Brief note

Mutarotase effect on colorimetric determination of blood glucose with β-D-glucose oxidase

Energy-consuming processes should be avoided while separating biomass components. This study investigated the dewatering and extraction of components from Sorghum bicolor silage using liquefied NH₃ (NH₃(l)). Using a plug-flow-type reactor, NH₃(l) was passed through the specimen at 20 °C and 0.85 MPa. When the NH₃/wet sample weight was 4.7, a 31.0 wt% dry ammonia-treated sample (AT) was obtained as an extract, achieving a 96.5 wt% dewatering ratio. Glucose, xylose, and an acid-insoluble fraction were retained in the AT, whereas lactic and acetic acids were separated as an extract. Crystalline cellulose was transformed, and the increase in glucose and xylose yields from the enzymatic hydrolysis of the AT was similar levels to those from sulfuric acid hydrolysis. Therefore, NH₃(l) treatment can dewater S. bicolor silage without severe pretreatment. In addition, it can be used in biofuel and chemical production using enzymatically degradable ATs from which organic acids are separated as extracts.

The glycoside hydrolase family 55 (GH55) includes inverting exo-β-1,3-glucosidases and endo-β-1,3-glucanases, acting on laminarin, which is a β1-3/1-6-glucan consisting of a β1-3/1-6-linked main chain and β1-6-linked branches. Despite their different modes of action toward laminarin, endo-β-1,3-glucanases share with exo-β-1,3-glucosidases conserved residues that form the dead-end structure of subsite −1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-β-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-β-1,3-glucanases, degraded internal β-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. β1-3-Glucans lacking β1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal β1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain β1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but k_cat/K_m values to laminarioligosaccharides (≤4.21 s⁻¹ mM⁻¹) were much lower than to laminarin (5920 s⁻¹ mM⁻¹). These results indicate that β-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites −1 and −2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-β-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-β-1,3-glucanases.

Amylomaltase can be used to synthesize large ring cyclodextrins (LR-CDs), applied as drug solubilizer, gene delivery vehicle and protein aggregation suppressor. This study aims to determine the functional amino acid positions of Corynebacterium glutamicum amylomaltase (CgAM) involved in LR-CD synthesis by site-directed mutagenesis approach and molecular dynamic simulation. Mutants named Δ167, Y23A, P228Y, E231Y, A413F and G417F were constructed, purified, and characterized. The truncated CgAM, Δ167 exhibited no starch transglycosylation activity, indicating that the N-terminal domain of CgAM is necessary for enzyme activity. The P228Y, A413F and G417F produced larger LR-CDs from CD36-CD40 as compared to CD29 by WT. A413F and G417F mutants produced significantly low LR-CD yield compared to the WT. The A413F mutation affected all tested enzyme activities (starch tranglycosylation, disproportionation and cyclization), while the G417F mutation hindered the cyclization activity. P228Y mutation significantly lowered the k_cat of disproportionation activity, while E231Y mutant exhibited much higher k_cat and K_m values for starch transglycosylation, compared to that of the WT. In addition, Y23A mutation affected the kinetic parameters of starch transglycosylation and cyclization. Molecular dynamic simulation further confirmed these mutations’ impacts on the CgAM and LR-CD interactions. Identified functional amino acids for LR-CD synthesis may serve as a model for future modification to improve the properties and yield of LR-CDs.

The carnivorous teleost fish is often intolerant to high levels of postprandial plasma glucose. This study aimed to evaluate the effects of insulin-like growth factor-1 (IGF-1) and growth hormone (GH) administrations on plasma glucose levels and expression of glucose transporters (GLUTs) in various tissues of hybrid grouper, and hence to further clarify the hormone-GLUTs-plasma glucose regulating axis. Twenty-four experimental fish (average body weight: 77.5 ± 5.4 g) were selected and injected with recombinant human IGF-1 (0.2 μg/g body weight) and PBS (0.01 mol/L) in enterocoelia, respectively, and in the GH injected experiment, the same quantity of fish (average body weight: 103.8 ± 5.8 g) were administrated with GH at a dose of 0.5 μg/g body weight or with PBS at a dose of 0.01 mol/L. Results showed that plasma glucose level was significantly (P < 0.05) declined by the IGF-1 administration but elevated by the GH administration. Plasma IGF-1 concentration was significantly (P < 0.01) elevated by the IGF-1 administration, while GH concentration did not significantly (P ≥ 0.05) respond to the GH administration. The relative mRNA levels of insulin-like growth factor-1 receptor a (IGF-Ra) in liver and muscle were decreased significantly with the IGF-1 administration, and a similar variation tendency was also found in insulin-like growth factor-1 receptor b (IGF-Rb) in liver, muscle and adipose tissues. Besides, the relative mRNA level of insulin receptor (IRS) in liver was significantly increased in the IGF-1 administrated group. After the GH administration, the mRNA levels of hepatic growth factor receptor 2 (GHR2) and IGF-1 were significantly elevated. As for GLUTs, the relative mRNA levels of GLUT1 and GLUT2 in liver were obviously elevated by the IGF-1 administration, while the mRNA level of GLUT4 in muscle was reduced. In liver, the protein levels of GLUT1, 2 and 4 were significantly elevated by the IGF-1 administration, and in adipose, only GLUT1 was observed to have a significantly increased protein level. The mRNA expression of GLUTs was less affected by the GH administration. The protein level of GLUT1 in liver was significantly reduced by the GH administration, while in adipose, it was significantly increased. The protein level of GLUT2 in liver or adipose showed an opposite variation as that of GLUT1. Overall, IGF-1 had a hypoglycemic effect on hybrid grouper, and this probably was through up-regulating the protein levels of hepatic GLUT1, 2 and 4 and adipose GLUT1. GH showed an opposite role in regulating plasma glucose level as IGF-1.

Regenerated cellulose can be prepared by treatment with an ionic liquid (IL) and an anti-solvent such as water, which significantly enhances the enzymatic hydrolysis in comparison to crystalline cellulose. The IL-aqueous two-phase system (IL-ATPS) is consisted of IL-condensed top phase and salt-condensed bottom phase, which could be suitable to produce regenerated cellulose with smaller amount of IL. Using IL-ATPS with different pH, the enzymatic saccharification efficiency of crystalline cellulose was determined. The use of 1-allyl-3-methylimidazolium chloride resulted in relatively higher yield of glucose production as compared to 1-butyl-3-methylimidazolium chloride. The IL-ATPS showing optimal pH for cellulase was prepared with mixed salt (NaH₂PO₄/Na₂HPO₄ = 5/1 (wt/wt)), which provide a regenerated cellulose with the pH range of 4.8–4.9 in enzymatic reaction mixture. Using such regenerated cellulose as feed of saccharification, the final yield of glucose was about 70%.

Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation, is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression.