Preparative Biochemistry & Biotechnology最新文献

The excessive use of conventional antibiotics has resulted in significant aquatic pollution and a concerning surge in drug-resistant bacteria. Efforts have been consolidated to explore and develop environmentally friendly antimicrobial alternatives to mitigate the imminent threat posed by multi-resistant pathogens. Antimicrobial peptides (AMPs) have gained prominence due to their low propensity to induce bacterial resistance, attributed to their multiple mechanisms of action and synergistic effects. Microalgae, particularly cyanobacteria, have emerged as promising alternatives with antibiotic potential to address these challenges. The aim of this review is to present some AMPs extracted from microalgae, emphasizing their activity against common pathogens and elucidating their mechanisms of action, as well as their potential application in the aquaculture industry. Likewise, the biosynthesis, advantages and disadvantages of the use of AMPs are described. Currently, biotechnology tolls are used to enhance the action of these peptides, such as genetically modified microalgae and recombinant proteins. Cyanobacteria are also mentioned as major producers of peptides, among them, the genus Lyngbya is described as the most important producer of bioactive peptides with potential therapeutic use. The majority of cyanobacterial AMPs are of the cyclic type, meaning that they have cysteine and disulfide bridges, thanks to this, their greater antimicrobial activity and selectivity. Likewise, we found that large hydrophobic aromatic amino acid residues increase specificity, and improve antibacterial efficacy. However, based on the results of this review, it is possible to highlight that while microalgae show potential as a source of AMPs, further research in this field is necessary to achieve safe and competitive production. Therefore, the data presented here can aid in the selection of microalgal species, peptide structures, and target bacteria, with the goal of establishing biotechnological platforms for aquaculture applications.

Interleukin-2 has emerged as a potent protein-based drug to treat various cancers, AIDS, and autoimmune diseases. Despite its immense requirement, the production procedures are inefficient to meet the demand. Therefore, efficient production procedures must be adopted to improve protein yield and decrease procedural loss. This study analyzed cytoplasmic and periplasmic IL-2 expression for increased protein yield and significant biological activity. The study is focused on cloning IL-2 into a pET-SUMO and pET-28a vector that expresses IL-2 in soluble form and inclusion bodies, respectively. Both constructs were expressed into different E. coli expression strains, but the periplasmic and cytoplasmic expression of IL-2 was highest in overnight culture in Rosetta 2 (DE3). Therefore, E. coli Rosetta 2 (DE3) was selected for large-scale production and purification. Purified IL-2 was characterized by SDS-PAGE and western blotting, while its biological activity was determined using MTT bioassay. The results depict that the periplasmic and cytoplasmic IL-2 achieved adequate purification, yielding 0.86 and 0.51 mg/mL, respectively, with significant cytotoxic activity of periplasmic and cytoplasmic IL-2. Periplasmic IL-2 has shown better yield and significant biological activity in vitro which describes its attainment of native protein structure and function.

This study explored the impact of sodium acetate (Na-acetate) impact on lipid, carotenoid, and β-carotene production by the newly isolated strain Rhodotorula mucilaginosa. Batch and fed-batch bioreactor cultures were employed to optimize growth conditions and product yields. R. mucilaginosa fed with Na-acetate in the yeast medium was evaluated in the batch bioreactor culture. The following merits were accomplished for the cell dry weight (5.02 gL^-1), lipid content (65.73%), carotenoid (40.33 µgg^-1) and β-carotene (17.63 µgg^-1) consistently. The fed-batch reactor cultivation using yeast extract supplemented with Na-acetate yielded superior lipid content (68.58%), cell dry weight (5.92 gL^-1), carotenoid (48.36 µgg^-1), and β-carotene production (21.38 µgg^-1) compared to batch cultivation. The fatty acid methyl esters (FAMEs) are produced from the lipids suitable for biodiesel production. These findings highlight the potential of R. mucilaginosa as a promising organism for sustainable biofuel and high-value compound production. Further optimization of culture conditions and downstream processing could enhance the commercial viability of this approach.

With numerous advantages over conventional techniques, ultrasound-assisted extraction (UAE) has become a viable method for the effective extraction of biomolecules from prokaryotic and eukaryotic cells. The fundamentals and workings of UAE are examined in this review, focusing on current developments, including how these impact the extraction of proteins, lipids, enzymes, and other bioactive compounds. UAE not only enhances cell disruption and mass transfer, leading to improved extraction yields, but also preserves the integrity of the extracted bioactive molecules under optimized conditions, making it a preferred choice in Biochemistry and Biotechnology. Additionally, this review explores recent innovative approaches that combine ultrasound with other techniques like enzymatic digestion, supercritical CO₂, deep eutectic solvents, and Three-Phase Partitioning (UA-TPP) etc, to further enhance extraction efficiency. The differences in extraction effectiveness between prokaryotic and eukaryotic cells are attributed to cellular structure and ultrasonic conditions. Overall, this review highlights UAE's promise as a viable and efficient substitute for biomolecule extraction concerning prokaryotic and eukaryotic cells while bringing up areas that need additional research and development.

Isolation of high-quality RNA from abaca is very challenging due to the presence of polyphenols, polysaccharides, and its high fiber content. In this study, we compared six extraction methods across three tissue types and different developmental stages (in-vitro-grown young versus field-grown mature tissue). The Invitrogen PureLink RNA kit proved to be the most efficient in extracting RNA from young abaca tissues (leaves, pseudostem, and corm). The quality of RNA extracted from young tissues was further assessed by RNA-seq applications, with raw sequencing reads mapping back to the M. textilis reference genome at rates of 86.0%-90.4%. The SDS-TRIzol-method modified with an added on-column DNAse I treatment was used to extract RNA from mature tissues (leaves, midrib, and pseudostem). RNA isolated from five M. textilis cultivars and across three mature tissue types showed RNA yield per 100 mg of fresh weight ranges from 0.57 to 10.94 µg and RNA integrity number (RIN) scores of more than 7.0 for all tissue types. Our improved SDS-Trizol method for RNA extraction described here is simple and yields good quality RNAs from mature abaca tissues while the PureLink RNA kit is suitable for extracting RNA from young abaca samples amenable to RT-qPCR and next-generation sequencing studies.

The world faces pressing environmental challenges, including greenhouse gas emissions, global warming, climate change, and rising sea levels. Alongside, these issues, the depletion of fossil fuels has intensified the search for alternative energy sources. Algal biomass presents a promising long-term solution to these global problems. The quest for sustainable energy has driven significant research into algal biofuels as a viable alternative to fossil fuels. Algae offers several advantages as a feedstock for biofuel production, including high biomass yield, rapid growth rates, cost-effective cultivation, carbon dioxide fixation capabilities, and the potential to grow on non-arable land using non-potable water. This manuscript provides an overview of algal biomass cultivation using renewable feedstocks, identifies potential algal strains for biofuel production, and explores bioengineering advancements in algae. Additionally, strain improvement strategies to enhance biofuel yields are discussed. The review also addresses large-scale algal biomass cultivation for biofuel production, assesses its commercial viability, examines challenges faced by the biofuel industry, and outlines prospects for biofuel production using highly potent algal strains. By overcoming and addressing these challenges, algal biofuels have the potential to become a cornerstone of sustainable energy solutions.

Sorbitol, known as D-Glucitol, is a hexose sugar alcohol that occurs naturally in various fruits, including berries, cherries, plums, pears, and apples. It is noteworthy that sorbitol can be metabolized by microbes, plants, and humans through distinct pathways. Nevertheless, in bacteria like Escherichia coli (E. coli), sorbitol is not the primary carbon source and its utilization is generally suppressed due to carbon catabolite repression. In this context, Escherichia coli has been engineered to enable the use of sorbitol as the sole carbon source for producing recombinant proteins. This modification involves a two-plasmid system where the sorbitol-6-phosphate dehydrogenase (srlD) gene is upregulated under an araBAD promoter, while the recombinant protein is expressed from a second plasmid under the tac promoter. The overexpression of srlD in the engineered E. coli strain enhances the utilization of sorbitol as the sole carbon source. When cultured in a medium supplemented solely with sorbitol, the engineered E. coli strain exhibits a 3.6 times higher specific growth rate and yields substantially higher concentration of recombinant protein compared to the wild-type strain. Additionally, the engineered strain demonstrates a higher Y_P/X ratio than the wild-type strain.

The microbial agents based on plant growth promoting rhizobacteria (PGPR) have become a hot topic in agricultural research, while the optimization of fermentation conditions for PGPR-based microbial agents still lack systematic research. The single-factor and orthogonal experiments were conducted to determine the optimal fermentation conditions of Pseudomonas synxantha M1. The results indicated that the glycerol and shaker speed was the most significant factors that influence the number of bacteria of P. synxantha M1 fermentation liquid. The viable bacteria count of microbial agent reached 7.1 × 10¹² cfu/mL at 36 h, which OD₆₀₀ value increased by 116.40% compared to before optimization, and promote the growth of highland barley. Significant differences of metabolites of fermentation liquid was observed in different fermentation times, including organic acids, lipids, and organoheterocyclic compounds using liquid chromatography tandem mass spectrometry (LC-MS/MS). In addition, the fermentation liquid was found to contain indoleacetic acid, glutathione and xanthine at the end of fermentation, which might contribute for the growth of plants as bioactive substances.

Using an engineered Escherichia coli to produce lipase and can easily achieve high-level expression. The investigation of biochemical processes during lipase fermentation, approached from a metabolomics perspective, will yield novel insights into the efficient secretion of recombinant proteins. In this study, the lipase batch fermentation was carried out first with enzyme activity of 36.83 U/mg cells. Then, differential metabolites and metabolic pathways were identified using an untargeted metabolomics approach through comparative analysis of various fermentation periods. In total, 574 metabolites were identified: 545 were up-regulated and 29 were down-regulated, mainly in 153 organic acids and derivatives, 160 organoheterocyclic compounds, 64 lipids and lipid-like molecules, and 58 organic oxygen compounds. Through metabolic pathways and network analysis, it could be found that tryptophan metabolism was of great significance to lipase production, which could affect the secretion and synthesis of recombinant protein. In addition, the promotion effects of cell growth by varying concentrations of indole acetic acid serve to validate the results obtained from tryptophan metabolism. This study offers valuable insights into metabolic regulation of engineered E. coli, indicating that its fermentation bioprocess can be systematically designed according to metabolomics findings to enhance recombinant protein production.

Aspergillus aculeatus pectinase was immobilized on magnetic nanoparticles coated with calcium alginate for pectin hydrolysis in guava juice by a stirred electromagnetic reactor (SER). The average crystallite size estimated by the Scherrer formula was 33.7 nm. The reaction rate in SER (701.7 U/mL) was almost twice that of the static process (362.5 U/mL). Both processes displayed a sigmoidal trend with positive cooperativity (n) of 5 and 4, respectively. Both free and immobilized pectinase showed great performance in the pH range of 4.0-7.0. After immobilization, pectinase acted optimally at 50 °C. Pectin hydrolysis was performed for over 10 successive cycles in SER losing only 30% of its initial activity. Thermodynamic activation parameters of the reaction revealed higher spontaneity and efficiency when hydrolysis was performed in SER. Pectin hydrolysis in guava juice displayed 41% turbidity and 85.5% viscosity reduction. The electromagnetic reactor displayed great potential for performing hydrolysis of pectin in guava juice. The biocatalyst showed good features for further applications in food industries.