Biotechnology Letters最新文献

Objectives: To expand the availability of promiscuous oleate hydratases (OAHs) for the asymmetric hydration of unactivated alkenes via sequence-based genome mining combined with targeted amino acid substitution.

Results: From 100 screened OAHs, 13 candidates were chosen, all exhibiting hydration activity toward oleic acid. These enzymes also showed significant activity with 1-decene (2 mM), with AuOAH, CkOAH, AiOAH, and CeaOAH producing (S)-2-decanol at concentrations of 822, 603, 495, and 461 μM, respectively. AuOAH, CeaOAH, and CkOAH further demonstrated notable activity with short-chain 1-heptene (2 mM), generating (S)-2-heptanol concentrations of 156, 115, and 133 μM, respectively. AuOAH, sourced from Acinetobacter ursingii for its relatively high activity and broad substrate range, was purified and characterized, showing turnover rates of 0.43-3.21 nmol min^-1 mg^-1 for 1-alkenes (C7-C13). The optimization of reaction conditions for whole-cell asymmetric hydration of 1-decene in recombinant E. coli (AuOAH) demonstrated that exogenous illumination with 561.5 nm light (9.4 µmol m^-2 s^-1) increased 1-decene conversion by approximately 1.5-fold. Similar light-induced enhancements (1.3-2.2-fold) were observed in OAHs from various sources. Under optimized conditions, recombinant E. coli (AuOAH) achieved 13.2-78.7% conversion for various unactivated alkenes (C7-C13) in an aqueous/organic two-phase system, with ee values ≥ 98%.

Conclusions: This study significantly enriches the enzymatic toolbox for asymmetric alkene hydration and illustrates the beneficial effect of light illumination on OAH-catalyzed hydration.

Arctigenin, a high-value lignan with broad bioactivities, suffers from low yield in conventional extraction. This study developed a green and efficient process integrating natural deep eutectic solvent (NADES)-pretreated Aspergillus niger fermentation with ultrasound-assisted extraction. Under optimized conditions (liquid-solid ratio 26:1 mL/g, 38 h fermentation, pH 5.6, 430 W ultrasound at 58 °C, 42% NADES water content), the arctigenin yield reached 54.91 mg/g, 14.64-fold higher than in the raw herb (3.75 mg/g). NADES enhanced fungal growth, membrane permeability, and extracellular enzyme activity, facilitating bioconversion and extraction. This strategy effectively overcomes limitations of low arctigenin content and poor extraction efficiency, offering a promising approach for producing rare herbal compounds and insight into NADES-assisted whole-cell catalysis.

α-Amylases (EC 3.2.1.1) are endoenzymes that hydrolyze α-1,4-glycosidic bonds in starch to produce maltooligosaccharides with broad industrial applications (food, textile, fermentation, biofuels). Most α-amylases act only on gelatinized starch, but Bacillus siamensis JJC33M secretes a native enzyme (AmyJ33-ABC) active on both gelatinized and raw starch. The growth of B. siamensis JJC33M was evaluated, showing µ = 0.55 h⁻¹, Y_p/s = 0.13 g/g, Y_x/s = 0.24 g/g, Y_p/x = 0.55 g/g, and Q_p = 0.063 g/Lh, values comparable with other native production systems. AmyJ33-ABC was partially purified and characterized. The enzyme displayed optimal activity at pH 5.0 and 80 °C, with K_m = 1.47 mg/mL, V_max = 39.37 U/mg, and catalytic efficiency K_cat/K_m = 22.31 s⁻¹ mg⁻¹ mL, comparable with another native systems. At optimal conditions, it hydrolyzed 57.5% of gelatinized potato starch, generating glucose, maltose, maltotriose, maltotetraose, and minor maltooligosaccharides up to DP7. Structural modeling confirmed the canonical GH13 fold (A/B and C domains) and revealed three aromatic-rich surface-binding sites (SBS) located near the catalytic triad. These SBS may explain the enzyme activity on raw starch despite lacking a carbohydrate-binding module (CBM). AmyJ33-ABC combines dual activity on gelatinized and raw starch, acidic pH preference, and high-temperature optimum. These distinctive features highlight its potential for starch bioconversion in bakery, syrup, and related industries.

Chronic wounds, with prolonged healing time, remain a formidable challenge due to lack of angiogenesis, antimicrobial effect, and biocompatibility of conventional wound dressings. The current study focuses on developing novel copper-doped mesoporous bioactive glass nanoparticles (Cu-MBGNs) doped with Propolis/ Sodium Alginate (ALG) based hydrogel to promote angiogenesis, tissue regeneration, biocompatibility, and antimicrobial efficacy for advanced wound healing applications. Hydrogel was synthesized using a solution-casting method. It was characterized using material and biological characterization techniques. Scanning electron microscopy (SEM) displayed an interconnected porous network with dispersed Cu-MBGNs facilitating hydrogel swelling/deswelling and degradation behavior (75%). Fourier Transform Infrared Spectroscopy (FTIR) confirmed crosslinking between propolis and ALG via hydrogen bonding. Hydrogel exhibited an antimicrobial effect against Escherichia coli and Staphylococcus gallinarum. Cu⁺² promoted vasculogenesis by modulating vascular endothelial growth factor release and cytocompatibility. These results suggest that Propolis/ALG/Cu-MBGNs hydrogel offers cost-effective and sustainable solution for enhanced wound healing.

The precise measurement of microRNAs (miRNAs) is essential for diagnosing newborn pneumonia. This paper presents a simple, sensitive and accurate fluorescence-based technique for miRNA identification, utilizing a λ-exonuclease (λ-Exo)-driven DNA walker and split G-quadruplex (split-G4)-facilitated signal amplification. In this biosensor, target miRNA initiates the DNA walker by unfolding the Walker-probe, hence perpetually facilitating the reassembly of split-G4. The reformed intact G4 structure is distinctly identified by the commercially accessible fluorescent dye thioflavin T (ThT), facilitating highly sensitive, label-free miRNA identification. Additionally, the DNA walking process is motivated by the λ-Exo, which endows the biosensor with a greatly elevated signal amplification efficiency. This method demonstrates low background noise and good dependability owing to its reliance on split-G4-generated signals. Furthermore, the technique has been effectively utilized on clinical specimens, indicating its capability for disease diagnosis.

Bovine enterokinase light chain (bEkL) is a serine protease, widely used for the specific cleavage of affinity tags from various recombinant proteins. However, getting soluble expression in Escherichia coli is a challenging task given the presence of multiple cysteines and four disulfide bonds. Strategies that have only been partially successful involve mutating the gene or covalent attachment of solubility tags. We demonstrate a simpler and more efficient production method that combines different strategies like co-expressing the GroES-GroEL chaperone in E. coli SHuffle cells, lowering temperatures to 18 °C post-induction, and ensuring the sufficient accumulation of GroES-GroEL in the cytoplasm before inducing the bEkL gene. A trade-off exists between producing too little GroES-GroEL and promoting inclusion body formation (of bEkL) or expressing too much GroES-GroEL thereby reducing bEkL yields. This optimum level was determined by varying the time difference between the two inductions, and the best results obtained when the co-expressed pGro7 plasmid was induced 2 h before bEkL induction, thus avoiding inclusion body formation. Interestingly, when we delayed the induction of GroES-GroEL to an OD₆₀₀ of 4, which in turn further delayed the induction of bEkL to an OD₆₀₀ of 10, we observed a slowdown in expression rates, but a further improvement in soluble yields. These yields increased over a 36 h long period post-induction at 18 °C in TB medium, where nutrient starvation was prevented by the addition of a concentrated pulse of substrate 20 h post-induction. This slow and steady buildup of soluble bEkL in the cellular cytoplasm allowed us to reach a concentration of 10 mg L^-1 with a high specific activity of approximately 5,000 AU µg^-1. Finally, Ni-NTA affinity chromatography was used to purify the soluble bEkL, and we obtained > 90% homogenous bEkL protein product. The enzymatic activity of this protein was tested using a fusion protein, containing an enterokinase recognition site, as a substrate which showed that the net increase in activity was around 20-fold compared to the initial expression levels obtained with SHuffle cells.

Microbial exopolysaccharides (EPSs) exhibit distinct physiological properties, including anti-inflammatory, anti-tumor, and anti-microbial activities that may have a range of industrial, pharmaceutical, and medical uses. This study aimed to separate, purify, and examine the properties of the Rhizobium sp. HR1101-4 EPS. The optimum parameter set (time, temperature, pH, sugar) for the production of EPS was determined. The highest yield was obtained at a temperature of 39 °C, a pH range of 8-10, and using mannose (0.2%) as the carbon source. The results of FT-IR and the NMR analysis revealed that the EPS is a mixture of hexoses with possible pentose sugars. Moreover, this structure showed positive antibiofilm activity against five pathogenic bacteria; Acinetobacter sp., Staphylococcus aureus, Shigella sonnei, Salmonella sp., and Shigella flexneri, as well as antioxidant activity (42%). Furthermore, the high water-holding (355%) and oil-holding capacity (170%) of the EPS may enhance the nutritional value and rheological characteristics of food products. The improved emulsifying activity of mustard oil suggested potential use for stabilizing emulsions. Additionally, the EPS showed promising flocculating activity, indicating its potential application in treatment of industrial wastewater. This also could be employed as a safe rheological agent and a nutritious food-grade additive for industrial use, paving the way for manufacturing innovation in near future.

The CRISPR/Cas9-based technology has been used for sequential gene editing in E. coli. The plasmids carrying the sgRNA and/or Cas9 genes need to be cured after each round of editing. Curing of these plasmids, particularly the sgRNA plasmid, limits the efficiency of sequential gene editing. In this study, a lethal endotoxin (ccdB) based counterselection was established for improving the overall efficiency of sequential gene editing in E. coli. This approach was validated for sequential editing (deletion) of cstA and ppsA genes in HBUT-P2 strain (W derivative). The experimental results showed that the transformation efficiency of sgRNA plasmid (pTargetF-tcr-P_L-ccdB-N20) reached 10⁸-10⁹ cfu/μg_-DNA, resulting in a 100% and 93.75% recombination rate for cstA and ppsA gene, respectively. Upon completion of cstA gene editing, the sgRNA plasmid (pTargetF-tcr-P_L-ccdB-N20 (cstA)) was effectively cured through ccdB based counterselection at 42 °C, with a 43.75% efficiency. At the end of sequential editing of ppsA gene, both Cas9 (25A) and sgRNA (pTargetF-tcr-P_L-ccdB-N20 (ppsA)) plasmids were cured simultaneously through the sacB and ccdB based counterselections by incubating the cells on LB-sucrose (5%) plate at 42 °C, achieving a curing rate of 100% for Cas9 plasmid (25A), 37.5% for sgRNA plasmid (pTargetF-tcr-P_L-ccdB-N20 (ppsA)), and 37.5% for both Cas9 and sgRNA plasmids. Moreover, this approach was further validated through efficient site-specific insertion of the csc operon into the slmA gene in DH5α (K12 derivative) and S322 (C derivative) strains. These results demonstrated that the endotoxin (ccdB) based counterselection improved the transformation efficiency of sgRNA plasmid, the recombination rate of the editing target gene, the curing rate of sgRNA plasmid, and the overall efficiency of sequential gene editing.

The success of bioremediation of produced water relies on the use of hydrocarbon-degrading bacteria. Hence, the selection of highly tolerant endogenous strains from produced water is crucial to designing successful bioremediation. However, the employed isolation and screening approaches are, in general, long. Integrative and rapid approaches based on microbiological and molecular techniques are now required due to the frequent fluctuation of the composition of the produced water. Here, enrichment cultures at high toxicity followed by protein profiling using MALDI-TOF MS were shown to be efficient in clustering the endogenous hydrocarbon-degrading bacteria and help select the potential candidates. Several bacterial strains (n = 18) were isolated from produced water sampled from Qatar's North Field natural gas production. Fourteen strains were identified as Bacillus cereus (n = 14), and one as Staphylococcus hominis (n = 1) using MALDI-TOF MS. Three strains were identified as Aneurinibacillus humi (n = 2) and Aneurinibacillus aneurinilyticus (n = 1) through ribotyping. The strains were further differentiated based on their protein profiles using MALDI-TOF MS and multivariate statistical analyses. Multivariate analyses (composite correlation index, principal component analysis, and dendrogram) demonstrated substantial diversity among the isolates, highlighting their potential as robust candidates for bioremediation and produced water treatment.

D-pantothenate is one of the essential micronutrients required by the organism. Stereoselective D-lactonohydrolase (D-lac) can hydrolyze DL-pantolactone into D-pantoic acid, the precursor of D-pantothenate. In this study, the D-lac gene from Fusarium moniliforme was heterogeneously expressed in Escherichia coli. Subsequently, coaxial 3D printing was employed, using sodium alginate containing the recombinant cells and CaCl₂ as printing materials. The coaxial nozzle was used to print hollow filaments for the immobilization of recombinant cells. After optimizing the printing conditions, the printed immobilization cells exhibited higher stability and a wider range of reaction conditions (such as pH and temperature) compared to the free cells. After 20 reaction cycles, the enzyme activity maintained approximately 80% of original. The printed hollow filaments were confirmed by scanning electron microscope (SEM). The microchannel structures and large specific surface of filaments may facilitate substrate exchange and enhance catalytic efficiency. The results indicated that the 3D printed hollow filaments can be used as a potential material for enzyme immobilization.