Protein-Glutaminase Engineering Based on Isothermal Compressibility Perturbation for Enhanced Modification of Soy Protein Isolate

Abstract

Protein-glutaminase plays a significant role in future food (e.g., plant-based meat) processing as a result of its ability to improve the solubility, foaming, emulsifying, and gel properties of plant-based proteins. However, poor stability, activity, high pressure, and high shear processing environments hinder its application. Therefore, we developed an application-oriented method isothermal compressibility perturbation engineering strategy to improve enzyme performance by simulating the high-pressure environment. The best variant with remarkable improvement in specific activity and half-time, N16M/Q21H/T113E, exhibited a 4.28-fold increase compared to the wild type in specific activity (117.18 units/mg) and a 1.23-fold increase in half-time (472 min), as one of the highest comprehensive performances ever reported. The solubility of the soy protein isolate deaminated by the N16M/Q21H/T113E mutant was 55.74% higher than that deaminated by the wild type, with a tinier particle size and coarser texture. Overall, this strategy has the potential to improve the functional performance of enzymes under complex food processing conditions.

Computer-Aided Reconstruction and Application of Bacillus halodurans S7 Xylanase with Heat and Alkali Resistance

“…All data were subjected to at least three biological replicates, and the error bars in the figure represent the SEM (standard error of the mean). , Statistical analysis was performed using OriginPro 2023, and statistical significance was set at P < 0.05.…”

Isothermal Compressibility Perturbation as a Protein Design Principle for T1 Lipase Stability–Activity Trade-Off Counteracting

“…19 Analysis of the root-mean square deviation (RMSD), radius of gyration (R g ), and solvent-accessible surface area (SASA) of the protein was performed using the built-in program of GROMACS. 20 β T was calculated according to Zheng et al 13 The binding free energy and energy decomposition per residue of wild-type and mutant protein−ligand complexes were calculated with the molecular mechanics Poisson−Boltzmann surface area (MM-PBSA) method.…”

“…According to Zheng et al, free energy calculation was performed using the FoldX suite and I-Mutant 2.0 software. 13 In addition, for STRUM (https://zhanglab.ccmb.med.umich.edu/STRUM/), 22 Mode I was selected to predict the single-point mutations, then the protein sequence and a list of mutants were input, and STRUM was run to obtain the prediction result.…”

“…In our previous studies, the isothermal compressibility perturbation engineering (ICPE) strategy could improve the properties of enzymes . Starting from the enzyme as a whole entity, we considered the fluctuation of the whole enzyme and secondary structures that make up the enzyme skeleton to identify high fluctuation regions.…”

“…9,10,12 In our previous studies, the isothermal compressibility perturbation engineering (ICPE) strategy could improve the properties of enzymes. 13 Starting from the enzyme as a whole entity, we considered the fluctuation of the whole enzyme and secondary structures that make up the enzyme skeleton to identify high fluctuation regions. The compressibility of an enzyme, which is characterized by its isothermal compressibility (β T ), is an index used to measure the plasticity of the high-fluctuation region.…”