond.…”

Section: Molecular Mechanism Underlying Thermostability Of Geocas9mentioning

confidence: 99%

Structure ofGeobacillus stearothermophilusCas9: insights into the catalytic process and thermostability of CRISPR-Cas9

Shen,

Zhang,

Liu

et al. 2024

Preprint

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CRISPR-Cas9 has been developed as a powerful gene editing tool, but the mechanism governing the intricate catalytic process remains incompletely resolved. Here, the cryo-electron microscopy structures of thermostable Cas9 fromGeobacillus stearothermophilus(GeoCas9) in complex with sgRNA and target DNA are reported. The structure of GeoCas9 in complex with sgRNA reveals a slit termed L1-crevice comprising HNH, RuvC, and L1 helix as a transient storage site of 5’ spacer of sgRNA. When 5’ spacer is extracted to pair with the target DNA, L1-crevice collapses to trigger the subsequent HNH domain translocation. In addition, structural and biochemical analyses suggest that the resilience of GeoCas9 at elevated temperature is related to the unique PI domain conformation. These results advance our understanding into the catalytic process of Cas9 and unveil the molecular mechanism that accounts for the superior thermal profile of GeoCas9.

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Section: Molecular Mechanism Underlying Thermostability Of Geocas9mentioning

confidence: 99%

Structure ofGeobacillus stearothermophilusCas9: insights into the catalytic process and thermostability of CRISPR-Cas9

Shen,

Zhang,

Liu

et al. 2024

Preprint

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“…This approach resulted in mutants with a T m of 79.48 °C, more than 12 °C higher than that of the wild type, which also had a higher t 1/2 of more than 10 h at 65 °C and an improved T opt of 75 °C compared to the wild-type enzyme, which had a t 1/2 of less than 5 min at the same temperature [ 23 ]. In another study, Wang et al [ 25 ] also adopted a rational design strategy which incorporated proline residue substitutions and constructed double mutant designs through homology modelling and computational structural analysis. The resultant mutant was able to retain comparable enzymatic activity while showing a t 1/2 increased up to 2-fold [ 25 ].…”

Section: Engineering Of Daeasesmentioning

confidence: 99%

“…In another study, Wang et al [ 25 ] also adopted a rational design strategy which incorporated proline residue substitutions and constructed double mutant designs through homology modelling and computational structural analysis. The resultant mutant was able to retain comparable enzymatic activity while showing a t 1/2 increased up to 2-fold [ 25 ].…”

Section: Engineering Of Daeasesmentioning

confidence: 99%

“…In this review, we discussed how new epimerases can be discovered [ 15 , 16 , 17 ], as well as methods to improve their stability and activity via protein engineering [ 21 , 22 , 24 , 25 , 28 ], the application of B. subtilis [ 11 , 25 , 29 ], and the potential of immobilization techniques to enhance performance [ 34 , 35 , 36 , 37 ]. These advancements have the potential to significantly reduce production time and costs associated with D -allulose biosynthesis.…”

Section: Summary and Future Visionmentioning

confidence: 99%

“…Proline residue substitution to reduce unfolded enzyme entropy Improved thermostability [25] DAEase from Clostridium cellulolyticum H10 E. coli BL21(DE3) Directed evolution, combinatorial mutagenesis Improved enzyme specificity, improved thermotolerance [21] DAEase from Clostridium cellulolyticum H10…”

Section: E Coli Bl21(de3)mentioning

confidence: 99%

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The Engineering, Expression, and Immobilization of Epimerases for D-allulose Production

Tan

Chen

et al. 2023

IJMS

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The rare sugar D-allulose is a potential replacement for sucrose with a wide range of health benefits. Conventional production involves the employment of the Izumoring strategy, which utilises D-allulose 3-epimerase (DAEase) or D-psicose 3-epimerase (DPEase) to convert D-fructose into D-allulose. Additionally, the process can also utilise D-tagatose 3-epimerase (DTEase). However, the process is not efficient due to the poor thermotolerance of the enzymes and low conversion rates between the sugars. This review describes three newly identified DAEases that possess desirable properties for the industrial-scale manufacturing of D-allulose. Other methods used to enhance process efficiency include the engineering of DAEases for improved thermotolerance or acid resistance, the utilization of Bacillus subtilis for the biosynthesis of D-allulose, and the immobilization of DAEases to enhance its activity, half-life, and stability. All these research advancements improve the yield of D-allulose, hence closing the gap between the small-scale production and industrial-scale manufacturing of D-allulose.

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Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis

Shen,

Xu,

et al. 2024

Bioprocess Biosyst Eng

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Improved thermostability of D-allulose 3-epimerase from Clostridium bolteae ATCC BAA-613 by proline residue substitution

Cited by 9 publications

References 37 publications

Structure ofGeobacillus stearothermophilusCas9: insights into the catalytic process and thermostability of CRISPR-Cas9

Structure ofGeobacillus stearothermophilusCas9: insights into the catalytic process and thermostability of CRISPR-Cas9

The Engineering, Expression, and Immobilization of Epimerases for D-allulose Production

Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis

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