Preparative Biochemistry & Biotechnology最新文献

In this paper, we have analyzed biodesulfurization of dibenzothiophene (DBT) and 4,6-dibenzothiophene (4,6-DMDBT) by 4S metabolic pathway using molecular simulations. Docking analysis revealed lower binding energies and inhibition constants (K_i) for 4,6-DMDBT and its metabolic intermediates with DSZ enzymes than DBT and its intermediates. The complexes of substrate and its metabolites with DSZ enzymes had higher stability for 4,6-DMDBT than DBT owing to lower RMSF values than apoprotein. The docking analysis revealed affinity of the inhibitors HBPS and HBP (for DBT) and DMHBPS and DMHBP (for 4,6-DMDBT) toward DSZ enzyme due to negative binding energies. Molecular dynamics simulations showed stability of several enzyme-inhibitor complexes. The inhibitory effect of DMHBPS on DSZC enzyme (K_i = 1.53 µM) and DMHBP on DSZB enzyme (K_i = 3.87 µM) was most marked. The inhibitory effect of HBP on DSZC and DSZB enzymes was moderate due to K_i of 6.36 and 7.93 µM, respectively. The inhibition effect of DMHBP on the DSZA enzyme was insignificant due to high K_i of 53.6 µM. In summary, higher stability of enzyme-substrate complexes and strong inhibition by DMHBPS and DMHBP (due to very low K_i) contribute to slower biodesulfurization of 4,6-DMDBT as compared to DBT.

Doramectin, a 16-membered macrocyclic lactone that is widely used in the treatment of mammalian parasitic diseases. Doramectin was produced by mutant Streptomyces avermitilis using cyclohexanecarboxylic acid as a precursor. As a semi-synthetic insecticidal agent produced, the production of doramectin was low, which could not be satisfy the demands of industrial fermentation. In this study, a high-yield mutant strain S. avermitilis DA-137 was screened from the starting strain S. avermitilis D-11 through a high-throughput screening strategy. S. avermitilis D-11 was treated with iterative atmospheric and room temperature plasma mutagenesis to induce mutations. Mutation strains were prescreened by spreading on enhanced doramectin-tolerance plates and were rescreened in 24-deep microtiter plates and via microplate readers to obtain high-producing strains. The resulting mutant strain S. avermitilis DA-137 produced 431.5 mg/L doramectin, a 187% increase compared to that of D-11, revealing mutagenesis and doramectin-tolerance screening is an efficient method to enhance doramectin production. Then, fermentation medium was optimized using the response surface method to improve doramectin production. In the optimized fermentation medium, the yield of doramectin was increased to 934.5 mg/L in shake flask. Furthermore, batch culture was carried out in a 50 L fermenter, and the yields of doramectin reached 1217 mg/L at 216 h, which was the highest yield reported to date. This study demonstrates a successful approach for enhancing doramectin production through high-throughput screening strategy and medium optimization, serving as a reference for increasing the yield of other macrocyclic lactone antibiotics.

Thermophilic Geobacillus kaustophilus HTA426 genome possesses a monoacylglycerol lipase (MAGL) gene. MAGLs can synthesize emulsifiers for use in the food and pharmaceutical industries from fatty acids and glycerol. They can also be used to analyze monoacylglycerol (MAG) levels in serum and food. The MAGL gene from strain HTA426 was artificially synthesized and heterologously expressed in Escherichia coli BL21(DE3). The recombinant His-tag fused MAGL (GkMAGL) was purified using a Ni²⁺-affinity column. The purified enzyme showed a temperature optimum at 65 °C and was stable up to 75 °C after 30 min incubation. In addition, the enzyme exhibited a pH optimum of 7.5 and was stable from pH 5.0 to 11.0. The enzyme hydrolyzed monoacylglycerols and showed the highest activity toward 1-monolauroylglycerol. The enzyme was stable in the presence of various organic solvents and detergents. The addition of Triton X-100 significantly increased GkMAGL activity. The thermal stability of the enzyme was higher than that of thermostable MAGL from Geobacillus sp. 12AMOR1 (12AMOR1_MAGL). Circular dichroism spectral analysis showed that the conformational stability of the GkMAGL was higher than that of 12AMOR1_MAGL at higher temperatures. These results indicate that the GkMAGL has useful features that can be used for various biotechnological applications.

Wheat straw contains a high amount of lignin, hindering the action of cellulase and hemicellulase enzymes, leading to difficulties in nutrient absorption by animals from straw feed. However, currently, the biological treatment of straw relies primarily on fungal degradation and cannot be directly utilized for the preparation of livestock feed. This study focuses on enzymatic co-fermentation of wheat straw to produce high-protein, low-cellulose biological feed, integrating lignin degradation with feed manufacturing, thereby simplifying the feed production process. After the optimization using Box-Behnken Design for the feed formulation, with a glucose oxidase addition of 2.46%, laccase addition of 3.4%, and malonic acid addition of 0.6%, the wheat straw feed prepared in this experiment exhibited a true protein content of 9.35%. This represented a fourfold increase compared to the non-fermented state, and the lignocellulose degradation rate of wheat straw reached 45.42%. These results not only highlight the substantial enhancement in protein content but also underscore the significant advancement in lignocellulose breakdown. This formulation significantly enhanced the palatability and nutritional value of the straw feed, contributing to the industrial development of straw feed.

Recently, kafirins from white sorghum [Sorghum bicolor (L) Moench] grain have shown promise as a source of biopeptides with anti-skin aging effects (anti-inflammatory, antioxidant, and inhibition of photoaging-associated enzymes). This study employed response surface methodology (RSM) to optimize the extraction and enzymatic hydrolysis of kafirins (KAF) for the production of peptides with anti-skin aging properties. The optimization of conditions (reaction time and enzyme/substrate ratio) for liquefaction with α-amylase and hydrolysis of KAF with alcalase was performed using 3² complete factorial designs. Subsequently, ultrafiltered peptide extracts were obtained with molecular weights of 1-3 kDa (KAF-UF₃) and lower than 1 kDa (KAF-UF₁), which mainly contain hydrophobic amino acids (proline, leucine, isoleucine, phenylalanine, and valine) and peptide fractions with molecular weights of 0.69, 1.14, and 1.87 kDa. Consequently, the peptide extracts protected immortalized human keratinocytes (HaCaT cells) from ultraviolet B radiation (UVB)-induced damage by preventing the decrease and/or restoring the activity of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)]. Furthermore, KAF-UF₃ and KAF-UF₁ inhibited (20-29%) elastase and collagenase overactivity in UVB-exposed murine fibroblasts (3T3 cells). Thus, KAF-UF₃ and KAF-UF₁ exhibited behavior similar to that observed with glutathione (GSH), suggesting their potential as functional peptide ingredients in skincare products.

This study aimed to enhance the extracellular polymeric substances (EPS) production of Virgibacillus dokdonensis VITP14 and explore its antioxidant potential. EPS and biomass production by VITP14 strain were studied under different culture parameters and media compositions using one factor at a time method. Among different nutrient sources, glucose and peptone were identified as suitable carbon and nitrogen sources. Furthermore, the maximum EPS production was observed at 5% of inoculum size, 5 g/L of NaCl, and 96 h of fermentation. Response surface methodology was employed to augment EPS production and investigate the optimal levels of nutrient sources with their interaction. The strain was observed to produce actual maximum EPS of about 26.4 g/L for finalized optimum medium containing glucose 20 g/L, peptone 10 g/L, and NaCl 50 g/L while the predicted maximum EPS was 26.5 g/L. There was a nine fold increase in EPS production after optimization study. Additionally, EPS has exhibited significant scavenging, reducing, and chelating potential (>85%) at their higher concentration. This study imparts valuable insights into optimizing moderately halophilic bacterial EPS production and evaluating its natural antioxidant properties. According to findings, V. dokdonensis VITP14 was a promising isolate that will provide significant benefits to biopolymer producing industries.

Proteases, enzymes that hydrolyze peptide bonds, have various applications in medicine, clinical applications, and pharmaceutical development. They are used in cancer treatment, wound debridement, contact lens cleaning, prion degradation, biofilm removal, and fibrinolytic agents. Proteases are also crucial in cardiovascular disease treatment, emphasizing the need for safe, affordable, and effective fibrinolytic drugs. Proteolytic enzymes and protease biosensors are increasingly used in diagnostic and therapeutic applications. Advanced technologies, such as nanomaterials-based sensors, are being developed to enhance the sensitivity, specificity, and versatility of protease biosensors. These biosensors are becoming effective tools for disease detection due to their precision and rapidity. They can detect extracellular and intracellular proteases, as well as fluorescence-based methods for real-time and label-free detection of virus-related proteases. The active utilization of proteolytic enzymatic biosensors is expected to expand significantly in biomedical research, in-vitro model systems, and drug development. We focused on journal articles and books published in English between 1982 and 2024 for this study.

To enhance the stability and light resistance of the yellow compounds in citrus pomace, our study successfully isolated and purified five compounds using ultrasonic-assisted extraction and column chromatography. The identified compounds include methyl linoleate, (2-ethyl)hexyl phthalate, 1,3-distearoyl-2-oleoylglycerol, 6,6-ditetradecyl-6,7-dihydroxazepin-2(3H)-one, and n-octadeca-17-enoic acid. The monomers extracted from fresh pomace, compounds 1 and 2, exhibit structural similarities to flavonoids and carotenoids. In contrast, the polymers isolated from fermented pomace, compounds 3, 4, and 5, share structural units with the fresh pomace compounds, indicating the transformation to stable polymeric forms. This suggests that the microbial fermentation process not only enhances the value of citrus pomace, but also provides a promising pathway for the synthesis of natural antioxidant yellow pigments with far-reaching theoretical and practical significance.

In the present study, an initial screening was conducted using 12 types of cell culture media, and four media with the best performance were selected for further study. The optimization of four media blend for YFV production was evaluated using an Augmented simplex centroid mixture design. Among all the different models that were investigated, the quadratic model was found to be the most appropriate model for exploring mixture design. It was found that M10 exhibited the greatest impact on YFV production, followed by M9, M4, and M1. The utilization of M1 and M4 media individually yielded higher compared to their blends with other media. The YFV titers were reduced when M1 media was combined with other media. The utilization of M9 and M10 media in combination resulted a higher viral yield compared to their respective concentrations. The optimal ratio for achieving a higher titer of YFV from primary CEFs was found to be approximately 38:62, with M9 and M10 being the most favorable media blend. The use of a media mixture led to a significant increase of virus titer up to 2.6 × 10⁸ PFU/ml or 2 log titer yield, which is equivalent to 1.92 × 10⁵ doses, without any changes to growth conditions or other process factors. This study concluded that the utilization of a mixture design could be efficiently employed to choose the optimal combination of media blends for enhanced viral production from cell culture.

This research performed cellulase production by Aspergillus fumigatus A4112 and evaluated its potential use in palm oil mill effluent (POME) hydrolysis to recover oil simultaneously with the generation of fermentable sugar useful for biofuel production under non-sterilized conditions. Empty fruit bunch (EFB) without pretreatment was used as carbon source. The combination of nitrogen sources facilitated CMCase production. The maximum activity (3.27 U/mL) was obtained by 1.0 g/L peptone and 1.5 g/L (NH₄)₂SO₄ and 20 g/L EFB at 40 °C for 7 days. High level of FPase activity (39.51 U/mL) was also obtained. Interestingly, the enzyme retained its cellulase activities more than 60% at ambient temperature over 15 days. In enzymatic hydrolysis, Triton X-100 was an effective surfactant to increase total oil recovery in the floating form. High yield of reducing sugar (50.13 g/L) and 21% (v/v) of floating oil was recoverable at 65 °C for 48 h. Methane content of the raw POME increased from 41.49 to 64.94% by using de-oiled POME hydrolysate which was higher than using the POME hydrolysate (59.82%). The results demonstrate the feasibility of the constructed process for oil recovery coupled with a subsequent step for methane yield enhancement in biogas production process that benefits the palm oil industry.