Biotechnology Letters最新文献

Phospholipase C enzymes (plcA, plcB, plcC) represent critical virulence determination in mycobacterium tuberculosis pathogenesis. These enzymes play pivotal roles in disrupting phagosomal maturation, inducing macrophage necrosis, and facilitating immune evasion mechanisms. Protein sequences of phospholipase C gene plcA, plcB,plcC were collected and screened against human to exclude homologous matches and minimize cross-reactivity. Linear B-cell epitopes and T cell epitopes were identified and evaluated for the ability to produce strong antigenicity, solubility, toxicity and allergenicity. Suitable segments were linked using EAAK for adjuvant fusion, GPGPG between T-cell epitopes, and AAY between cytotoxin T-cell epitopes, with the L7/L12 ribosomal proteins at N-terminus as immunostimulatory adjuvant, The full vaccine structures were modelled in 3-D and improved for accuracy, and interaction of tuberculosis related proteins were analysed. Immunological potent epitopes for all 3 phospholipase C enzymes with favourable physiochemical properties and structural stability were identified. Immune simulation predicted effective stimulation of both humoral and cell-mediated responses. Molecular docking revealed promising interactions with key targets, characterized by favourable binding energies and stable complex formation dominated by hydrogen bonds and electrostatic interactions, suggesting potential functional efficacy of the designed vaccine. The study presents a strong potential of rationally designed multi-epitope vaccine candidate targeting phospholipase C virulence factor of M. tuberculosis. The computational workflow established a rigorous selection process for immunologically relevant epitopes assembled into chimeric construct with predicted vaccine potential. Further experimental validation through invitro antigenicity assay and in-vivo immunization studies is needed to assess the translational potential of this computationally designed vaccine.

Chitin valorization through microbial bioprocessing relies on efficient utilization of its monomeric units as fermentation substrates. In this study, the effects of salt concentration and the mixing ratio of N-acetylglucosamine (GlcNAc) to glucosamine hydrochloride (GlcN·HCl) on the specific growth rate of our previously isolated V. natriegens N5.3 was investigated in the shake-flask. Batch and fed-batch fermentations using chitin-derived amino sugars were further performed to assess high-cell-density cultivation potential.Although the maximum specific growth rate ( ${\mu }_{\max }$ ) at 60 g/L NaCl was nearly two-fold lower than that at the optimal concentration of 15 g/L, strain N5.3 retained robust growth with ${\mu }_{\max }$ values of 0.37 h^-1 on GlcN·HCl and 0.66 h^-1 on GlcNAc. Fed-batch cultivation yielded a maximum cell dry weight (CDW) of 42.3 g/L within 9 h on GlcNAc, with ${\mu }_{\max }$ of 0.53 h^-1, but with a low biomass yield ( $Y_{X/S}$ = 0.16 g/g). In contrast, a substrate mixture containing 5% (w/w) GlcNAc and 95% (w/w) GlcN·HCl maintained a high ${\mu }_{\max }$ (0.49 h^-1) while substantially improving $Y_{X/S}$ (0.29 g/g), resulting in a CDW of 35.5 g/L after 9 h. Due to low solubility of both amino sugars, exponential feeding with non-sterilized powders was successfully applied. The absence of contamination demonstrate the feasibility of this approach. These results demonstrate that the mixture of GlcNAc:GlcN·HCl (1:19 ratio) is effective substrate for cultivation of V. natriegens N5.3. This provides a promising foundation for the microbial conversion of chitin-derived feedstocks into high-value products.

Laccase is an environmentally friendly biocatalyst widely used in wastewater treatment, the food industry, and biosensors. However, free laccase is susceptible to environmental factors such as pH and temperature. Immobilizing it on nanomaterials can significantly mitigate these issues. This approach also enhances the reusability of laccase, demonstrating broad application prospects. Four different morphologies of MnO₂ were compared, with δ-MnO₂ (sheet) demonstrating the most effective immobilization effect as a carrier. Under conditions of pH=5, 30 °C, and laccase concentration of 1 mg/L, the reaction achieves optimal immobilization after 4 h of adsorption. Characterization of δ-MnO₂ and immobilized laccase was performed using Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The enzymatic properties of immobilized laccase were also investigated. The results indicate that the optimal temperature for immobilized laccase is 75 °C, with an optimal pH of 3. Compared to free laccase, stability has been significantly enhanced. Furthermore, even after 30 days of storage at -4 °C the relative enzyme activity of the immobilized laccase remained as high as 74.51%. The kinetic constants demonstrate that immobilized laccase not only enhances the maximum reaction rate but also significantly improves substrate affinity. Compared to free laccase, the relative enzyme activity of MnO₂-immobilized laccase (MnO₂@Lac) is significantly enhanced. This may be attributed to the synergistic effect between MnO₂ nanoparticles and laccase during substrate conversion. This study creates favorable conditions for the further application of immobilized laccase in the treatment of organic pollutants.

Strains of Penicillium sp. are known to produce various enzymes with industrial relevance. The primary objective of the study is to determine the glycanolytic enzyme-producing potentialities of three Penicillium sp. strains, viz. PDF4, XDF1(i), and XDF7(iii), and to optimize the enzyme production and activity at different pH and temperatures. The efficacy of enzyme production and enzyme activities was tested both qualitatively and quantitatively using different glycan and lignocellulosic substrates under varying pH and temperature conditions. Among the strains, Penicillium oxalicum strain XDF7(iii) (ITS: OR555781; NL: OR555751) was the highest pectinase producer (0.303547 μmol mL^-1min^-1) at pH 3.0 in YP-pectin medium. It also exhibited the highest xylanase activity (0.768501 μmol mL^-1min^-1) in YP-xylan medium at pH 5.0. In contrast, all strains were poor producers of CMCase in all culture conditions. The strain XDF7(iii) also effectively utilized both Musambi peel (MP) and Sugarcane bagasse (SB) and recorded the highest xylanase activity (1.0912764 μmol mL^-1min^-1) and pectinase activity (0.8576 μmol mL^-1min^-1) on the 3^rd day of incubation on MP. Thus, the study concludes that these new environmental strains of Penicillium sp. can produce a high amount of industrial enzymes, such as xylanase and pectinase, under standard fermentation conditions. Furthermore, the low-cost lignocellulose biomass MP could be utilized for large-scale xylanase and pectinase production.

Biosynthesis of the fragrance compound cinnamyl isobutyrate was achieved in Escherichia coli through acyltransferase selection and artificial pathway construction.The acyltransferase CATec3 Y20F responsible for cinnamyl isobutyrate formation was identified. Genes involved in cinnamyl alcohol biosynthesis, including Arabidopsis thaliana phenylalanine ammonia-lyase (AtPAL), Hypericum calycinum cinnamate:CoA ligase (HcCNL), Lolium perenne cinnamyl-CoA reductase (LpCCR1), and the branched-chain α-keto acid dehydrogenase complex (BKD) for isobutyryl-CoA biosynthesis, were introduced along with CATec3 Y20F into E. coli BL21 (DE3). The resulting recombinant strain, BD02, produced 2.6 mg/L cinnamyl isobutyrate and 428.9 mg/L cinnamyl acetate. Subsequently, alsS (encoding acetolactate synthase) from Bacillus subtilis and ilvC (encoding ketol-acid reductoisomerase)/ilvD (encoding dihydroxyacid dehydratase) from E. coli were overexpressed to increase the isobutyryl-CoA level, generating the recombinant strain BD03, which achieved a cinnamyl isobutyrate titer of 7.5 mg/L and 112.4 mg/L of cinnamyl acetate. Thereafter, the panB gene (encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase), the pfkA gene (encoding phosphofructokinase), and the mdh gene (encoding malate dehydrogenase) were knocked out to enhance the supply of isobutyryl-CoA, resulting in the recombinant strain BD04. The strain BD04 produced 17.7 mg/L of cinnamyl isobutyrate and 67.2 mg/L of cinnamyl acetate. De novo biosynthesis of cinnamyl isobutyrate was achieved for the first time in engineered E. coli, thereby establishing a proof-of-concept for its microbial biosynthesis. This process was accompanied by the generation of cinnamyl acetate, which provides a reference for the microbial synthesis of diverse cinnamyl esters.

To evaluate a novel β-1, 4-endo-glucanase, BsEgl5A, from the bacterium Bacillus subtilis ZS57 isolated from forest soil. The β-1, 4-endo-glucanase (BsEgl5A) gene was cloned and heterogeneously expressed in Escherichia coli. BsEgl5A contained a catalytic domain of glycoside hydrolase family 5 and shared 71% identity with β-1, 4-endo-glucanase from Bacillus sp. IARI-SP-4. The purified endoglucanase had a molecular weight of 55 kDa, as estimated by SDS-PAGE and western blotting. The enzyme exhibited maximum activity at 55℃ and pH 5.0, and showed remarkable stability at 70℃ and pH 3.0-7.0. The activity of BsEgl5A was improved by Mn²⁺ ions and inhibited in the presence of Cu²⁺ ions. BsEgl5A was highly active towards laminarin and barley β-glucan, moderately towards CMC-Na. BsEgl5A degraded cellotetrose, cellopentose and cellohexaose to cellotriose and cellobiose. Moreover, BsEgl5A reduced both the filtration time and the viscosity of the brewer mash. Among tested agricultural straws, BsEgl5A showed highest reducing sugars production (3.54 mg/mL) with corn straw. These characteristics make BsEgl5A as useful candidate for degradation of plant biomass to simple sugars in brewing industry and biofuel production.

Heme, an iron-incorporated porphyrin compound, serves as the prosthetic group for numerous proteins involved in diverse biological processes. The prokaryotic heme biosynthetic pathway features a complex cascade of reactions, in which glutamyl-tRNA reductase (GluTR) catalyzes the formation of 5-aminolevulinic acid (ALA) that represents a critical rate-limiting step and determines ultimate heme yield. In this study, OsGluTR^A510V showed enhanced heme synthesis capacity in Oryza sativa and was used for developing microbial cell factories dedicated to free heme production. Through systematic protein engineering involving site-directed mutagenesis and N-terminal modification, OsGluTR^A510V was optimized to improve the structural stability and catalytic efficiency. It yielded the recombinant enzyme GluTR^{A510V/S189T/KK}, which achieved a maximum heme titer of 13.14 mg/L in Escherichia coli, representing a 7.6-fold improvement over that of GluTR^A510V. To establish heme production in Bacillus subtilis, GluTR^{A510V/S189T/KK} was introduced into the ΔhmoAB-hemX chassis, a modified B. subtilis host lacking key heme biosynthesis inhibitors (hmoA, hmoB, and hemX). This engineered system elevated the heme yield from 0.77 to 3.86 mg/L, achieving a 5.0-fold improvement. This study demonstrates a combinatory metabolic engineering strategy that reconstitutes the heme synthetic route in B. subtilis, enabling efficient production of food-grade free heme through enzyme engineering and chassis optimization.

The aim of this study was to produce fungal chitosan from a potential fungus using a cost-effective substrate (sugar beet molasses) and optimize the growth conditions using Taguchi L9 orthogonal array (OA). The obtained fungal chitinous chitosan (FC) was then used for the microencapsulation of crocin. Optimal conditions were found as 120 mL/L molasses, initial pH at 6 and 5 g/L magnesium sulphate. The dried biomass was weighed as 22.7 g/L, while 8.1 g/L alkali insoluble material (AIM), 5.3 g/L FC and 2.7 g/L native chitosan (NC) were obtained. Deacetylation degree (DD) of the obtained chitosan was calculated as 80.27 and 78.81% for NC and FC, respectively. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was employed for molecular weight detection of the chitosan samples. Molecular weights were found for FC and commercial chitosan (CC) as 145 and 219 kDa, respectively. The solubility of NC and FC in 1% acetic acid was found as 84 and 78% respectively. The fungus was isolated from jujube fruit which was identified as Penicillium expansum HNP11 (GenBank Accession Number: PQ057454). To deepen the research, antimicrobial activity was carried out. The zeta potential of crocin loaded alginate-chitosan microparticles was about - 49 mV and loading capacity was found as 46%. Cytotoxicity of FC was found lower than CC at low concentrations. Consequently, P. expansum has higher antimicrobial activity and minimal toxic structure and Taguchi orthogonal array contributes economic chitosan production.

Polyunsaturated fatty acids (PUFAs) have attracted significant attention for their roles in human health, particularly in cardiovascular protection. As a promising source of PUFAs, microalgae have become a research hotspot due to their rapid growth and ability to accumulate large amounts of PUFAs under stress conditions. This paper reviewed the recent progress in microalgal PUFAs research, focusing on analyzing PUFA content in different microalgal species, the effects of environmental factors on PUFAs synthesis, as well as related biochemical pathways. Additionally, this paper explored strategies for enhancing PUFA production in microalgae using metabolic engineering and gene editing, and outlined current research challenges.