- Purification and kinetics of the PHB depolymerase of Microbacterium paraoxydans RZS6 isolated from a dumping yard. [Journal Article]
- PlosPLoS One 2019; 14(6):e0212324
- Poly-β-hydroxybutyrate (PHB) depolymerase is known to decompose PHB, biodegradable polymers and therefore has great commercial significance in the bioplastic sector. However, reports on PHB depolymer…
Poly-β-hydroxybutyrate (PHB) depolymerase is known to decompose PHB, biodegradable polymers and therefore has great commercial significance in the bioplastic sector. However, reports on PHB depolymerases from isolates obtained from plastic-contaminated sites that reflect the potential of the source organism is scarce. In this study, we evaluated the production of extracellular PHB depolymerase from Microbacterium paraoxydans RZS6 isolated from the plastic-contaminated site in the municipal area of Shahada, Maharashtra, India, for the first time. The isolate was identified using 16S rRNA gene sequencing, gas chromatographic analysis of fatty acid methyl esters (GC-FAME), and BIOLOG method. Ithydrolyzed PHB on minimal salt medium (MSM) containing PHB as the only source of carbon. The isolate produced PHB depolymerase at 45°C during 48 h of incubation. The enzyme was purified most efficiently using octyl-sepharose CL-4B column, with the highest purification yield of 6.675 Umg-1mL-1. The activity of the enzyme was enhanced in the presence of Ca2+ and Mg2+ ions but inhibited by Fe2+ (1 mM) ions and mercaptoethanol (1000 rpm). the nzyme kinetic analysis revealed that the enzyme was a metalloenzyme; requiring Mg2+ ions, that showed optimum enzyme activity at 30°C (mesophilic) and under neutrophilic (pH 7) conditions. Scale-up from the shake-flask level to a laboratory-scale bioreactor further enhanced the enzyme yield by 0.809 UmL-1. The molecular weight of the enzyme (40 kDa), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, closely resembled the PHB depolymerase of Aureobacterium saperdae. Our findings highlighted the applicability of M. paraoxydans as a producer of extracellular PHB depolymerase having potential of degrading PHB under diverse conditions.
- Dynamic formation of cellular aggregates of chondrocytes and mesenchymal stem cells in spinner flask. [Journal Article]
- CPCell Prolif 2019 Jun 17; :e12587
- CONCLUSIONS: Cellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large-scale production of cellular aggregates for cell-based therapy, such as cartilage regeneration.
- Interactive effects between cadmium stabilized by palygorskite and mobilized by siderophores from Pseudomonas fluorescens. [Journal Article]
- EEEcotoxicol Environ Saf 2019 Jun 12; 181:265-273
- The application of palygorskite (PAL) for potentially toxic trace elements (Cd2+, Ni2+, etc.) remediation in polluted soil can substantially reduce the bioavailability and toxicity of these hazard ma…
The application of palygorskite (PAL) for potentially toxic trace elements (Cd2+, Ni2+, etc.) remediation in polluted soil can substantially reduce the bioavailability and toxicity of these hazard materials. However, the secretion of organic acids and siderophores by microorganisms might result in the re-mobilization of cadmium (Cd) in PAL-bound forms (PAL-Cd). In this study, the interactive effects between Cd stabilized by PAL and mobilized by siderophores from Pseudomonas fluorescens were performed with four flask-shaking experimental treatments, namely, strain with or without an ability of siderophores production respectively associated with or without PAL-Cd. The GC-MS and UHPLC-MS test methods were used to analyze the concentrations of metabolites. Results showed that the Cd mobilized by strain with siderophores production was 22.1% higher than that of strain without the ability of siderophores production (p < 0.05). The mobilization of Cd in PAL in turn significantly reduced the siderophores production of Pseudomonas fluorescens by 25.1% (p < 0.05). The numbers of metabolites significantly up-regulated and down-regulated were 9 and 22 in strain groups with PAL-Cd addition compared with the groups without PAL-Cd, respectively. Metabolomics analysis revealed that the mobilized Cd affects the signal transduction pathway and primary metabolic processes, reduces the metabolic capacity of pentose phosphate pathway, glycolysis and tricarboxylic acid cycle pathway. These changes inhibit the ability of strain to biosynthesize amino acids during the mobilization processes, further reducing the capacity of Pseudomonas fluorescens to produce siderophores. This study provides a useful information on how to select soil Cd-stabilizing materials in a targeted manner and how to avoid Cd re-mobilization by siderophores.
- Promoter engineering enables overproduction of foreign proteins from a single copy expression cassette in Bacillus subtilis. [Journal Article]
- MCMicrob Cell Fact 2019 Jun 14; 18(1):111
- CONCLUSIONS: A strong promoter was engineered for efficient chromosomally integrated expression of heterologous proteins.
- Infrared-assisted extraction followed by high performance liquid chromatography to determine angoroside C, cinnamic acid, and harpagoside content in Scrophularia ningpoensis. [Journal Article]
- BCBMC Complement Altern Med 2019 Jun 14; 19(1):130
- CONCLUSIONS: The results show that the IRAE-HPLC is a simple, accurate, and green analytical preparatory method for the potential extraction and quantification of angoroside C, cinnamic acid, and harpagoside in Scrophularia ningpoensis.
- Systematic Optimization of Limonene Production in Engineered Escherichia coli. [Journal Article]
- JAJ Agric Food Chem 2019 Jun 14
- Limonene, a cyclic monoterpene, is widely used in food and cosmetics industries as well as in agriculture. In the work described herein, employing a systematic optimization strategy, we constructed a…
Limonene, a cyclic monoterpene, is widely used in food and cosmetics industries as well as in agriculture. In the work described herein, employing a systematic optimization strategy, we constructed an efficient platform for producing limonene via the heterologous mevalonate pathway in Escherichia coli. By site-directed mutation of EfMvaS and tuning the initial translation of EfMvaE and EfMvaSA110G through ribosome binding site engineering, the upstream module for overproducing mevalonate was obtained. Expression of MmMK with ScPMK, ScPMD, and ScIDI under FAB80 promoter resulted in an efficient midstream module to produce 181.73 mg/L of limonene. Subsequently, coexpression of SlNPPS and MsLS in the downstream module led to a great improvement of limonene production to 694.61 mg/L. Finally, metabolically engineered strain ELIM78 produced 1.29 g/L of limonene in 84 h by fed-batch fermentation in a shake-flask. This is the first report on limonene biosynthesis in E. coli using neryl pyrophosphate synthase, which has promising potential for producing other monoterpenes.
- Metabolic engineering of Corynebacterium glutamicum S9114 based on whole-genome sequencing for efficient N-acetylglucosamine synthesis. [Journal Article]
- SSSynth Syst Biotechnol 2019; 4(3):120-129
- Glucosamine (GlcN) and its acetylated derivative N-acetylglucosamine (GlcNAc) are widely used in the pharmaceutical industries. Here, we attempted to achieve efficient production of GlcNAc via genomi…
Glucosamine (GlcN) and its acetylated derivative N-acetylglucosamine (GlcNAc) are widely used in the pharmaceutical industries. Here, we attempted to achieve efficient production of GlcNAc via genomic engineering of Corynebacterium glutamicum. Specifically, we ligated the GNA1 gene, which converts GlcN-6-phosphate to GlcNAc-6-phosphate by transferring the acetyl group in Acetyl-CoA to the amino group of GlcN-6-phosphate, into the plasmid pJYW4 and then transformed this recombinant vector into the C. glutamicum ATCC 13032, ATCC 13869, ATCC 14067, and S9114 strains, and we assessed the GlcNAc titers at 0.5 g/L, 1.2 g/L, 0.8 g/L, and 3.1 g/L from each strain, respectively. This suggested that there were likely to be significant differences among the key genes in the glutamate and GlcNAc synthesis pathways of these C. glutamicum strains. Therefore, we performed whole genome sequencing of the S9114 strain, which has not been previously published, and found that there are many differences among the genes in the glutamate and GlcNAc synthesis pathways among the four strains tested. Next, nagA (encoding GlcNAc-6-phosphate deacetylase) and gamA (encoding GlcN-6-phosphate deaminase) were deleted in C. glutamicum S9114 to block the catabolism of intracellular GlcNAc, leading to a 54.8% increase in GlcNAc production (from 3.1 to 4.8 g/L) when grown in a shaker flask. In addition, lactate synthesis was blocked by knockout of ldh (encoding lactate dehydrogenase); thus, further increasing the GlcNAc titer to 5.4 g/L. Finally, we added a key gene of the GlcN synthetic pathway, glmS, from different sources into the expression vector pJYW-4-ceN, and the resulting recombinant strain CGGN2-GNA1-CgglmS produced the GlcNAc titer of 6.9 g/L. This is the first report concerning the metabolic engineering of C. glutamicum, and the results of this study provide a good starting point for further metabolic engineering to achieve industrial-scale production of GlcNAc.
- The Morphological Effects of ZnO Upon the Antimicrobial and Deodorant Activities of Polyethylene Terephthalate/ZnO Composite Filaments. [Journal Article]
- JNJ Nanosci Nanotechnol 2019 Dec 01; 19(12):7721-7728
- In this study, the morphological effects of ZnO on the antimicrobial and deodorant activities of synthetic fibers were investigated. Three different polyethylene terephthalate (PET)/ZnO filaments wer…
In this study, the morphological effects of ZnO on the antimicrobial and deodorant activities of synthetic fibers were investigated. Three different polyethylene terephthalate (PET)/ZnO filaments were prepared by incorporating various ZnO nanostructures (rods, plates, and spheres) into PET filaments via a melt-spinning process. The antimicrobial activity of the as-prepared fibers was evaluated by the shake-flask method using two types of bacteria (Staphylococcus aureus and Klebsiella pneumoniae). The deodorant activity of the as-prepared fibers was evaluated by the gas detection tube method. All the PET/ZnO filaments exhibited excellent antimicrobial activity with a bacterial reduction value of 99.9%. The PET/ZnO rod filament showed the best deodorant performance of 60.0%. Both the antimicrobial and deodorant activities of the PET/ZnO filaments were influenced by the morphology of ZnO. However, the morphology of ZnO had a different effect on each functionality of the PET/ZnO filaments. The antimicrobial activity of the PET/ZnO filaments was mainly affected by the physical properties of ZnO rather than its morphology. By contrast, the deodorant activity of the PET/ZnO filaments was highly influenced by the morphology of ZnO.
- Modeling and optimization of tannase production with Triphala in packed bed reactor by response surface methodology, genetic algorithm, and artificial neural network. [Journal Article]
- 3B3 Biotech 2019; 9(7):259
- In this research, optimization of the production medium to enhance tannase production by Bacillus gottheilii M2S2 in laboratory-scale packed bed reactor was studied. Amount of substrate Triphala, moi…
In this research, optimization of the production medium to enhance tannase production by Bacillus gottheilii M2S2 in laboratory-scale packed bed reactor was studied. Amount of substrate Triphala, moisture content, aeration rate, and fermentation period was chosen for optimization study. During one variable at a time optimization, the highest tannase activity of 0.226 U/gds was shown with Triphala as a substrate at the fermentation period of 32 h. Furthermore, the optimum conditions predicted by response surface methodology (RSM) and genetic algorithm (GA) were found to be 11.532 g of substrate Triphala, 47.071% of the moisture content, and 1.188 L/min of an aeration rate with uppermost tannase activity of 0.262 U/gds. In addition, the single hidden layer feedforward neural network (SLFNN) and the radial basis function neural network (RBFNN) of an artificial neural network (ANN) were adopted to compare the prediction performances of the RSM and GA. It revealed that the ANN models (SLFNN, R 2 = 0.9930; and RBFNN, R 2 = 0.9949) were better predictors than the RSM (R 2 = 0.9864). Finally, the validation experiment exhibited 0.265 U/gds of tannase activity at the optimized conditions, which is an 11-fold increase compared to unoptimized media in shake flask.
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- A design of experiments approach for the rapid formulation of a chemically defined medium for metabolic profiling of industrially important microbes. [Journal Article]
- PlosPLoS One 2019; 14(6):e0218208
- Geobacillus thermoglucosidans DSM2542 is an industrially important microbe, however the complex nutritional requirements of Geobacilli confound metabolic engineering efforts. Previous studies have ut…
Geobacillus thermoglucosidans DSM2542 is an industrially important microbe, however the complex nutritional requirements of Geobacilli confound metabolic engineering efforts. Previous studies have utilised semi-defined media recipes that contain complex, undefined, biologically derived nutrients which have unknown ingredients that cannot be quantified during metabolic profiling. This study used design of experiments to investigate how individual nutrients and interactions between these nutrients contribute to growth. A mathematically derived defined medium has been formulated that has been shown to robustly support growth of G. thermoglucosidans in two different environmental conditions (96-well plate and shake flask) and with a variety of lignocellulose-based carbohydrates. This enabled the catabolism of industrially relevant carbohydrates to be investigated.