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

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

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

Given the widely existing stability–activity trade-off in enzyme evolution, it is still a goal to obtain enzymes embracing both high activity and stability. Herein, we employed an isothermal compressibility (βT) perturbation engineering (ICPE) strategy to comprehensively understand the stability–activity seesaw-like mechanism. The stability and activity of mutants derived from ICPE uncovered a high Pearson correlation (r = 0.93) in a prototypical enzyme T1 lipase. The best variant A186L/L188M/A190Y exhibited a high Tm value up to 78.70 °C, catalytic activity of 474.04 U/mg, and a 73.33% increase in dimethyl sulfoxide resistance compared to the wild type, one of the highest comprehensive performances reported to date. The elastic activation mechanism mediated by conformational change with a ΔβT range of −6.81 × 10–6 to −1.90 × 10–6 bar–1 may account for the balancing of stability and activity to achieve better performing enzymes. The ICPE strategy deepens our understanding of stability–activity trade-off and boosts its applications in enzyme engineering

Computer-Aided Reconstruction and Application of <i>Bacillus halodurans</i> S7 Xylanase with Heat and Alkali Resistance

β-1,4-Endoxylanase is the most critical hydrolase for xylan degradation during lignocellulosic biomass utilization. However, its poor stability and activity in hot and alkaline environments hinder its widespread application. In this study, BhS7Xyl from Bacillus halodurans S7 was improved using a computer-aided design through isothermal compressibility (βT) perturbation engineering and by combining three thermostability prediction algorithms (ICPE-TPA). The best variant with remarkable improvement in specific activity, heat resistance (70 °C), and alkaline resistance (both pH 9.0 and 70 °C), R69F/E137M/E145L, exhibited a 4.9-fold increase by wild-type in specific activity (1368.6 U/mg), a 39.4-fold increase in temperature half-life (458.1 min), and a 57.6-fold increase in pH half-life (383.1 min). Furthermore, R69F/E137M/E145L was applied to the hydrolysis of agricultural waste (corncob and hardwood pulp) to efficiently obtain a higher yield of high-value xylooligosaccharides. Overall, the ICPE-TPA strategy has the potential to improve the functional performance of enzymes under extreme conditions for the high-value utilization of lignocellulosic biomass

Effects of BNIP-3 silencing on cadmium-induced apoptosis, mitochondrial cyt c release to the cytoplasm, and changes in caspase-9 and caspase-3 expression.

<p>(a) Flow cytometry assessment of the rPT cell apoptosis rate after 12-h cadmium and/or BNIP-3 siRNA treatment. (b) Percentage of apoptotic cells. (c, e) Representative images of cyt c, caspase-9, and caspase-3 western blots. (d, f) Quantitative analysis of cyt c, caspase-9, and caspase-3; grayscale of the control was set at 1. Results are from three independent experiments (mean ± SEM, n = 3). *<i>P</i> < 0.05, **<i>P</i> < 0.01, and ^<i>##</i><i>P</i> < 0.01 as compared to control.</p

Cadmium induced mitochondrial cyt c release to the cytoplasm and subsequent caspase-9 and caspase-3 activation in rPT cells.

<p>(a, c) Representative western blots of cyt c, cleaved caspase-9, and cleaved caspase-3. (b, d) Quantitative analysis of cyt c, cleaved caspase-9, and cleaved caspase-3 western blots; grayscale of the control was set at 1. Quantitative analysis was performed with images from three independent experiments (mean ± SD, n = 3). *<i>P</i> < 0.05, **<i>P</i> < 0.01, and ^<i>##</i><i>P</i> < 0.01 as compared to control.</p

Cadmium induced BNIP-3 expression and cytoplasmic AIF and Endo G translocation to the nucleus after 12-h cadmium treatment.

(a, c, e) Representative images of BNIP-3, AIF, and Endo G western blots. (b, d, f) Quantitative analysis of BNIP-3, AIF, and Endo G; grayscale of the control was set at 1. (g) Cadmium treatment (12 h) triggered AIF nuclear translocation dose-dependently. rPT cells were stained with anti-AIF antibodies and Alexa Fluor 488–labeled goat anti-rabbit IgG. AIF nuclear translocation was evaluated under fluorescence microscopy with DAPI staining. Scale bar: 50 μm. Effects of BNIP-3 silencing on changes in BNIP-3 expression and cytoplasmic AIF and Endo G translocation to the nucleus after 12-h cadmium treatment in the absence or presence of BNIP-3 small interfering RNA (siRNA). (h, j, l) Representative images of BNIP-3, AIF, and Endo G western blots. (i, k, m) Quantitative analysis of BNIP-3, AIF, and Endo G; grayscale of the control was set at 1. Results are from three independent experiments (mean ± SD, n = 3). **P #P ##P < 0.01 as compared to control.</p

Effects of Z-VAD-FMK on cadmium-induced rPT cell apoptosis.

<p>(a) Representative images of cleaved caspase-3 western blot after 12-h cadmium treatment in the absence or presence of Z-VAD-FMK. (b) Quantitative analysis of cleaved caspase-3; grayscale of the control was set at 1. (c) Flow cytometry assessment of the rPT cell apoptosis rate after 12-h cadmium treatment with/without Z-VAD-FMK. (d) Percentage of apoptotic cells. Results are expressed as the mean ± SD of three separate experiments, each performed in triplicate (n = 9). **<i>P</i> < 0.01 as compared to control.</p

Effect of Z-VAD-FMK on BNIP-3 expression and cytoplasmic AIF translocation to the nucleus after 12-h cadmium treatment.

<p>(a) Representative images of BNIP-3 western blot. (b) Quantitative analysis of BNIP-3; grayscale of the control was set at 1. (c) Twelve-hour cadmium treatment with/without Z-VAD-FMK triggered AIF nuclear translocation. rPT cells were stained with anti-AIF antibodies and Alexa Fluor 488–labeled goat anti-rabbit IgG. AIF nuclear translocation was evaluated under fluorescence microscopy with DAPI staining. Scale bar: 50 μm. Results are from three independent experiments (mean ± SD, n = 3). **<i>P</i> < 0.01 as compared to control.</p

Confocal microscopy of MPTP opening after 12-h cadmium (2.5 μM) treatment in the absence or presence of CsA (5 μM).

<p>(a) Representative confocal images of cadmium-induced rPT cells. (b) Quantification of calcein fluorescence. The calcein fluorescence values were quantified relative to the control, where the fluorescence value was set at 100%. (c) Flow cytometry detection of the apoptosis rate after 12-h cadmium (2.5 μM) treatment in the absence or presence of CsA (5 μM) with annexin V–FITC/PI staining. (d) Percentage of apoptotic cells. Results are the mean ± SD of three separate experiments, each performed in triplicate (n = 9). **<i>P</i> < 0.01 as compared to control.</p