Preparative Biochemistry & Biotechnology最新文献

This study aimed to optimize the ultrasonic/microwave-assisted extraction (UAME) technique of Elaeagnus angustifolia polysaccharides (EAP-UM) by response surface methodology (RSM). The optimum conditions include: solid-liquid ratio of 1:23 g/mL, ultrasonic power of 252 W, microwave power of 417 W, and extraction time of 11 min. Under these conditions, the yield of polysaccharides reached 7.87%, superior to ultrasound-assisted extraction (UAE, 5.74%), microwave-assisted extraction (MAE, 6.86%), and hot water extraction (HWE, 4.91%). UAME could also result in higher carbohydrate, protein, and uronic acid contents compared to other methods. The properties of EAP-UM were further analyzed by infrared spectra (IR) and scanning electron microscopy (SEM). Moreover, EAP-UM exhibited a remarkable free radical scavenging ability (DPPH, IC_50, 79 µg/mL) and reduced power. EAP-UM also showed α-amylase inhibiting activity (IC₅₀, 2.826 mg/mL). UAME has the advantage of high extraction rate and short extraction time, and the obtained polysaccharide EAP-UM had better properties. These findings may serve as a reference for the development and application of E. angustifolia.

Staphylococcus aureus has developed resistance to most conventional antibiotics and is a causative agent of serious infections. Alternative therapies are urgently needed. Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, including Escherichia coli (E. coli) and Streptococcus pyogenes (S. pyogenes), and represent a potential solution. While several bacteriocins have shown promise, their synergy with bacteriocins from other bacterial species remains largely unexplored. This work used agar diffusion on Muller-Hinton Agar (MHA) with S. aureus as a test bacterium to evaluate E. coli, S. pyogenes and their combined bacteriocins. The bacteriocins of S. pyogenes showed the maximum antimicrobial activity of zone of inhibition (ZOI), 24.93 mm, compared to that of E. coli bacteriocin, which was 19.28 mm, and that of the combined ones at 100% concentration, 22.6 mm. The combined bacteriocins at 50% concentration showed a reduced activity of 18.35 mm. These observations suggest that the bacteriocins produced by S. pyogenes have higher specificity and activity against S. aureus, making them effective therapeutic agents in the fight against multidrug-resistant infections.

Fungal lipases are the leading industrial biocatalyst due to their broad applications, but high cost limits their commercial usage. The low-cost agri-residues substrates can reduce the cost of lipase production. However, the compatibility of agri-residue with fungal species, recovery process of lipase and stability of the enzyme are crucial steps. The aim of the present work was optimization of lipase production from a suitable combination of fungal culture with a locally available vegetable oilseed cake (mustard/groundnut/almond/cottonseed) in solid-state fermentation process and its direct immobilization. The enzyme produced using selected combination of Rhizopus oryzae and mustard oilseed cake was optimized by Plackett-Burman design, one-factor-at-a-time and central composite design (CCD). The highest enzyme activity of 25.08 U/gds was obtained by CCD at urea 2.11% w/w, inoculum size 1.18% v/w, and moisture content 69.99% w/w. The crude enzyme from the extract was immobilized on functionalized magnetic nanoparticles with the results of protein loading 68.88 ± 3.54 µg/mg of MNPs and activity recovery of 60.33 ± 3.03%. This study can be helpful to explore the suitability of locally available agri-residue for production of lipase and utilization of enzyme in different industrial applications.

L-asparaginase (asparagine amidohydrolase) contributes to 40% of the total enzyme demands worldwide and is one-third of the global requirement as an anti-cancerous drug in treating acute lymphocytic leukemia (ALL), a type of leukemia. This protein breaks down L-asparagine into aspartic acid and ammonia those involved in ALL, rely on for growth and survival. Both non-recombinant and recombinant L-asparaginase can be produced by bacteria when a suitable substrate and method (solid-state fermentation (SSF) or submerged fermentation (SmF) which are techniques to grow microorganisms under controlled conditions), is provided. Between both L-asparaginase's isozymes, asparaginase type II displays higher specific action against L-asparagine and precisely shows antitumor activity. The applied methods in purification of L-asparaginase in the frame of three phases of protein purification strategy known as CIPP (including capture, intermediate purification, and polishing phase) are discussed in this review. Depending on whether the production of the enzyme is intracellular or extracellular, various steps in each phase, like removal of insoluble material, extraction, concentration, and purification, must followed. In this review, authors summarize the upstream processes in L-asparaginase production and the various applied chromatographic and non-chromatographic methods in each step of CIPP, in downstream processes.

Halophilic bacteria are promising candidates for biofuel production because of their efficient cellulose degradation. Their cellulases exhibit high activity, even in the presence of inhibitors and under extreme conditions, making them ideal for biorefinery applications. In this study, we isolated a strain of Halomonas elongata (Kadal6) from decomposed cotton cloth on a Rameshwaram seashore. Morphological, biochemical, and 16S rRNA analyses revealed that Kadal6 was 99.93% similar to the cellulase-producing strain, H. elongata MH25661. The tolerance of the cellulase to inhibitors was assessed through molecular docking with a cellulase model of MH25661 generated by I-TASSER and experimentally using response surface methodology (RSM) with Kadal6. A molecular docking study indicated a high inhibition constant for ethanol, hydroxymethylfurfural (HMF), and furfural. Cellulase from H. elongata Kadal6 (CellHe) showed a maximum inhibition rate of 44.27% at 55 °C, 15% ethanol, and 6.5 g/L furfural and HMF. The enzyme retained 50% of its activity in the presence of these inhibitors, and remained unaffected at 1 g/L furfural and HMF, although inhibition occurred at 3 g/L. H. elongata cellulase demonstrated significant tolerance to inhibition both in vitro (RSM) and in silico, indicating its potential for biorefinery applications in harsh environments.

Chinese hamster ovary (CHO) cells represent the most common host system for the expression of high-quality recombinant proteins. The development of stable CHO cell lines used in industrial recombinant protein production often relies on dihydrofolate reductase (DHFR) and glutamine synthetase (GS) amplification systems. Conventional approaches to develop stable cell lines lead to heterogeneous cell populations. Consequently, it is desirable to adopt innovative strategies to increase the efficiency of clone selection to reduce the time and effort invested in the cell line development process. Attenuating the selection marker gene is an effective strategy for isolating high-producing cells. In this study, we evaluated the efficiency of an attenuated glutamine synthetase selection system for the expression of human tissue plasminogen activator (t-PA) in CHO cells. We introduced an AU-rich element (ARE) at the 3'UTR of the glutamine synthetase coding sequence and employed a weak promoter (mSV40) for the expression of this gene. Subsequently, we analyzed the effect of ARE on the GS RNA levels, and recombinant t-PA expression. Our results demonstrate that the use of ARE significantly enhances the detection of high expressing cells compared to the control. Additionally, the t-PA expression level in GS-ARE clones was approximately 900-fold greater than those without the ARE.

In this study, the potential of pea whey wastewater (PWW) as a substrate for the biosynthesis of docosahexaenoic acid (DHA) was investigated by culturing the strain Aurantiochytrium limacinum SFD-1502. The results showed that culturing SFD-1502 in PWW alone resulted in poor growth, possibly due to an insufficient carbon source. The addition of glucose and monosodium glutamate to PWW resulted in a significant improvement in cell growth, and the dry weight of the cells reaching 43.45 ± 0.39 g/L g/L, comparable to that of the control (using artificial seawater fermentation medium), despite the lipid content in the cells and the DHA proportion in the lipids were slightly lower than those of the control. Subsequent studies demonstrated that the presence of raffinose family oligosaccharides, a higher concentration of arginine, and a lower concentration of Na⁺ relative to artificial seawater in PWW resulted in the reduction of cellular lipids and the proportion of DHA. Furthermore, the chemical oxygen demand (COD) of PWW was reduced by approximately 60% during the fermentation. Consequently, the utilization of PWW in A. limacinum culture for DHA production is a viable and cost-effective strategy.

Myo-inositol is an active sugar alcohol which has important physiological functions. In this study, an engineered strain that could simultaneously utilize glucose and xylose to produce myo-inositol was constructed, and its fermentation performance was determined. Firstly, the ptsG gene was deleted to make E. coli BL21 capable of utilizing glucose and xylose simultaneously as mixed carbon source. Galp and glk genes were introduced to promote the glucose absorption after ptsG knockout. Secondly, the ino1 gene from Saccharomyces cerevisiae SC288 was introduced and the suhB gene was overexpressed to construct the complete biosynthetic pathway of myo-inositol in E. coli BL21. Ultimately, when 20 g/L glucose and xylose with a ratio of 3:2 were used as the mixed carbon source, the consumption rate of the total sugar was the fastest, and the yield of myo-inositol was 0.63 g/L in 50 mL/250 mL culture system. When the fermentation system was expanded to 1 L shake flask, the yield of myo-inositol was 0.69 g/L. This study contributes to the production of myo-inositol with mixed sugar as the carbon source.

The biomethanization of lignocellulosic wastes remains an inefficient and complex process due to lignin structures that hinder the hydrolysis step, therefore, some treatments are required. This work describes the addition of an enriched microbial consortium in the biomethanization of rice straw. The experiment was carried out in lab batch reactors following two strategies: (i) pretreatment of rice straw for 48 h using the enriched microbial consortium (dilution 1:100), and (ii) addition of this enriched microbial consortium (dilution 1:100) directly to the anaerobic reactors (bioaugmentation). The kinetic behavior was described using three models. As a result, the microbial consortium molecular characterization showed 58 different bacterial species, predominantly the Lactobacillaceae family (45.7%), and the Clostridiaceae family (19.1%), which were responsible for the positive effect obtained by bioaugmentation with methane yield increases of 16% (290 L_NCH₄/kgVS) respect to the control. All kinetic models applied fitted the experimental data for cumulative methane production, although the modified Hill model showed the best fit. Bioaugmentation strategies demonstrate their effectiveness in lignocellulosic biodegradation, but the novelty of this research lies in the application of an enriched microbial consortium obtained by the authors through soil isolation techniques, which are very inexpensive and affordable for developing countries.

The study employed batch shake flasks to evaluate the impact of various nitrogen sources, phosphate levels, and sodium acetate (Na-acetate) on the Rhodotorula mucilaginosa growth and metabolite production. Adding Na-acetate to the medium resulted in significant improvements in critical metabolites. In shake flask experiments, this led to a cell dry weight (CDW) of 1.65 ± 0.94 g L^-1, with lipids comprising 66.53% of the biomass. While β-carotene and carotenoid were 5.84 ± 0.05 and 37.66 ± 2.13 µg g^-1, respectively. Subsequent experiments in a batch reactor with Na-acetate supplementation further improved these metrics. CDW increased to 5.02 ± 0.83 g L^-1, and lipid content to 65.73 ± 0.81%. Carotenoid production rose to 40.33 ± 1.84 µg g^-1, with β-carotene reaching 17.63 ± 0.32 µg g^-1. The most promising results were obtained using a fed-batch bioreactor strategy with Na-acetate. R. mucilaginosa achieved the highest yields across all parameters: 48.36 ± 1.14 µg g^-1 of total carotenoids, 21.38 ± 1.14 µg g^-1 of β-carotene, and a lipid content of 68.58 ± 1.95%.