Journal of bioscience and bioengineering最新文献

As an industrial enzyme that catalyzes the formation and cleavage of ester bonds, carboxylesterase has attracted attention in fine chemistry, pharmaceutical, biological energy and bioremediation fields. However, the weak thermostability limits their further developments in industrial applications. In this work, a novel carboxylesterase (EstF) from Streptomyces lividans TK24, belonging to family XVII, was acquired by successfully heterologous expressed and biochemically identified. The EstF exhibited optimal activity at 55 °C, pH 9.0 and excellent catalytic performances (K_m = 0.263 mM, k_cat/K_m = 562.3 s⁻¹ mM⁻¹ for p-nitrophenyl acetate (pNPA₂) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 °C and 2.86 h at 100 °C. Furthermore, the EstF was modified to obtain EstF_P144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstF_P144G displayed a 5.10-fold increase of half-life at 100 °C versus wild-type without affecting catalytic performance. Structural analysis implied that the glycine introduction could release a steric strain and induce cooperative effects between distal residues to increase the thermostability. Therefore, the thermostable EstF and EstF_P144G with prominently catalytic characteristics have potential industrial applications and the introduction of a single glycine strategy opens up alternative avenues for the thermostability engineering of other enzymes.

Xyloglucan in plant cell walls has complex side-chain structures; Aspergillus oryzae produces various enzymes to degrade and assimilate xyloglucan. In this study, we identified and characterized α-1,2-l-fucosidase (AfcA) which is involved in xyloglucan degradation in A. oryzae. AfcA expression was induced in the presence of xyloglucan oligosaccharides. AfcA showed specific activity toward α-(1→2)-linked l-fucopyranosyl residues attached to the side chains of xyloglucan oligosaccharides and milk oligosaccharides, but not toward α-(1→3)-, α-(1→4)-, and α-(1→6)-linked l-fucopyranosyl residues. As fucopyranosyl residues in the side chains of xyloglucan oligosaccharides prevent the degradation of xyloglucan oligosaccharides by isoprimeverose-producing oligoxyloglucan hydrolase and β-galactosidase, the cooperative action of AfcA, isoprimeverose-producing oligoxyloglucan hydrolase, and β-galactosidase play a key role in degrading fucosylated xyloglucan in A. oryzae.

Flavonoids comprise a group of natural compounds with diverse bioactivities; however, their low water solubility and limited bioavailability often impede their potential health benefits for humans. In this study, five derivatives, namely 2′,5′-dihydroxyflavanone (1), 2′-dihydroxyflavanone-5′-O-4″-O-methyl-β-d-glucoside (2), 2′-dihydroxyflavanone-6-O-4″-O-methyl-β-d-glucoside (3), 2′-dihydroxyflavanone-3′-O-4″-O-methyl-β-d-glucoside (4) and hydroxyflavanone-2′-O-4″-O-methyl-β-d-glucoside (5), were biosynthesized from 2′-hydroxyflavanone through microbial transformation using Beauveria bassiana ATCC 7159. Product 1 was identified as a known compound while 2–5 were structurally characterized as new structures through extensive 1D and 2D NMR analysis. The water solubility of biotransformed products 1–5 was enhanced by 30–280 times compared to the substrate 2′-hydroxyflavanone. Moreover, the antioxidant assay revealed that 1 and 2 exhibited improved 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity relative to the substrate, decreasing the logIC₅₀ from 8.08 ± 0.11 μM to 6.19 ± 0.08 μM and 7.15 ± 0.08 μM, respectively. Compound 5 displayed significantly improved anticancer activity compared to the substrate 2′-hydroxyflavanone against Glioblastoma 33 cancer stem cells, decreasing the IC₅₀ from 25.05 μM to 10.59 μM. Overall, fungal biotransformation represents an effective tool to modify flavonoids for enhanced water solubility and bioactivities.

Respiratory syncytial virus (RSV) infection is an acute respiratory infection caused by RSV. It occurs worldwide, and for over 50 years, several attempts have been made to research and develop vaccines to prevent RSV infection; effective preventive vaccines are eagerly awaited. The RSV fusion (F) protein, which has gained attention as a vaccine antigen, causes a dynamic structural change from the preF to postF state. Therefore, the structural changes in proteins must be regulated to produce a vaccine antigen that can efficiently induce antibodies with high virus-neutralizing activity. We successfully discovered several mutations that stabilized the antigen site Ø in the preF state, trimerized it, and improved the level of protein expression through observation and computational analysis of the RSV-F protein structure and amino acid mutation analysis of RSV strains. The four RSV-F protein mutants that resulted from the combination of these effective mutations stably conserved a wide range of preF- and trimeric preF-specific epitopes with high virus-neutralizing activity. Absorption assay using human serum revealed that mutants constructed bound to antibodies with virus-neutralizing activity that were induced by natural RSV infection, whereas they hardly bound to anti-postF antibodies without virus-neutralizing activity. Furthermore, mouse immunization demonstrated that our constructed mutants induced a high percentage of antibodies that bind to the preF-specific antigen site. These characteristics suggest that the mutants constructed can be superior vaccine antigens from the viewpoint of RSV infection prevention effect and safety.

Omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3) are widely used as additives in fish feed in the aquaculture sector. To date, the supply of omega-3 PUFAs have heavily depended upon fish oil production. As the need for omega-3 PUFAs supply for the growing population increases, a more sustainable approach is required to keep up with the demand. The oleaginous diatom Fistulifera solaris is known to synthesize EPA with the highest level among autotrophically cultured microalgae, however, this species does not accumulate significant amounts of DHA, which, in some cases, is required in aquaculture rather than EPA. This is likely due to the lack of expression of essential enzymes namely Δ5 elongase (Δ5ELO) and Δ4 desaturase. In this study, we identified endogenous Δ5ELO genes in F. solaris and introduced recombinant expression cassettes harboring Δ5ELO into F. solaris through bacterial conjugation. As a result, it managed to induce the synthesis of docosapentaenoic acid (DPA; C22:5n-3), a direct precursor of DHA. This study paves the way for expanding our understanding of the omega-3 PUFAs pathway using endogenous genes in the oleaginous diatom.

The α-1,3-glucanase Agl-EK14 from Flavobacterium sp. EK-14 comprises a signal peptide (SP), a catalytic domain (CAT), a first immunoglobulin-like domain (Ig1), a second immunoglobulin-like domain (Ig2), a ricin B-like lectin domain (RicinB), and a carboxy-terminal domain (CTD). SP and CTD are predicted to be involved in extracellular secretion, while the roles of Ig1, Ig2, and RicinB are unclear. To clarify their roles, domain deletion enzymes Agl-EK14ΔRicinB, Agl-EK14ΔIg2RicinB, and Agl-EK14ΔIg1Ig2RicinB were constructed. The insoluble α-1,3-glucan hydrolytic, α-1,3-glucan binding, and fungal cell wall hydrolytic activities of the deletion enzymes were almost the same and lower than those of Agl-EK14. Kinetic analysis revealed that the K_m values of the deletion enzymes were similar and uniformly higher than those of Agl-EK14. These results suggest that the deletion of RicinB causes a decline in binding and hydrolytic activity and increases the K_m value. To confirm the role of RicinB, Ig1, Ig2, and RicinB were fused with green fluorescent protein (GFP). As a result, RicinB-fused GFP (GFP-RicinB) showed binding to insoluble α-1,3-glucan and Aspergillus oryzae cell walls, whereas Ig1- and Ig2-fused GFP did not. These results indicated that RicinB is involved in α-1,3-glucan binding. The fusion protein GFP-Ig1Ig2RicinB was also constructed and GFP-Ig1Ig2RicinB showed strong binding to the cell wall of A. oryzae compared to GFP-RicinB. Gel filtration column chromatography suggested that the strong binding was due to GFP-Ig1Ig2RicinB loosely associated with itself.

The synthesis of carbocyclic-ddA, a potent antiviral agent against hepatitis B, relies significantly on (1R,3R)-3-hydroxycyclopentanemethanol as a key intermediate. To effectively produce this intermediate, our study employed a chemoenzymatic approach. The selection of appropriate biocatalysts was based on substrate similarity, leading us to adopt the CrS enoate reductase derived from Thermus scotoductus SA-01. Additionally, we developed an enzymatic system for NADH regeneration, utilising formate dehydrogenase from Candida boidinii. This system facilitated the efficient catalysis of (S)-4-(hydroxymethyl)cyclopent-2-enone, resulting in the formation of (3R)-3-(hydroxymethyl) cyclopentanone. Furthermore, we successfully cloned, expressed, purified, and characterized the CrS enzyme in Escherichia coli. Optimal reaction conditions were determined, revealing that the highest activity occurred at 45 °C and pH 8.0. By employing 5 mM (S)-4-(hydroxymethyl)cyclopent-2-enone, 0.05 mM FMN, 0.2 mM NADH, 10 μM CrS, 40 μM formic acid dehydrogenase, and 40 mM sodium formate, complete conversion was achieved within 45 min at 35 °C and pH 7.0. Subsequently, (1R,3R)-3-hydroxycyclopentanemethanol was obtained through a simple three-step chemical conversion process. This study not only presents an effective method for synthesizing the crucial intermediate but also highlights the importance of biocatalysts and enzymatic systems in chemoenzymatic synthesis approaches.

Mesenchymal stem cells (MSCs) are promising candidates for use in novel cell therapies, although such live cell products are highly complex compared with traditional drugs. For example, difficulties such as the control of manufacturing conditions hinder the manufacture of stable cell populations that maintain their therapeutic potency. Here, assuming that medium selection significantly affects cell potency, we focused on the culture media as a critical manufacturing factor influencing the therapeutic efficacy of MSCs. We therefore performed a tube formation assay to quantify the angiogenic activities of conditioned media used to culture human umbilical vein endothelial cells compared with unconditioned media. Comprehensive molecular genetic analysis using microarrays was applied to determine the effects of these media on signal transduction pathways. We found that activation of the vascular endothelial growth factor (VEGF) signaling pathway differed, and that VEGF concentration was dependent on the composition of the conditioned media. These results indicate that the activation level of cell signaling pathways which contribute to therapeutic efficacy may vary depending on the media components affecting MSCs during their cultivation. Moreover, they indicate that therapeutic efficacy will likely depend on how cells are handled during manufacture. These findings will enhance our understanding of the quality control measures required to ensure the efficacy and safety of cell therapy products.

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles.

Shewanella vesiculosa HM13, a psychrotrophic gram-negative bacterium isolated from the intestinal contents of horse mackerel, produces abundant extracellular membrane vesicles (EMVs) by budding the outer membrane. The EMVs of this bacterium carry a single major cargo protein, P49, of unknown function, which may be useful as a carrier for the secretory production of heterologous proteins as cargoes of EMVs. In this study, to increase the utility of S. vesiculosa HM13 as a host for EMV-mediated protein production, we improved its EMV productivity by weakening the linkage between the outer membrane and underlying peptidoglycan layer. In gram-negative bacteria, the outer membrane is connected to peptidoglycans predominantly through Braun's lipoprotein (Lpp), and the formation of this linkage is catalyzed by an l,d-transpeptidase (Ldt). We constructed gene-disrupted mutants of Lpp and Ldt and assessed their EMV productivity. The EMVs of the lpp- and ldt-disrupted mutants grown at 18 °C were evaluated using nanoparticle tracking analysis, and their morphologies were observed using transmission electron microscopy. As a result, an approximately 2.5-fold increase in EMV production was achieved, whereas the morphology of the EMVs of these mutants remained almost identical to that of the parent strain. In accordance with the increase in EMV production, the mutants secreted approximately 2-fold higher amounts of P49 than the parent strain into the culture broth as the EMV cargo. These findings will contribute to the development of an EMV-based secretory production system for heterologous proteins using S. vesiculosa HM13 as a host.