Thermal stability enhancement: Fundamental concepts of protein engineering strategies to manipulate the flexible structure

Rahban, Mahdie; Zolghadri, Samaneh; Salehi, Najmeh; Ahmad, Faizan; Haertlé, Thomas; Rezaei‐Ghaleh, Nasrollah; Sawyer, Lindsay; Saboury, Ali Akbar

doi:10.1016/j.ijbiomac.2022.06.154

Cited by 51 publications

(36 citation statements)

References 209 publications

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“…Loop 18 was exactly opposite the substrate pocket of Th APT3 and was directly exposed to the solvent (Figure S6a). Surface-exposed loops, which connect α-helices and/or β-sheets, are highly flexible and play an important role in enzyme function . Loop 18 , which was the most flexible region of the entire enzyme molecule (Figure S6b), connected α-helix 7 (where the catalytic residue Ser 225 is located) and α-helix 8 .…”

Section: Resultsmentioning

confidence: 99%

“…Surfaceexposed loops, which connect α-helices and/or β-sheets, are highly flexible and play an important role in enzyme function. 32 Loop 18 , which was the most flexible region of the entire enzyme molecule (Figure S6b), connected α-helix 7 (where the catalytic residue Ser 225 is located) and α-helix 8 . Local structural fluctuations driven by heat can cause local unfolding, which is susceptible to intermolecular trapping and cleavage.…”

Section: Transplantation and Optimization Of Wt-loop 18mentioning

confidence: 99%

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Improving the Activity and Stability of Serine Protease ThAPT3 by Alleviating Self-Cleavage and Its Application in Deproteinization of Shrimp Shells

Wang

Dong

Zhou

et al. 2023

J. Agric. Food Chem.

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The self-cleavage properties of proteases result in low activity and instability, which limit their industrial application. In this study, the serine protease ThAPT3 from Torrubiella hemipterigena was successfully expressed in Komagataella phaffii. We investigated the self-degradation mechanism of ThAPT3 and presented a rational strategy to alleviate self-cleavage. A major selfdegradation site (Leu 238 -Met 239 ) and a primary autolysis region were identified. The autolysis regions (loop 18 , α 8 -helix, and loop 19 ) were redesigned and optimized using loop transplantation, energy calculations, surface cavity optimization, and loop anchoring. A triple-superposition mutant, ThAPT3-M9 (M 239 GKDGAVAAGLC 250 → M 239 TLNRTTAANAC 250 /A251E/A254Q/R259L/ A267E/S280N), was obtained. Compared to the wild type, the autolysis of M9 was significantly alleviated, and its half-life at 60 °C was increased approximately 39-fold (from 1.6 to 62.4 min). The optimal temperature and specific activity of M9 increased by 5 °C (from 60 to 65 °C) and 62% (4985 vs 3078 U/mg), respectively. M9 showed significant advantages in shrimp shell deproteinization.

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Section: Resultsmentioning

confidence: 99%

Section: Transplantation and Optimization Of Wt-loop 18mentioning

confidence: 99%

Improving the Activity and Stability of Serine Protease ThAPT3 by Alleviating Self-Cleavage and Its Application in Deproteinization of Shrimp Shells

Wang

Dong

Zhou

et al. 2023

J. Agric. Food Chem.

View full text Add to dashboard Cite

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“…This is also true for enzymatic reactions, however, the protein fold of enzymes limits the maximal applicable temperature. The catalytic activity of most enzymes increases up to a temperature of 50 °C, above this temperature the denaturation of the enzyme starts and reduces activity by irreversible denaturation (134,135,138).…”

Section: Temperature Of the Enzymatic Hydrolysismentioning

confidence: 99%

Enzymatic Pretreatment of Plant Cells for Oil Extraction

Vovk

Karnpakdee

Ludwig

et al. 2023

Food Technol. Biotechnol. (Online)

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Oil from oilseeds can be extracted by mechanical extraction (pressing), aqueous extraction, or by extraction with organic solvents. Although solvent extraction is the most efficient method, organic solvents are a potential hazard to the life and health for workers as well as to the environment, when solvent vapors are released and act as air pollutant with a high ozone forming potential. Pressing is safer, environmentally friendly, and it preserves valuable natural components in the resulting oils. The problems associated with pressing are the high energy consumption and the lower yield on oil extraction, because the applied mechanical force does not completely destroy the structural cell components storing the oil. In seed cells, the oil is contained in the form of lipid bodies (oleosomes) that are surrounded by a phospholipid monolayer with a protein layer on the surface. These lipid bodies are further protected by the seed cell walls consisting mainly of polysaccharides such as pectins, hemicelluloses and cellulose, but also of glycoproteins. The use of hydrolases to degrade these barriers is a promising pretreatment strategy to support mechanical extraction and improve the oil yield. It is advisable to use a combination of enzymes different activities when considering the multicompartment and multicomponent structure of oilseed cells. This article gives an overview on the microstructure and composition of oilseed cells, reviews enzymes capable to destroy oil containing cell compartments, and summarizes the main parameters of enzymatic treatment procedures, such as the composition of the enzyme cocktail, the amount of enzyme and water used, temperature, pH, and the duration. Finally, it analyzes the efficiency of proteolytic, cellulolytic, and pectolytic enzyme pretreatment to increase the yield of mechanically extracted oil from various types of vegetable raw materials with the main focus on oilseeds.

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“…It is generally believed that the flexible region is one of the key regions contributing to poor enzyme stability. Flexible region modification strategies mainly include iterative saturation mutations, substitution with rigid sequences, flexible region deletion, and introduction of glycosylation . However, the flexible region usually consists of multiple amino acid residues, making the iterative saturation mutation within these regions a ton of work .…”

Section: Introductionmentioning

confidence: 99%

“…Flexible region modification strategies mainly include iterative saturation mutations, 16 substitution with rigid sequences, 16 region deletion, 17 and introduction of glycosylation. 18 However, the flexible region usually consists of multiple amino acid residues, making the iterative saturation mutation within these regions a ton of work. 16 On the other hand, substitution or deletion of the flexible regions does not always work smoothly.…”

Section: ■ Introductionmentioning

confidence: 99%

Enhanced Thermostability and Catalytic Activity of Streptomyces mobaraenesis Transglutaminase by Rationally Engineering Its Flexible Regions

Yang

Wang

et al. 2023

J. Agric. Food Chem.

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Streptomyces mobaraenesis transglutaminase can catalyze the cross-linking of proteins, which has been widely used in food processing. In this study, we rationally modified flexible regions to further improve the thermostability of FRAPD-TGm2 (S2P-S23V-Y24N-E28T-S199A-A265P-A287P-K294L), a stable mutant of the transglutaminase constructed in our previous study. First, five flexible regions of FRAPD-TGm2 were identified by molecular dynamics simulations at 330 and 360 K. Second, a script based on Rosetta Cartesian_ddg was developed for virtual saturation mutagenesis within the flexible regions far from the substrate binding pocket, generating the top 18 mutants with remarkable decreases in folding free energy. Third, from the top 18 mutants, we identified two mutants (S116A and S179L) with increased thermostability and activity. Finally, the above favorable mutations were combined to obtain FRAPD-TGm2-S116A-S179L (FRAPD-TGm2A), exhibiting a half-life of 132.38 min at 60 °C (t 1/2(60 °C)) and a specific activity of 79.15 U/mg, 84 and 21% higher than those of FRAPD-TGm2, respectively. Therefore, the current result may benefit the application of S. mobaraenesis transglutaminase at high temperatures.

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Thermal stability enhancement: Fundamental concepts of protein engineering strategies to manipulate the flexible structure

Cited by 51 publications

References 209 publications

Improving the Activity and Stability of Serine Protease ThAPT3 by Alleviating Self-Cleavage and Its Application in Deproteinization of Shrimp Shells

Improving the Activity and Stability of Serine Protease ThAPT3 by Alleviating Self-Cleavage and Its Application in Deproteinization of Shrimp Shells

Enzymatic Pretreatment of Plant Cells for Oil Extraction

Enhanced Thermostability and Catalytic Activity of Streptomyces mobaraenesis Transglutaminase by Rationally Engineering Its Flexible Regions

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