Improving the Thermostability and Catalytic Activity of an Inulosucrase by Rational Engineering for the Biosynthesis of Microbial Inulin

Ni, Dawei; Zhang, Shuqi; Kırtel, Onur; Xu, Wei; Chen, Qiuming; Öner, Ebru Toksoy; Mu, Wanmeng

doi:10.1021/acs.jafc.1c04852

Cited by 23 publications

(7 citation statements)

References 54 publications

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“…Consensus sequence design, a widely accepted approach to improving the thermostability of biocatalysts, , was performed using thermophilic proteins as align models to search for the hot spots that affect the Cp KR thermostability. The EnzymeMiner platform, a user-friendly bioinformatic tool, was employed to retrieve thermophilic enzymes sharing a certain degree of identity with Cp KR. , Using the sequence of Cp KR as a query and identifying Ser133, Tyr171, and Lys175 as the essential residues for catalysis and cofactor binding, four proteins from thermophiles were obtained (Table S1).…”

Section: Resultsmentioning

confidence: 99%

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Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

et al. 2023

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The flexible regions represented by loops have been the focus of protein stability engineering. However, residues located in specific rigid regions are also crucial for protein stability. The role of dynamic correlations between mutations in these two regions on protein stabilization remains inadequately understood. In this work, a ketoreductase CpKR originated from Candida parapsilosis was rationally engineered to improve its stability by comprehensively redesigning the flexible and rigid regions. Hot spots were successively identified and validated in the rigid regions with low B-factor or root-mean-square fluctuation values, as well as in relatively flexible areas of the protein. Based on the non-additive and cooperative mutational effects, a combinatorial variant (M2) with beneficial mutations in these two distinct regions showed a 4630-fold increased half-life at 40 °C and a 12 °C higher apparent melting temperature as compared with those of the wild type. The variant M2 also showed improved pH compatibility, better organic substance tolerance, and enhanced performance in asymmetric reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate (1a), the precursor of cardiovascular drug Diltiazem. The crystal structures and molecular dynamics simulations demonstrated that additional interaction networks in both the rigid and flexible regions modulated protein stability, and the dynamic correlation between these two regions mediated the observed synergistic combination. These results showed that both the flexible and rigid regions, especially the cross-correlated areas, are crucial for enhancing protein stability, and comprehensively redesigning these two regions is a powerful and attractive approach for acquiring stable enzymes.

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Section: Resultsmentioning

confidence: 99%

“…Consensus sequence design, a widely accepted approach to improving the thermostability of biocatalysts, 40,41 was performed using thermophilic proteins as align models to search for the hot spots that affect the CpKR thermostability.…”

Section: Combination Of Stabilizing Mutations Identified Frommentioning

confidence: 99%

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

et al. 2023

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“…reuteri 121 (50 °C) and IS from L . gasseri (55 °C) [ 27 , 28 ]. To further test the stability of Psor-LS at different temperatures, the enzyme was incubated at 45, 55, and 65 °C, respectively.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Thermostable Levansucrase from Pseudomonas orientalis That Allows Unique Product Specificity at Different Temperatures

Guang

Zhang

et al. 2023

Polymers

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The biological production of levan by levansucrase (LS, EC 2.4.1.10) has aroused great interest in the past few years. Previously, we identified a thermostable levansucrase from Celerinatantimonas diazotrophica (Cedi-LS). A novel thermostable LS from Pseudomonas orientalis (Psor-LS) was successfully screened using the Cedi-LS template. The Psor-LS showed maximum activity at 65 °C, much higher than the other LSs. However, these two thermostable LSs showed significantly different product specificity. When the temperature was decreased from 65 to 35 °C, Cedi-LS tended to produce high-molecular-weight (HMW) levan. By contrast, Psor-LS prefers to generate fructooligosaccharides (FOSs, DP ≤ 16) rather than HMW levan under the same conditions. Notably, at 65 °C, Psor-LS would produce HMW levan with an average Mw of 1.4 × 106 Da, indicating that a high temperature might favor the accumulation of HMW levan. In summary, this study allows a thermostable LS suitable for HMW levan and levan-type FOSs production simultaneously.

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“…Many studies have confirmed that the catalytic properties can be further improved by combining mutation sites that affect the same catalytic property [ 32 , 49 ]. Since S33T, T174V, and N218L variants enhanced different catalytic properties of rAprY, we performed combinational mutagenesis to further improve the catalytic properties of rAprY.…”

Section: Resultsmentioning

confidence: 99%

Combined Computer-Aided Predictors to Improve the Thermostability of Nattokinase

Chen

Zhu

et al. 2023

Foods

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Food-derived nattokinase has strong thrombolytic activity and few side effects. In the field of medicine, nattokinase has been developed as an adjuvant drug for the treatment of thrombosis, and nattokinase-rich beverages and health foods have also shown great potential in the field of food development. At present, the poor thermostability of nattokinase limits its industrial production and application. In this study, we used several thermostability-prediction algorithms to predict nattokinase from Bacillus mojavensis LY-06 (AprY), and screened two variants S33T and T174V with increased thermostability and fibrinolytic activity. The t1/2 of S33T and T174V were 8.87-fold and 2.51-fold those of the wild type AprY, respectively, and their enzyme activities were also increased (1.17-fold and 1.28-fold, respectively). Although the thermostability of N218L was increased by 2.7 times, the fibrinolytic activity of N218L was only 73.3% of that of wild type AprY. The multiple-point mutation results showed that S33T-N218L and S33T-T174V-N218L variants lost their activity, and the T174V-N218L variant did not show any significant change in catalytic performance, while S33T-T174V increased its thermostability and activity by 21.3% and 24.8%, respectively. Although the S33T-T174V variant did not show the additive effect of thermostability, it combined the excellent transient thermostability of S33T with the better thrombolytic activity of T174V. Bioinformatics analysis showed that the overall structure of S33T and T174V variants tended to be stable, while the structure of S33T-T174V variant was more flexible. Local structure analysis showed that the increased rigidity of the active center region (positions 64–75) and the key loop region (positions 129–130, 155–163, 187–192, 237–241, and 268–270) determined the increased thermostability of all variants. In addition, the enhanced flexibility of S33T-T174V variant in the Ca1 binding region (positions 1–4, 75–82) and the peripheral region of the catalytic pocket (positions 210–216) may account for the inability to superpose its thermostability. However, the increased flexibility in the active central region (positions 210–216) and the interaction region with Ca1 binding site (positions 1–4, 210–216) of the S33T-T174V variant may contribute to the decreased thermostability of the S33T-T174V variant compared with the corresponding single-point mutation. We explored the effective strategy to enhance the thermostability of nattokinase, and the resulting variants have potential industrial production and application.

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Improving the Thermostability and Catalytic Activity of an Inulosucrase by Rational Engineering for the Biosynthesis of Microbial Inulin

Cited by 23 publications

References 54 publications

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

Identification of a Thermostable Levansucrase from Pseudomonas orientalis That Allows Unique Product Specificity at Different Temperatures

Combined Computer-Aided Predictors to Improve the Thermostability of Nattokinase

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