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Biotechnology and Bioengineering
John Wiley & Sons
Biotechnology and Bioengineering

John Wiley & Sons

0006-3592

Biotechnology and Bioengineering/Journal Biotechnology and BioengineeringSCIISTPEI
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    Biotechnology and Bioengineering: Volume 119, Number 9, September 2022

    3页

    Plant extract‐based synthesis of metallic nanomaterials, their applications, and safety concerns

    Aziz UllahSung In Lim
    32页
    查看更多>>摘要:Abstract Nanotechnology has attracted the attention of researchers from different scientific fields because of the escalated properties of nanomaterials (NMs) compared with the properties of macromolecules. NMs can be prepared through different approaches involving physical and chemical methods. The development of NMs through plant‐based green chemistry approaches is more advantageous than other methods from the perspectives of environmental safety, animal, and human health. The biomolecules and metabolites of plants act as reducing and capping agents for the synthesis of metallic green NMs. Plant‐based synthesis is a preferred approach as it is not only cost‐effective, easy, safe, clean, and eco‐friendly but also provides pure NMs in high yield. Since NMs have antimicrobial and antioxidant potential, green NMs synthesized from plants can be used for a variety of biomedical and environmental remediation applications. Past studies have focused mainly on the overall biogenic synthesis of individual or combinations of metallic NMs and their oxides from different biological sources, including microorganisms and biomolecules. Moreover, from the viewpoint of biomedical applications, the literature is mainly focusing on synthetic NMs. Herein, we discuss the extraction of green molecules and recent developments in the synthesis of different plant‐based metallic NMs, including silver, gold, platinum, palladium, copper, zinc, iron, and carbon. Apart from the biomedical applications of metallic NMs, including antimicrobial, anticancer, diagnostic, drug delivery, tissue engineering, and regenerative medicine applications, their environmental remediation potential is also discussed. Furthermore, safety concerns and safety regulations pertaining to green NMs are also discussed.

    Advances in early diagnosis of cervical cancer based on biosensors

    Hadi Esmaeili Gouvarchin GhalehAlireza ShahriaryMorteza IzadiMahdieh Farzanehpour...
    8页
    查看更多>>摘要:Abstract Human papillomavirus (HPV) is a causative agent of cervical cancer among women worldwide. Serological and molecular tests are commonly used to detect and identify HPV, but all the detection methods for HPV have some limitations. Nowadays, considerable advancements in nanosensors have enabled monitoring of hybridization procedures dynamically for HPV detection. Biosensors, as effective, quick, economical, and highly sensitive tools, can be used in the diagnosis of HPV as an alternative technique instead of other detection methods. Biosensor detection methods of HPV in use from 2000 to 2021 were investigated using several databases?such as Google Scholar, Scopus, PubMed, and the Scientific Information Database. Furthermore, a manual search of the references of the retrieved articles was performed. On analyzing the most recently developed biosensors for HPV identification, we observed that three biosensor systems, electrochemical, optical, and piezoelectric systems, are the main transducers used in the development of HPV biosensors. The aim of this review is to examine recent research on biosensors for the detection of HPV and perform a comparison with other diagnostic methods. Considering the importance of rapid HPV detection in the control of infection and development of public health measures, improvement of biosensors as an economical and quick method can be very useful in the diagnosis of HPV.

    Angiogenic and osteogenic effects of flavonoids in bone regeneration

    Abinaya ShanmugavadivuKalimuthu BalagangadharanNagarajan Selvamurugan
    18页
    查看更多>>摘要:Abstract Bone is a highly vascularized tissue that relies on a close spatial and temporal interaction between blood vessels and bone cells. As a result, angiogenesis is critical for bone formation and healing. The vascular system supports bone regeneration by delivering oxygen, nutrients, and growth factors, as well as facilitating efficient cell–cell contact. Most clinical applications of engineered bone grafts are hampered by insufficient vascularization after implantation. Over the last decade, a number of flavonoids have been reported to have osteogenic–angiogenic potential in bone regeneration because of their excellent bioactivity, low cost, availability, and minimal in vivo toxicity. During new bone formation, the osteoinductive nature of certain flavonoids is involved in regulating multiple signaling pathways contributing toward the osteogenic–angiogenic coupling. This review briefly outlines the osteogenic–angiogenic potential of those flavonoids and the mechanisms of their action in promoting bone regeneration. However, further studies are needed to investigate their delivery strategies and establish their clinical efficacy.

    High‐level production of wild‐type and oxidation‐resistant recombinant alpha‐1‐antitrypsin in glycoengineered CHO cells

    Izel KoyuturkSurbhi KediaAnna RobothamAlexandra Star...
    14页
    查看更多>>摘要:Abstract Alpha‐1‐antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma‐purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host‐specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild‐type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N‐glycans analogous to human plasma‐derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α?1,6‐fucosylation (Fut8) and α?2,3‐sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α?2,6‐sialyltransferase (ST6Gal1) using a cumate‐inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5‐ to 6.5‐fold higher with the cumate‐inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8?g/L of wild‐type and mutein forms of A1AT, respectively, with N‐glycans analogous to the plasma‐derived clinical product Prolastin‐C. Supplementation of N‐acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi‐antennary and disialylated A2G2S2 N‐glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin‐C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

    Validation and scalability of homemade polycaprolactone macrobeads grafted with thermo‐responsive poly(N‐isopropylacrylamide) for mesenchymal stem cell expansion and harvesting

    Linh T. B. NguyenTimothée BaudequinZhanfeng CuiHua Ye...
    14页
    查看更多>>摘要:Abstract In this study, polycaprolactone (PCL) macrobeads were prepared by an oil‐in‐water (o/w) emulsion solvent evaporation method with poly(vinyl alcohol)?(PVA) as an emulsifier and conjugated to poly(N‐isopropylacrylamide) (PNIPAAm) to be used as cell carriers with noninvasive cell detachment properties (thermo‐response). Following previous studies with PCL‐PNIPAAm carriers, our objectives were to confirm the successful conjugation on homemade macrobeads and to show the advantages of homemade production over commercial beads to control morphological, biological, and fluidization properties. The effects of PCL concentration on the droplet formation and of flow rate and PVA concentration on the size of the beads were demonstrated. The size of the beads, all spherical, ranged from 0.5?to 3.7?mm with four bead categories based on production parameters. The morphology and size of the beads were observed by scanning electron microscopy to show surface roughness enhancing cell attachment and proliferation compared to commercial beads. The functionalization steps with PNIPAAm were then characterized and confirmed by Fourier transform infrared?spectroscopy, scanning electron microscopy, and energy dispersion spectroscopy. PNIPAAm‐grafted macrobeads allowed mesenchymal stem cells (MSCs) to spread and grow for up to 21 days. By reducing the temperature to 25°C, the MSCs were successfully detached from the PCL‐PNIPAAm beads as observed with fluorescence microscopy. Furthermore, we validated the scalability potential of both macrobeads production and conjugation with PCL, to produce easily kilograms of thermo‐responsive macrocarriers in a lab environment. This could help moving such approaches towards clinically and industrially relevant processes were cell expansion is needed at very large scale.

    Transcriptomic analysis reveals mode of action of butyric acid supplementation in an intensified CHO cell fed‐batch process

    Markus SchulzeYadhu KumarMerle RattayJulia Niemann...
    15页
    查看更多>>摘要:Abstract Process intensification is increasingly used in the mammalian biomanufacturing industry. The key driver of this trend is the need for more efficient and flexible production strategies to cope with the increased demand for biotherapeutics predicted in the next years. Therefore, such intensified production strategies should be designed, established, and characterized. We established a CHO cell process consisting of an intensified fed‐batch (iFB), which is inoculated by an N‐1 perfusion process that reaches high cell concentrations (100?×?106?c?ml?1). We investigated the impact of butyric acid (BA) supplementation in this iFB process. Most prominently, higher cellular productivities of more than 33% were achieved, thus 3.5?g?L?1 of immunoglobulin G (IgG) was produced in 6.5 days. Impacts on critical product quality attributes were small. To understand the biological mechanisms of BA in the iFB process, we performed a detailed transcriptomic analysis. Affected gene sets reflected concurrent inhibition of cell proliferation and impact on histone modification. These translate into subsequently enhanced mechanisms of protein biosynthesis: enriched regulation of transcription, messenger RNA processing and transport, ribosomal translation, and cellular trafficking of IgG intermediates. Furthermore, we identified mutual tackling points for optimization by gene engineering. The presented strategy can contribute to meet?future requirements in the continuously demanding field of biotherapeutics production.

    Monitoring and control of the release of soluble O2 from H2O2 inside porous enzyme carrier for O2 supply to an immobilized d‐amino acid oxidase

    Sabine SchelchJuan M. BolivarBernd Nidetzky
    14页
    查看更多>>摘要:Abstract While O2 substrate for bio‐transformations in bulk liquid is routinely provided from entrained air or O2 gas, tailored solutions of O2 supply are required when the bio‐catalysis happens spatially confined to the microstructure of a solid support. Release of soluble O2 from H2O2 by catalase is promising, but spatiotemporal control of the process is challenging to achieve. Here, we show monitoring and control by optical sensing within a porous carrier of the soluble O2 formed by an immobilized catalase upon feeding of H2O2. The internally released O2 is used to drive the reaction of d‐amino acid oxidase (oxidation of d‐methionine) that is co‐immobilized with the catalase in the same carrier. The H2O2 is supplied in portions at properly timed intervals, or continuously at controlled flow rate, to balance the O2 production and consumption inside the carrier so as to maintain the internal O2 concentration in the range of 100–500?μM. Thus, enzyme inactivation by excess H2O2 is prevented and gas formation from the released O2 is avoided at the same time. The reaction rate of the co‐immobilized enzyme preparation is shown to depend linearly on the internal O2 concentration up to the air‐saturated level. Conversions at a 200?ml scale using varied H2O2 feed rate (0.04–0.18?mmol/min) give the equivalent production rate from d‐methionine (200?mM) and achieve rate enhancement by ~1.55‐fold compared to the same oxidase reaction under bubble aeration. Collectively, these results show an integrated strategy of biomolecular engineering for tightly controlled supply of O2 substrate from H2O2 into carrier‐immobilized enzymes. By addressing limitations of O2 supply via gas‐liquid transfer, especially at the microscale, this can be generally useful to develop specialized process strategies for O2‐dependent?biocatalytic reactions.

    Characterization and application of a thermophilic Argonaute from archaeon Thermococcus thioreducens

    Mengjun FangZhipeng XuDi HuangMuhammad Naeem...
    11页
    查看更多>>摘要:Abstract Prokaryotic Argonaute proteins (pAgos) play an important role in host defense against invading genetic elements. The functional diversities make pAgos very promising in development of novel nucleic acid manipulation tools and attract increasing attentions. Here, we reported the in vitro characterization of an Argonaute protein from archaeon Thermococcus thioreducens (TtrAgo) and its example of application in hepatitis B virus DNA detection. The results showed that TtrAgo functions as a programmable DNA endonuclease by utilizing both short 5′‐phosphorylated and 5′‐hydroxylated single‐stranded DNA guides, and presents high efficiency and accuracy at optimal temperatures ranging from 75°C to 95°C. In addition, TtrAgo also possesses stepwise cleavage activity like PfAgo (Pyrococcus furiosus) and chopping activity toward double‐stranded DNA similar to MjAgo (Methanocaldococcus jannaschii). This study increases our understanding of pAgos and expands the Ago‐based DNA detection toolbox.

    Engineering of reaction specificity, enantioselectivity, and catalytic activity of nitrilase for highly efficient synthesis of pregabalin precursor

    Xia‐Feng LuHong‐Juan DiaoZhe‐Ming WuZi‐Long Zhang...
    14页
    查看更多>>摘要:Abstract Simultaneous evolution of multiple enzyme properties remains challenging in protein engineering. A chimeric nitrilase (BaNITM0) with high activity towards isobutylsuccinonitrile (IBSN) was previously constructed for biosynthesis of pregabalin precursor (S)‐3‐cyano‐5‐methylhexanoic acid ((S)‐CMHA). However, BaNITM0 also catalyzed the hydration of IBSN to produce by‐product (S)‐3‐cyano‐5‐methylhexanoic amide. To obtain industrial nitrilase with vintage performance, we carried out engineering of BaNITM0 for simultaneous evolution of reaction specificity, enantioselectivity, and catalytic activity. The best variant V82L/M127I/C237S (BaNITM2) displayed higher enantioselectivity (E?=?515), increased enzyme activity (5.4‐fold) and reduced amide formation (from 15.8% to 1.9%) compared with BaNITM0. Structure analysis and molecular dynamics simulations indicated that mutation M127I and C237S restricted the movement of E66 in the catalytic triad, resulting in decreased amide formation. Mutation V82L was incorporated to induce the reconstruction of the substrate binding region in the enzyme catalytic pocket, engendering the improvement of stereoselectivity. Enantio‐ and regio‐selective hydrolysis of 150?g/L IBSN using 1.5?g/L Escherichia coli cells harboring BaNITM2 as biocatalyst afforded (S)‐CMHA with >99.0% ee and 45.9% conversion, which highlighted the robustness of BaNITM2 for efficient manufacturing of pregabalin.