Engineering in Life Sciences最新文献

Combined cross-linked enzyme aggregates (combi-CLEAs) represent a promising carrier-free immobilized enzyme technology. This study describes the preparation of combi-CLEAs comprising leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) for the regeneration of cofactor nicotinamide adenine dinucleotide necessary for 2-aminobutyric acid production. Different from traditional methods using ammonium sulfate or organic reagents as precipitant, this work utilized low concentrations of calcium ions to purify and precipitate the histidine-tagged enzymes. We developed a simple and environmentally friendly protocol for combi-CLEAs formation, involving precipitation with 10 mM calcium ions at an enzyme activity ratio of 1:2 for LeuDH and FDH, respectively, followed by cross-linking with 0.15% (w/v) glutaraldehyde at 20°C for 2 h at pH 7.5. The optimal catalytic reaction temperature and pH value for the combi-CLEAs were determined to be a temperature of 37°C and a pH of 7.5. The combi-CLEAs demonstrated enhanced thermal and pH tolerance compared to the free enzyme mixture. Moreover, the combi-CLEAs showed good operational stability, retaining 40% of its initial activity after seven cycles of reuse. These findings suggest that the combi-CLEAs of LeuDH and FDH are an efficient and cost-effective option for 2-aminobutyric acid production.

Retraction: Y.A. Arani, Z. Noormohammadi, B. Rasekh, F. Yazdian, and H. Kazemi, “Evaluation of SDS-Coated Iron Nanostructure on the Gene Expression of Bio Surfactant-Producing Genes by Pseudomonas Aeruginosa,” Engineering in Life Sciences 22, no. 9 (2022): 584–593, https://doi.org/10.1002/elsc.202200002.

The above article, published online on 24 August 2022, in Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the journal Editors-in-Chief, An-Ping Zeng and Ralf Takors; and Wiley Periodicals LLC. Following an investigation by the publisher, the parties have concluded that this article was accepted solely on the basis of a compromised peer review process. In addition, a third party informed the publisher that Figures 4 and 5 were reproduced from two articles published either previously or in the same year, and were used here in a different scientific context. The publisher has investigated and confirmed this, and found additional image manipulation in Figure 4. Therefore, the article must be retracted. Corresponding author Behnam Rasekh disagrees with this decision.

The enhanced surface cylindrical flask (ESCF) consists of an eight-striped inner arrangement holding 16 standard microscopic slides placed inside a cylindrical vessel. The specially designed spatula-accessible slides can be withdrawn from the vessel during cultivation without disturbing biofilm formation through an innovative window-flap accessibility mechanism. The vessel and its accessories were three-dimensional (3D) printed by applying a fused deposition modeling technique utilizing biodegradable polylactic acid. Biofilms of clinically relevant bacteria namely Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were successfully grown in the ESCF and observed through confocal laser scanning microscopy. Advantages of the device include an enhanced surface area for biofilm formation, ease of insertion and removal of microscopic slides, convenient fitting into standard rotary shaker platforms, creation of anoxic/microaerophilic environment inside the vessel as well as the feasibility of pH, dissolved gases, and metabolite measurements in the liquid surrounding the biofilm. The ESCF will find widespread application in medical, industrial, and environmental disciplines.

Antimicrobial peptides (AMPs) are host defense peptides that act against a broad spectrum of microorganisms. AMPs are of high interest as medicinal products, antimicrobial coatings, and for controlling biofilm formation. Applications and research of many AMPs are still hampered by insufficient titers and lack of production platforms that can tolerate high titers of AMPs. Corynebacterium glutamicum is an excellent microbial host for protein secretion and has been barely explored as a host for AMP production. Here, we report the successful production and secretion of two AMPs (amounts of up to 130 mg/L for liquid chromatography peak I [LCI] and 54 mg/L for Psoriasin) by C. glutamicum with low amounts of secreted byproducts.

Recombinant adeno-associated virus (rAAV) vector production is a complex process in which the robust cultivation of human embryonic kidney cells (HEK293) plays a critical role in generating high-quality viral vectors. Tracking the viable cell concentration (VCC) during upstream production is essential for process monitoring and for implementing actions that ensure optimal process management. The advent of inline capacitance probes has introduced a crucial process analytical technology (PAT) tool for real-time VCC measurement. Here, we present the development and application of a method for real-time monitoring of VCC in HEK293-based rAAV vector production. In a first step, BioPAT Viamass probes were used to record capacitance data of individual 10 L rAAV-8 batches within a frequency range of 50 kHz–20 MHz. Based on the capacitance data, a linear single-frequency model and an orthogonal partial least square (OPLS) multifrequency model for VCC prediction were developed. Subsequently, these models were deployed inline, and predictions were exposed into BioPAT MFCS bioprocess control software, enabling real-time VCC monitoring in subsequent rAAV-8 production batches. In addition, the continuous VCC signal was used as input for an exponential cell growth model that was deployed inline to provide accurate real-time forecasting of the transfection time point. To the best of our knowledge, this is the first example of inline deployment of VCC and Time-Till-Transfection predictive models to the bioprocess control system for real-time monitoring and forecasting of these parameters in HEK-cell-based transient rAAV vector production.

For about 100 years, the shake flask has been established for biotechnological cultivations as one of the most important cultivation systems in early process development. Its appeal lies in its simple handling and highly versatile application for a wide range of cell types—from bacteria to mammalian cells. In recent decades, extensive research has been conducted on the shake flask, to not perform processes blindly but to gain a deeper understanding of the various process parameters, phenomena, and their impact on the process. Although the characterization of the shake flask is now well-established in literature, many publications show that this knowledge is often inadequately applied. Therefore, this review provides an overview of the current state of knowledge on various topics related to the shake flask. We first present the key process parameters and their influence on different physical phenomena, such as power input, the largely unknown in-phase/out-of-phase phenomenon, as well as temperature and mass transfer. Then, the most common online monitoring systems that have been established for shake flasks are discussed. Finally, various pitfalls that often arise from inadequate knowledge of handling shake flask cultivations are discussed and guidance on how to avoid them is provided.

This study emphasizes the critical importance of closely monitoring and controlling the sialic acid content in therapeutic glycoproteins, including EPO, interferon-γ, Orencia, Enbrel, and others, as the level of sialylation directly impacts their pharmacokinetics (PK), immunogenicity, potency, and overall clinical performance due to its influence on protein clearance via hepatic asialoglycoprotein receptors (ASGPR). The ASGPR recognizes and binds to glycoproteins exposed to terminal galactose or N-acetylgalactosamine residues, leading to receptor-mediated endocytosis. Recent studies have demonstrated that sialylation of O-linked glycan plays a role in protecting against macrophage galactose lectin (MGL)-mediated clearance. In addition to the impact on serum half-life, sialylation can influence other clinical outcomes, including immunogenicity, potency, and cytotoxicity. Therefore, the level of sialic acid is a critical quality attribute (CQA), and monitoring and regulating sialylation has become a regulatory requirement to ensure desired clinical performance. To achieve consistent levels of sialic acid-to-protein ratio, the time of upstream harvest and conductivity of downstream wash buffers must be tightly regulated based on the sialic acid content. Therefore, the utilization of process analytical technology (PAT) tools for generating real-time or near-real-time sialic acid content is a business-critical requirement. This work demonstrates the utility of an integrated PAT system for near real-time online sialic acid measurements. The system consists of a micro-sequential injection analyzer (µSIA) interfaced with SegFlow and an ultra performance liquid chromatography (UPLC). The fully automated architecture exemplifies the execution of online sampling, automatic sample preparation, and subsequent online UPLC analysis. This carefully orchestrated PAT framework effectively supports the requirements of QbD-driven continuous bioprocessing.

Sustainable chemical production from C₁ gaseous substrates, such as syngas or CO₂/H₂, can be achieved through gas fermentation. In gas fermentation, acetogenic bacteria are able to utilize oxidized inorganic carbon sources as the sole carbon source and electron acceptor, while reduced inorganic species are used as the electron donor. Clostridium ljungdahlii, a model acetogen, is only capable of reducing CO₂ to acetate and ethanol, with H₂ as electron donor. In order to expand the product profile of this bacterium, five alcohol acetyltransferases (AATs) from yeast were heterologously expressed in C. ljungdahlii to evaluate its potential to produce ethyl acetate. When growing on CO₂ and H₂, up to 7.38 ± 0.43 mg/L of ethyl acetate were produced. Using fructose as the main carbon and energy source, up to 23.15 ± 1.28 mg/L of ethyl acetate were produced. Ethanol and fumarate supplementation were able to boost ethyl acetate titers (up to 37.51 ± 9.44 mg/L). Hence, ethyl acetate production was enabled in C. ljungdahlii at low titers, which could be explained by the high energetic cost of operation of AATs, and their shown promiscuity. However, we also show that this opens the door to more complex esterification reactions of higher added value biomolecules.

Polyporus umbellatus is a rare porous fungus that exhibits notable pharmacological activities. Particularly, due to its diuretic properties, it is considered an important source of targeted drugs for the treatment of kidney disease. Extensive research has been conducted on this fungus, focusing not only on its challenging cultivation techniques but also on its diverse array of medicinal ingredients, including polysaccharides and steroids. These active compounds demonstrate considerable variability and exhibit a wide range of medicinal properties. As a result, extracting, separating, and purifying these active compounds has become a subject of interest. This review aims to provide a comprehensive overview of the types, structures, and physicochemical properties of these active compounds. Additionally, the medicinal effects of P. umbellatus are thoroughly examined, offering valuable insights into the utilization of its resources and the rational development of medical fungi.