Spatial-Temporal Heterogeneity in Large Three-Dimensional Nanofibrillar Cellulose Hydrogel for Human Pluripotent Stem Cell Culture.

One approach to cell expansion is to use large hydrogel for growing a large number of cells. Nanofibrillar cellulose (NFC) hydrogel has been used for human induced pluripotent stem cell (hiPSCs) expansion. However, little is known about the status of hiPSCs at the single cell level inside large NFC hydrogel during culture. To understand the effect of NFC hydrogel property on temporal-spatial heterogeneity, hiPSCs were cultured in 0.8 wt% NFC hydrogel with different thicknesses with the top surface exposed to the culture medium. The prepared hydrogel exhibits less restriction in mass transfer due to the presence of macropores and micropores interconnecting the macropores. More than 85% of cells at different depths survive after 5 days of culture inside 3.5 mm thick hydrogel. Biological compositions at different zones inside the NFC gel were examined over time at a single-cell level. A dramatic concentration gradient of growth factors estimated in the simulation along 3.5 mm NFC hydrogel could be a reason for the spatial-temporal heterogeneity in protein secondary structure and protein glycosylation and pluripotency loss at the bottom zone. pH change caused by the lactic acid accumulation over time leads to changes in cellulose charge and growth factor potential, probably another reason for the heterogeneity in biochemical compositions. This study may help to develop optimal conditions for producing high-quality hiPSCs in large nanofibrillar cellulose hydrogel at scale.

The fermentation optimization for alkaline protease production by Bacillus subtilis BS-QR-052.

Introduction: Proteases exhibit a wide range of applications, and among them, alkaline proteases have become a prominent area of research due to their stability in highly alkaline environments. To optimize the production yield and activity of alkaline proteases, researchers are continuously exploring different fermentation conditions and culture medium components. Methods: In this paper, the fermentation conditions of the alkaline protease (EC 3.4.21.14) production by Bacillus subtilis BS-QR-052 were optimized, and the effect of different nutrition and fermentation conditions was investigated. Based on the single-variable experiments, the Plackett-Burman design was used to explore the significant factors, and then the optimized fermentation conditions, as well as the interaction between these factors, were evaluated by response surface methodology through the Box-Behnken design. Results and discussion: The results showed that 1.03% corn syrup powder, 0.05% MgSO4, 8.02% inoculation volume, 1:1.22 vvm airflow rate, as well as 0.5% corn starch, 0.05% MnSO4, 180 rpm agitation speed, 36°C fermentation temperature, 8.0 initial pH and 96 h incubation time were predicted to be the optimal fermentation conditions. The alkaline protease enzyme activity was estimated to be approximately 1787.91 U/mL, whereas subsequent experimental validation confirmed it reached 1780.03 U/mL, while that of 500 L scale-up fermentation reached 1798.33 U/mL. This study optimized the fermentation conditions for alkaline protease production by B. subtilis through systematic experimental design and data analysis, and the activity of the alkaline protease increased to 300.72% of its original level. The established model for predicting alkaline protease activity was validated, achieving significantly higher levels of enzymatic activity. The findings provide valuable references for further enhancing the yield and activity of alkaline protease, thereby holding substantial practical significance and economic benefits for industrial applications.

d-Glucose Isomerization with PAMAM Dendrimers as Environmentally Friendly Catalysts.

The isomerization of d-glucose to d-fructose plays a key role in the biochemical and chemical conversion of biomass, and it is therefore desirable to develop and improve catalysts for this reaction. In this study, the environmentally friendly polymer poly(amidoamine) (PAMAM) dendrimer's properties as catalysts for this isomerization are investigated. The experimental results showed that the PAMAM dendrimers, which have basic terminal groups, can effectively promote the d-glucose isomerization reaction. Under the optimized reaction conditions, d-fructose was generated with a 20% maximum yield and above 90% selectivity. 13C and 2H isotope experiments by NMR were carried out to explore the reaction mechanism. When the reaction was performed in D2O, the C1 signal of d-fructose changed to a triplet, which confirmed that the C1 carbon binds to a deuterium atom, i.e., isotopic exchange. It was also found that the deuterium atom at the C2 position of d-glucose-2-d1 cannot transfer to d-fructose. These data indicate that PAMAM dendrimers catalyze d-glucose isomerization through a mechanism, which includes deprotonation, formation of ene-diol intermediate, and proton exchange with the solvent.