Improving thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations

Yuan, Su-Su; Yan, Renxiang; Lin, Bin; Li, Renkuan; Ye, Xiuyun

doi:10.1016/j.bbrc.2023.02.064

Cited by 10 publications

(5 citation statements)

References 37 publications

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“…The thermal stability of amylase can be significantly enhanced through structural-based rational design approaches. These strategies involve reducing the free energy associated with enzyme molecule folding, as well as incorporating targeted amino acid substitutions and mutations (Ben Ali et al, 2006;Deng et al, 2014;Yuan et al, 2023). During the dynamic motion of enzyme molecules, conformational motion pathways play a pivotal role in determining the patterns and rate of protein motion.…”

Section: Discussionmentioning

confidence: 99%

“…The hydrogen bond network formed between substrate molecules and amino acids within the catalytic center of the enzymes play a vital role in influencing their activity and stability (Sebastiani et al, 2023;Yuan et al, 2023). Hydrophilic amino acids could increase the hydrogen bonding with substrates and vice versa.…”

Section: Discussionmentioning

confidence: 99%

“…The conformational flexibility and rigidity of the enzyme molecule play a decisive role in determining its overall stability (Dušan and Shina Caroline Lynn, 2018;Maria-Solano et al, 2018;Zhu et al, 2023). The generally used method was to substitute and mutate the amino acids in the surface of the protein to enhance the stability of amylase (Ben Ali et al, 2006;Deng et al, 2014;Yuan et al, 2023). Conformational motion pathways are the profile of the dynamic motion of enzyme molecules might act as switch plays a pivotal role in regulation of many essential biological pathways (Oyen et al, 2015;Hong et al, 2023).…”

Section: Discussionmentioning

confidence: 99%

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Modifying the amino acids in conformational motion pathway of the α-amylase of Geobacillus stearothermophilus improved its activity and stability

Hu,

Hong,

et al. 2023

Front. Microbiol.

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Amino acids along the conformational motion pathway of the enzyme molecule correlated to its flexibility and rigidity. To enhance the enzyme activity and thermal stability, the motion pathway of Geobacillus stearothermophilus α-amylase has been identified and molecularly modified by using the neural relational inference model and deep learning tool. The significant differences in substrate specificity, enzymatic kinetics, optimal temperature, and thermal stability were observed among the mutants with modified amino acids along the pathway. Mutants especially the P44E demonstrated enhanced hydrolytic activity and catalytic efficiency (kcat/KM) than the wild-type enzyme to 95.0% and 93.8% respectively, with the optimum temperature increased to 90°C. This mutation from proline to glutamic acid has increased the number and the radius of the bottleneck of the channels, which might facilitate transporting large starch substrates into the enzyme. The mutation could also optimize the hydrogen bonding network of the catalytic center, and diminish the spatial hindering to the substrate entry and exit from the catalytic center.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Modifying the amino acids in conformational motion pathway of the α-amylase of Geobacillus stearothermophilus improved its activity and stability

Hu,

Hong,

et al. 2023

Front. Microbiol.

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“…The release behavior of the encapsulated Rhodamine B was then explored by α-amylasecatalyzed enzymatic disintegration of the network polysaccharide assembly. When the mixture of the assembly with 0.2 mol/L acetate buffer was stirred in the presence of α-amylase (from Bacillus amyloliquefaciens) for 4 h at 40 • C (Figure 10b); optimal temperatures for hydrolysis of soluble starch by α-amylase from this source were reported to be 40-60 • C [25], the color gradually changed to red, as shown in Figure 11a, indicating that Rhodamine B was released into the acetate buffer by enzymatic disintegration. A fluorescence peak at 575 nm, assigned to Rhodamine B, was then detected in the fluorescence spectrum of the reaction mixture excited at 550 nm (Figure 11b).…”

Section: Runmentioning

confidence: 99%

Enzymatic Assembly of Chitosan-Based Network Polysaccharides and Their Encapsulation and Release of Fluorescent Dye

Totani,

Nakamichi,

Kadokawa

2024

Molecules

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We prepared network polysaccharide nanoscopic hydrogels by crosslinking water-soluble chitosan (WSCS) with a carboxylate-terminated maltooligosaccharide crosslinker via condensation. In this study, the enzymatic elongation of amylose chains on chitosan-based network polysaccharides by glucan phosphorylase (GP) catalysis was performed to obtain assembly materials. Maltoheptaose (Glc7) primers for GP-catalyzed enzymatic polymerization were first introduced into WSCS by reductive amination. Crosslinking of the product with the above-mentioned crosslinker by condensation was then performed to produce Glc7-modified network polysaccharides. The GP-catalyzed enzymatic polymerization of the α-d-glucose 1-phosphate monomer from the Glc7 primers on the network polysaccharides was conducted, where the elongated amylose chains formed double helices. Enzymatic disintegration of the resulting network polysaccharide assembly successfully occurred by α-amylase-catalyzed hydrolysis of the double helical amyloses. The encapsulation and release of a fluorescent dye, Rhodamine B, using the CS-based network polysaccharides were also achieved by means of the above two enzymatic approaches.

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“…To further determine the effect of the altered overall structure of the variants on their thermostability and catalytic activity, we determined the changes in the composition of the secondary structure and the number of bonds formed in each variant. Previous studies have suggested that the α-helix and β-sheet determine the rigidity of the enzyme, while the more flexible loop structure affects the thermostability of the enzyme [ 52 , 53 ]. Ban et al demonstrated that the increased proportion of α-helix in the secondary structure is an important reason for the increased thermostability of 1,4-α-glucan branching enzyme in glycerol solutions containing potassium/sodium ions [ 54 ].…”

Section: Understanding the Mechanism For Enhanced Thermostability And...mentioning

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 thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations

Cited by 10 publications

References 37 publications

Modifying the amino acids in conformational motion pathway of the α-amylase of Geobacillus stearothermophilus improved its activity and stability

Modifying the amino acids in conformational motion pathway of the α-amylase of Geobacillus stearothermophilus improved its activity and stability

Enzymatic Assembly of Chitosan-Based Network Polysaccharides and Their Encapsulation and Release of Fluorescent Dye

Combined Computer-Aided Predictors to Improve the Thermostability of Nattokinase

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