Applied Biochemistry and Biotechnology最新文献

This study assessed the impact of organic loading rate (OLR) on methane (CH₄) production in the anaerobic co-digestion (AcoD) of sugarcane vinasse and molasses (SVM) (1:1 ratio) within a thermophilic fluidized bed reactor (AFBR). The OLR ranged from 5 to 27.5 kg COD.m^-3.d^-1, with a fixed hydraulic retention time (HRT) of 24 h. Organic matter removal varied from 56 to 84%, peaking at an OLR of 5 kg COD.m^-3.d^-1. Maximum CH₄ yield (MY) (272.6 mL CH₄.g^-1COD_rem) occurred at an OLR of 7.5 kg COD.m^-3.d^-1, while the highest CH₄ production rate (MPR) (4.0 L CH₄.L^-1.d^-1) and energy potential (E.P.) (250.5 kJ.d^-1) were observed at an OLR of 20 kg COD.m^-3.d^-1. The AFBR exhibited stability across all OLR. At 22.5 kg COD.m^-3.d^-1, a decrease in MY indicated methanogenesis imbalance and inhibitory organic compound accumulation. OLR influenced microbial populations, with Firmicutes and Thermotogota constituting 43.9% at 7.5 kg COD.m^-3.d^-1, and Firmicutes dominating (52.7%) at 27.5 kg COD.m^-3.d^-1. Methanosarcina (38.9%) and hydrogenotrophic Methanothermobacter (37.6%) were the prevalent archaea at 7.5 kg COD.m^-3.d^-1 and 27.5 kg COD.m^-3.d^-1, respectively. Therefore, this study demonstrates that the organic loading rate significantly influences the efficiency of methane production and the stability of microbial communities during the anaerobic co-digestion of sugarcane vinasse and molasses, indicating that optimized conditions can maximize energy yield and maintain methanogenic balance.

Electro-acupuncture (EA) is identified as an effective therapeutic method for cerebral ischemia/reperfusion injury (CIRI), which is a combination of Chinese traditional acupuncture and modern electro-therapy. However, the downstream molecular mechanisms of EA in CIRI process remains largely unknown. The purpose of the present study is to unveil the therapeutic effect of EA on CIRI rat and its regulatory mechanisms. At first, we constructed middle cerebral artery occlusion (MCAO) rat models and then treated them with EA to observe the pathological changes. The results indicated that EA decreased the infarct volume (43.81 ± 3.34 vs 15.96 ± 2.22) and the neurological scores (3.33 ± 0.52 vs 1.67 ± 0.52) and suppressed the apoptosis in MCAO model rats. For ferroptosis analysis, EA decreased the Fe2 + (0.08 ± 0.01 vs 0.06 ± 0.01), MDA (36.61 ± 4.29 vs 21.72 ± 2.79), and LPS (5.25 ± 0.69 vs 2.89 ± 0.42) contents and increased the GSH (4.94 ± 1.04 vs 11.69 ± 1.88) content in MCAO model rats. We next detected whether succinylation mediated EA-treated I/R injury. According to immunoprecipitation and western blot analysis, EA treatment could lower both levels of succinylation and KAT3B in MCAO rats. Moreover, mechanism experiments unveiled that KAT3B promoted the succinylation of the ferroptosis-related protein ACSL4 at K661 site and thus stabilizing ACSL4. Finally, EA-treated MCAO rats were further injected with KAT3B expression vector. The results showed that KAT3B overexpression increased the infarct volume (31.44 ± 3.92 vs 7.94 ± 2.84) and the neurological scores (2.67 ± 0.51 vs 1.33 ± 0.51) and promoted the apoptosis in EA treated MCAO model rats. For ferroptosis analysis, KAT3B overexpression increased the Fe2 + (0.08 ± 0.01 vs 0.05 ± 0.01), MDA (29.24 ± 4.30 vs 22.06 ± 1.89), and LPO (5.07 ± 0.45 vs 2.88 ± 0.49) contents and decreased the GSH (7.86 ± 1.09 vs 11.06 ± 1.76) content in EA treated MCAO model rats. Collectively, our study demonstrates that EA plays a therapeutic role in CIRI through suppressing KAT3B-induced stabilization of ACSL4 to inhibit ferroptosis. These findings contribute to our understanding of the molecular mechanisms underlying the neuroprotective effects of EA and open new avenues for the development of innovative therapeutic strategies for CIRI.

Soybean molasses, which contains high levels of raffinose family oligosaccharides (RFOs) such as stachyose and raffinose, is subjected to a process of bio-purification to remove sucrose while maintaining the RFOs, consequently increasing its value. This study employed morphological observation, physiological and biochemical studies, and molecular biology techniques to identify YA176, a yeast strain renowned for its effective bio-purification of soy molasses. Through single-factor and orthogonal experiments, optimal bio-purification conditions were established. YA176, belonging to Wickerhamomyces anomalus, demonstrated robust growth across a wide range of temperature and pH levels, coupled with remarkable tolerance to glucose, sucrose, and NaCl up to 41.2%, 47.3%, and 10%, respectively. Under these optimized conditions, YA176 efficiently utilized sucrose while preserving 93.3% of raffinose and 78.6% of stachyose, ensuring the retention of functional RFOs. In summary, yeast strain YA176 exhibits exceptional bio-purification abilities, making it an ideal candidate for producing functional RFOs from soy molasses.

The current study aimed to produce an amyloglucosidase enzyme from the fungal consortium. The best amylolytic fungal consortia were identified as Alternaria alternata and Aspergillus niger through the 18S rDNA technique. Fermentation kinetics and various nutritional and cultural parameters were analyzed. Maximum production was obtained in M4 media, pH 5.5, 30 °C, and 4 mL inoculum at 150 rpm after 72 h of incubation. Along with that, sodium nitrate at 2.5%, maltose, beef extract 1%, zinc sulfate (0.1%), and Tween 80 (0.1%) supported the maximum amyloglucosidase production. Amyloglucosidase was partially purified up to 1.6 purification fold with a specific activity of 1.84 Umg^-1 in a stepwise manner by ammonium sulfate purification, dialysis, and ion exchange chromatography. The AMG enzyme also revealed maximum activity at 50 °C with 5.0 pH. Upon the kinetic analysis, the specific yield coefficient Yp/x and volumetric rates Qp and Qx were also found to be significant in the above optimized conditions. The Km value 0.33 mg mL^-1 and Vmax 26.31 U mL^-1 were obtained at 1% soluble starch substrate. Thermodynamic parameters for soluble starch hydrolysis were as follows: ΔH = 48.78 kJ mol^-1, (Ea) =  - 46.0 kJ mol^-1, and ΔS =  - 43.10 J mol^-1 K^-1. This finding indicates the indigenously isolated fungal consortium can be the best candidate for industrial applications.

Sucrose isomerase is an important food enzyme that catalyzes the isomerization of sucrose into isomaltulose, a functional sugar widely used in food industry, while the production level of sucrose isomerase in food safe host strains was much lower than industrial requirement. Bacillus subtilis is an excellent host strain for recombinant protein expression, which owns the characteristics of powerful secretory capability and generally recognized as safe state. In this study, the expression of sucrose isomerase in B. subtilis was improved through expression element optimization and fermentation optimization. Firstly, the extracellular chaperone PrsA was overexpressed to enhance extracellular folding of sucrose isomerase, which improved the recombinant expression level by 80.02%. Then, the protein synthesis level was optimized through promoter screening, improving the recombinant expression level by 60.40%. On the basis of strain modification, the fermentation conditions including nitrogen source, carbon source, metal ion, pH and temperature were optimized successively in shake-flask. Finally, the 3 L bioreactor cultivation condition was optimized and yielding a sucrose isomerase activity of 862.86 U/mL, the highest level among the food safety strains. This study provides an effective strategy to improve the expression level of food enzymes in B. subtilis.

Enzymatic biofuel cells (EBFC) are promising sources of green energy owing to the benefits of using renewable biofuels, eco-friendly biocatalysts, and moderate operating conditions. In this study, a simple and effective EBFC was presented using an enzymatic composite material-based anode and a nonenzymatic bimetallic nanoparticle-based cathode respectively. The anode was constructed from a glassy carbon electrode (GCE) modified with a multi-walled carbon nanotube (MWCNT) and ferrocene (Fc) as a conductive layer coupled with the enzyme glucose oxidase (GOx) as a sensitive detection layer for glucose. A chitosan layer was also applied to the electrode as a protective layer to complete the composite anode. Chronoamperometry (CA) results show that the MWCNT-Fc-GOx/GCE electrode has a linear relationship between current and glucose concentration, which varied from 1 to 10 mM. The LOD and LOQ were calculated for anode as 0.26 mM and 0.87 mM glucose, respectively. Also the sensitivity of the proposed sensor was calculated as 25.71 $\mu$ A/mM. Moreover, the studies of some potential interferants show that there is no significant interference for anode in the determination of glucose except ascorbic acid (AA), uric acid (UA), and dopamine (DA). On the other hand, the cathode consisted of a disposable pencil graphite electrode (PGE) modified with platinum-palladium bimetallic nanoparticles (Nps) which exhibit excellent conductivity and electron transfer rate for the oxygen reduction reaction (ORR). The constructed EBFC was optimized and characterized using various electroanalytical techniques. The EBFC consisting of MWCNT-Fc-GOx/GCE anode and Pt-PdNps/PGE cathode exhibits an open circuit potential of 285.0 mV and a maximum power density of 32.25 µW cm^-2 under optimized conditions. The results show that the proposed EBFC consisting of an enzymatic composite-based anode and bimetallic nanozyme-based cathode is a unique design and a promising candidate for detecting glucose while harvesting power from glucose-containing natural or artificial fluids.

Aspergillus oryzae is an ideal cell factory for protein expression with powerful protein processing and secretion capabilities. The current study aimed to explore the homologous expression of A. oryzae lipase AOL (GenBank: KP975533) by constructing an auxotrophic A. oryzae △pyrG△nptB and subsequently characterizing the immobilization and catalytic properties of recombinant lipase. Initially, the pyrG gene knocked out in wild-type A. oryzae by homologous recombination, followed by the creation of a uridine/uracil auxotroph transformation. Through this system, the protease gene nptB was precisely knocked out, leading to a substantial decrease in extracellular (39.04%) and intracellular (90.07%) protease activity. The A. oryzae △nptB△pyrG strain was used as host for homologous expression of lipase AOL. After transformation of linearized lipase-expression cassette, the engineered A. oryzae AOL-8 was screened out with the lipase gene copy number of 14, exhibiting extracellular and intracellular lipase activities of 1.75 U/mL and 46.4 U/g, respectively. Subsequently, the production and immobilization of the recombinant lipase, via physical adsorption on macroporous resin XRZ04B, were achieved through submerged fermentation of the AOL-8 strain. The results of esterification catalytic properties of immobilized recombinant lipase indicated that the lipase exhibited optimal catalytic activity with lauric acid and methanol as substrates, a reaction temperature of 35 °C, and n-hexane as the preferred solvent medium; its highest conversion rate can reach at 72.3%.

Glutathione remains one of the most efficient antioxidant compounds in living systems, and the biological abilities of hydrazides have been well documented in literature. This study highlights the phytochemical constituents of garlic and the separation of the bioactive benzoic acid, 4-chloro- 1-(4-methoxyphenyl) hydrazide (BA4C) using gas chromatography-mass spectroscopy (GC–MS) technique. Preliminary phytochemical screening reveals the presence of alkaloids, saponins, flavonoids, tannins, terpenoids, steroids and phenols. Computationally, compound BA4C was optimized using the B3LYP/aug-cc-PVDZ DFT method. Spectroscopic studies of the compound involved analysis of the vibrational FT-IR frequencies and the modes of vibrations. Frontier molecular orbitals analysis records an energy gap of 4.3391 eV; NBO studies reveal that the compound has strong perturbation energies of 246 kcal/mol and 269 kcal/mol among its intramolecular interactions such as (uppi)*C₁₂ – C₁₃ to (uppi)*C₁₄ – C₁₅ and (uppi)*C₁₁ – C₁₆ to (uppi)*C₁₄ – C₁₅, respectively. According to the visualization of non-covalent interactions, steric repulsions were observed at the core of the phenyl and benzene rings. However, other regions of the compound depict a significant balance of forces between steric repulsions and van der Waals forces. To significantly deduce the reducing power of compound BA4C, electrons were found to be highly localized at the methoxy and hydrazide moieties significantly implying their propensity to donate electrons to oxidized systems. Furthermore, ADMET analysis reveals that the compound has two hydrogen donors. Most significantly, the compound binds to NADPH dehydrogenase (5V4P) and glutathione reductase (1XAN) with binding energies of − 6.0 kcal/mol and − 8.0 kcal/mol showing considerable favourable binding feasibility as well as forming plural hydrogen bonds with the amino acid residues. Notably, BA4C was bonded at the active site of 1XAN, which implies the ability of the compound for the reduction of oxidized glutathione.

Graphical Abstract

The aim of this study is to determine the effect of the digestibility of cassava starch by the enzymes extracted from corn malt, which will constitute one of the answers to the problem of integrating local products into the process in a modern brewery. Cassava starch solutions of different concentrations (E0: 0 g/L; E1: 1 g/L; E2: 1.1 g/L; E3: 1.2 g/L; E4: 1.3 g/L; E5: 1.4 g/L and E6: 1.5 g/L) were prepared and subjected to two treatments (gelatinized and non-gelatinized) and 5 mL of each were placed in a test tube. Three millilitres (3 mL) of the solution containing amylases extracted from malt corn was then added to each of the test tubes containing the cassava flour solutions. All the treatments were subjected to three temperature stages (50 °C for 15 min, 90 °C for 20 min, and 100 °C for 75 min). Twenty-eight (28) objects (two duplicates) were experimented in a complete factorial design (2 treatments × 2 temperature levels). The results obtained showed that gelatinization had no effect, which could be due to the high optimum temperatures of corn enzyme activity. The concentrations also did not have significant differences which shows that these concentrations can well be used on an industrial scale to digest cassava starch by corn malt enzymes.