Biotechnology Letters最新文献

In enzyme engineering, a lot of studies have focused on engineering the active site to broaden substrate specificity or enhance transaminase activity; however, relatively little is known about the mechanisms by which substrates are recognized and enter the binding pocket. Transaminases play a crucial role in the synthesis of chiral amines due to their exceptional stereoselectivity and catalytic efficiency. In this study, we explored how the pedal-like loop at the active site influences (R)-transaminase (ATA) activity and substrate recognition by modulating the substrate channel. The pedal-like loop at the active site was swapped with loops from other well-characterized transaminases, and the best-performing variant exhibited a 5.2-fold increase in activity toward (R)-phenylethylamine ((R)-PEA) and an 11.8-fold increase in activity toward isopropylamine (IPA). Additionally, some variants showed significant changes in substrate preference. Homology modeling and molecular docking analysis provided compelling evidence that the pedal-like loop is a critical determinant of both substrate recognition and catalytic activity in (R)-ATA.

Neurturin, a neurotrophic growth factor, has been identified as a potential treatment or reversal agent for neurodegenerative conditions. Although Escherichia coli is an appropriate host for recombinant protein expression, the production of proteins with disulfide bonds, such as neurturin, in this strain is frequently accompanied by the formation of inclusion bodies. In this study, the Rosetta-gami strain, which is well-suited for the accurate formation of disulfide bonds was employed for the soluble production of neurturin. Response surface methodology (RSM) was also used to investigate the effects of IPTG concentration, post-induction time and temperature on the soluble production of neurturin. The results showed that the highest yield of neurturin production occurred in the presence of 0.8 mM of IPTG after 5.5 h at 26 ºC. Fractional Factorial Design was used in the subsequent stage to screen the effects of culture medium components on the protein production. The best concentrations of yeast extract, tryptone and MgSO₄ to have a significant effect on total protein concentration were determined by RSM design to be 15 g/l for both tryptone and yeast extract and 2.2 g/l for MgSO₄. Finally, an experiment was carried out under optimized conditions to evaluate the yield of the process. The results demonstrated a notable enhancement in neurturin production following optimization, with an increase of 8.6-fold compared to the normal condition.

Metagenomics is increasingly recognized as a vital technique for exploring uncultured microorganisms, with one key application being the discovery of novel enzymes for industrial use. This study identified an endoglucanase gene from soil metagenome, termed ZFEG1801, which was expressed in E. coli BL21, purified, and characterized for its biochemical properties. The 72.8 kDa recombinant protein exhibited hydrolytic activity against sodium carboxymethyl cellulose (CMC) and konjac glucomannan (KG), with activities of 12.1 U/mg and 42.1 U/mg, respectively. The enzyme displayed optimal activity at pH 5 for CMC and pH 6 for KG, with broad pH stability ranging from 5 to 9. The optimal temperature was 40 °C, and it remained thermally stable between 20 and 40 °C, retaining over 60% of its activity. The enzyme activity remained stable in the presence of most metal ions; however, CMCase activity was inhibited by Cu²⁺, while glucomannanase activity was inhibited by Mn²⁺, Fe³⁺, and Ca²⁺. The catalytic efficiency towards both substrates was reduced by addition of SDS, DMSO, ethanol, isopropanol and acetonitrile. The V_max and K_m of the purified recombinant enzyme were 106.4 μmol/L/min and 4.9 mg/mL for CMC, and 833.3 μmol/L/min and 11.1 mg/mL for KG, respectively. The dual catalytic properties of ZFEG1801, broad pH stability and resistance to additives, demonstrate its potential for use in various biomass degradation processes.

Objectives: To enhance hyaluronic acid (HA) production in Escherichia coli by utilizing hasA genes from Streptococcus pyogenes and Streptococcus parauberis, and employing metabolic engineering strategies.

Results: The expression of the hasA (SpaHasA) gene from S. parauberis in E. coli K12 W3110 led to higher HA production compared to the other gene. Knockout of the zwf and pfkA genes in the engineered E. coli expressing SpaHasA gene, further increased HA production to 891 mg l^-1. Overexpression of the galU and ugd genes in the zwf and pfkA double mutant harboring the SpaHasA gene elevated HA output to 1017 mg l^-1. Using the same engineered E. coli strain, optimizing the MgSO₄ concentration in the culture medium enhanced production to 1187 mg l^-1, and in fed-batch fermentation, it achieved 2283 mg HA l^-1.

Conclusions: The hasA genes from various Streptococcus groups, especially S. parauberis, significantly boost HA production in E. coli, demonstrating their potential for microbial fermentation applications.

This work aimed to assess the SARS-CoV-2 structural proteins' expression and virus-like particles (VLP) production by Baculovirus/Insect cell platform using two levels of Multiplicity of Infection (MOI), and two culture media, one of them a serum-free medium and the other one chemically defined. Two SARS-CoV-2 VLP were obtained from Sf9 cells coinfection using in both cases, three monocistronic recombinant baculoviruses holding the genes of Nucleocapsid (N; MOI = 2 or 0.2), Membrane (M; MOI = 1 or 0.1), and Envelope (E; MOI = 1 or 0.1) viral proteins, and the fourth one was changed between a baculovirus bearing Spike protein (S; MOI = 3 or 0.3) or receptor-binding domain (RBD; MOI = 3 or 0.3) genes of SARS-CoV-2. Similar performance was verified for both culture media in SARS-CoV-2 VLP production bearing four structural virus proteins or RBD domain. The SARS-CoV-2 structural proteins' expression was comparable at different MOIs (tenfold) as well as SARS-CoV-2 VLP size (around 100 nm). The increase in specific death rates over the coinfection phase was confirmed in relatively high MOI assays. This finding was related to an exponential virus titer profile for high MOIs over the entire infection phase, meanwhile, a viral peak was observed at low MOIs, confirming a secondary infection. The SARS-CoV-2 VLP improved production carrying immunogenic S protein was confirmed concerning others holding RBD. However, the protein composition of produced VLP should be studied further to assess the VLP homogeneity when different culture media and MOIs are used.

Background: Lipoprotein (a) [Lp(a)] is a critical factor in cardiovascular health, composed of low-density lipoprotein-like particles bound to apolipoprotein (a). Elevated Lp(a) levels are associated with an increased risk of cardiovascular diseases (CVD), accelerating disease progression and raising CVD-related mortality. However, the lack of standardized measurement methods for Lp(a) contributes to diagnostic uncertainties in this area.

Method: A quantitative measurement method for serum Lp(a) was developed using fully automated latex-enhanced particle immunoturbidimetry, marking a significant advancement in diagnostic capabilities. Key parameters, including repeatability, stability, linearity, detection limit, interference, and method comparison, were evaluated to ensure the assay's reliability and accuracy.

Result: Lp(a) in samples was detected by carboxylated latex particles (95 nm in diameter) covalently coated with anti-Lp(a) antibodies. Lp(a) concentration was quantified by measuring the turbidity changes caused by agglutination at 600 nm. This method provides rapid, accurate, and fully automated measurements on the Hitachi 7100 automatic biochemical analyzer. With intra-batch precision CV% of 1.10% and inter-batch precision CV% of 1.79%, the method demonstrates reliable performance with Randox biochemical quality control samples. It has a detection limit of 7 mg/L and a high correlation coefficient (R² = 0.9946) within the 0-1500 mg/L range. Minimal interference from bilirubin, fat emulsion, hemoglobin, and ascorbic acid was observed. Additionally, it shows strong correlation (R² = 0.9972) with a commercially available latex-enhanced immunoturbidimetric Lp(a) assay reagent, confirming its comparability and clinical suitability.

Conclusion: The quantitative serum Lp(a) determination method based on latex-enhanced immunoturbidimetry offers numerous advantages. It provides rapid, accurate, and automated results, making it ideal for routine clinical testing. The method effectively measures Lp(a) in serum samples by leveraging the interaction between Lp(a) and latex particles.

The hydrophilic microenvironment surrounding an enzyme's active site can influence its catalytic activity. This study examines the effect of enhancing this environment in the Aspergillus niger fructosyltransferase, SucC. Bioinformatics analysis identified a cysteine residue (C66) near the catalytic triad (D64, D194, E271) as vital for maintaining the active site's structure and facilitating substrate transport. Simulated mutagenesis suggested that mutating cysteine to serine (C66S) could increase hydrophilicity without altering the structure significantly. This mutation was predicted to enhance substrate affinity, with binding energy changing from -3.65 to -4.14 kcal mol^-1. The C66S mutant, expressed in Pichia pastoris GS115, showed a 61.3% increase in specific activity, a 13.5% decrease in K_m (82.20/71.14 mM), and a 21.6% increase in k_cat (112.23/136.48 min^-1), resulting in a 40.1% increase in catalytic efficiency (1.37/1.92 min^-1 mM^-1). For fructooligosaccharides (FOS) production, C66S demonstrated enhanced transfructosylation, particularly in the initial stages of the reaction, achieving higher overall FOS yields. These findings highlight that modifying the active site hydrophilicity, without causing major structural changes, is a promising strategy for improving an enzyme's catalytic efficiency.

Phellinus linteus (PL) is a medicinal fungus known for producing hispolon, a bioactive compound with antioxidant, anti-inflammatory, and anticancer properties. However, the natural scarcity of PL and the unsuccessful cultivation of its fruiting bodies have led to the exploration of alternative methods for enhancing its bioactive compound production. In this study, static fermentation was employed, and Valproic acid (VPA), a histone deacetylase inhibitor (HDACi), was added to the culture medium to induce epigenetic modifications and enhance hispolon production. After 30 days of fermentation, the hispolon concentration was analyzed using high-performance liquid chromatography (HPLC), mycelial dry weight was measured, and the expression of hispolon synthesis-related enzymes was quantified using quantitative PCR (qPCR). Additionally, the anticancer potential of the fermented media was assessed in human breast adenocarcinoma HTB-26 cells using assays for cytotoxicity, reactive oxygen species (ROS) formation, apoptosis, antioxidant activity, and autophagy markers. The results revealed that the addition of 400 µM VPA increased hispolon production by 120% and mycelial dry weight by 41%, likely due to enhanced transcriptional accessibility. Furthermore, the PL fermentation media significantly inhibited HTB-26 cell growth through the induction of ROS formation, autophagy, and apoptosis. These findings suggest that VPA-enhanced static fermentation of PL offers a promising strategy for optimizing hispolon production and developing effective anticancer therapeutics.

MAPKs are one of the essential signal transduction complexes which are responsible for the perception of abiotic stress and for the producing of related transcripts for responding to abiotic stress. For systematical analyzes of the mitogen-activated protein (MAP) kinase gene families and their expression profiles in Solanum lycopersicum L. exposed to diverse heavy metal stresses, 17 SlMAPK genes were studied in comparison with their 159 orthologs from various plant species. The result of phylogenetic analysis revealed that SlMAPKs were divided into four different subgroups (A, B, C, and D) based on their nucleic acid and protein sequence alignments. SlMAPKs including A, B and C group had lower molecular weights and more hydrophobic structures than D group SlMAPKs, while possible extra phosphorylation sites predicted in D-group SLMAPKs. 24 cis regulating elements such as Box 4, TATA-box, ABRE and CAAT-box were detected in their upstream parts of DNA sequences. Also, it was determined that SlMAPKs show interactions with important proteins such as Guanine nucleotide-binding protein beta subunit, heterotrimeric G-protein, protein phosphatase 2C and HY5. The results from our gene expression analyzes, significant increases were found in the expressions of the selected SLMAPK gene with applications of a range of increasing heavy metal concentrations (e.g., by the application of the 400 mM Ni + Pb exposure, a 16-fold increase in the expression of SlMAPK gene was noted). Overall, SlMAPK genes and proteins known were re-evaluated, and the SlMAPKs interactions with some other important proteins were observed. Also, some predictions about the extra phosphorylation sites of SlMAPKs having effects on their functions were done. In addition, the expression levels of SlMAPK genes were monitored under different levels of heavy metal stress exposures.

Objective: This study aimed to prepare oxygen-microbubbles incorporating ferrous porphyrin to emulate the oxygen-capturing ability of hemoglobin porphyrin in red blood cells.

Results: We synthesized poly(2-hydroxyethyl aspartamide) (PHEA) grafted with ferrous porphyrins (Iron-P-PHEA) and created microbubbles using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. These microbubbles trapped oxygen and retained it over a 2 h period. The O₂-microbubbles demonstrated an enhanced photoacoustic effect as an ultrasound contrast agent, as confirmed by Doppler ultrasound testing.

Conclusions: The innovative strategy for O₂-microbubble preparation enhances the efficiency of targeted delivery in molecular optical and ultrasound imaging.