Shake Flask Assays Research Articles

High-Level Extracellular Expression of Hyaluronate Lyase HylP in Bacillus subtilis for Hyaluronan Degradation.

Hyaluronate lyase (HA lyase) has potential in the industrial processing of hyaluronan. In this study, HylP, an HA lyase from Streptococcus pyogenes phage (SPB) was successfully expressed in Bacillus subtilis. To improve the extracellular enzyme activity of HylP in B. subtilis, signal peptide engineering systematic optimization was carried out, and cultured it from shake flasks and fermenters, followed by purification, characterization, and analysis of degradation products. The results showed that the replacement of the signal peptide increased the extracellular enzyme activity of HylP from 1.0 × 104 U/mL to 1.86 × 104 U/mL in the shake flask assay, and using a 20L fermenter in a batch fermentation process, the extracellular enzyme activity achieved the level of 1.07 × 105 U/mL. HylP exhibited significant thermal and pH stability in the temperature range of 40°C and pH range of 4-8, respectively. The enzyme showed optimum activity at 40°C and pH 6, with significant activity in the presence of Na+, Mg2+, and Co2+ ions. Degradation analysis showed that HylP efficiently degraded hyaluronan as an endonuclease, releasing unsaturated disaccharides. These comprehensive findings underscore the substantial industrial potential of HylP for hyaluronan processing applications, offering valuable insights into enzyme characterization and optimization of expression for potential industrial utilization.

Ulva rigida Valorization into Poly(3-hydroxybutyrate), Organic Acids and Functional Ingredients.

Halomonas elongata 1H9T is a moderate halophilic strain able to produce poly(3-hydroxybutyrate) (P(3HB)), a biodegradable plastic, and gluconic acid, a valuable organic acid with wide industrial applications. In this work, the green alga Ulva rigida was used as platform to produce cultivation substrates for microbial conversion as well as functional ingredients, targeting its full valorization. The liquor obtained by autohydrolysis presented the highest concentration of oligosaccharides and protein, being an interesting feedstock to produce functional ingredients. The acid and/or enzymatic hydrolysis liquors are adequate as substrates for microbial processes. Shake flask assays with H. elongata revealed that the N-rich liquor produced after acidic treatment was the best suited for cell growth while the N-poor liquor produced by the enzymatic treatment of acid-pretreated algae residues produced the highest P(3HB) titers of 4.4 g/L. These hydrolysates were used in fed-batch cultivations as carbon and protein sources for the co-production of gluconic acid and polymer achieving titers of 123.2 g/L and 7.2 g/L, respectively. Besides gluconic acid, the Krebs cycle intermediate 2-oxoglutaric acid, also called alpha-ketoglutaric acid (KGA), was produced. Therefore, the co-production of P(3HB) and acids may be of considerable interest as an algal biorefinery valorization strategy.

Biodegradation of micro sized nylon 6, 6 using Brevibacillus brevis a soil isolate for cleaner ecosystem

The increasing accumulation of microplastic constitutes pathogens and hazardous chemicals posing a great threat to the ecosystem. Biodegradation is an eco-friendly strategy for the removal of these contaminants. The present investigation demonstrates bioremediation of nylon 6, 6 microplastics (NMPs) for the first time using a soil isolate called Brevibacillus brevis (B.brevis) by shake flask assay. 22 w/w % weight loss of nylon 6, 6 microplastics was noticed after 35 d of incubation with B.brevis. Upon interaction with microplastics, a change in the shape of the bacterium (rod to round) could be observed along with the size reduction of the microorganism. In the case of NMPs incubated in presence of this bacterium, irregular shapes of NMPs with cracks and holes could be visualized using SEM and TEM. TGA and FTIR analysis reported the disappearance of intermolecular hydrogen bonding of nylon 6, 6 posts microbial interaction. The release of various organic acids and enzyme/enzymatic activities of the bacterium was found to be higher in the presence of NMPs. The mass spectrometric analysis confirmed the release of adipic acid and hexamethylenediamine derivatives during aerobic biodegradation, indicating NMPs being the only carbon source for the growth of B.brevis in the reaction environment. The study addresses B.brevis as a viable bacterial source for the degradation of NMPs while at the same time attempting to understand the mechanism involved. A biodegradation pathway for B.brevis induced NMP degradation has been also proposed for the first time. This finding promises a potential approach for reducing the accumulation of nylon 6, 6 in the environment.

Upgrading end-of-line residues of the red seaweed Gelidium sesquipedale to polyhydroxyalkanoates using Halomonas boliviensis

Agar extraction from Gelidium and Gracilaria red seaweed species produces hundred thousand ton of carbohydrate-rich residues annually. Gelidium sesquipedale waste biomass obtained after agar extraction, still contained 44.2 % w/w total carbohydrates (dry-weight basis). These residues were biologically up-graded to poly-3-hydroxybutyrate (P3HB) after saccharification of their carbohydrate fraction to simple sugars. A combined hydrolysis treatment using sulfamic acid followed by enzymatic hydrolysis with cellulases produced a glucose-rich hydrolysate with a negligible content of inhibitors. With this treatment a sugar yield of circa 30 % (g glucose/g biomass) was attained. The algal hydrolysates were assessed as carbon source for the production of P3HB by the halotolerant bacteria Halomonas boliviensis. A cell concentration of 8.3 g L−1 containing 41 % (w/w) of polymer and a yield (YP/S) of 0.16 gpolymer/gglucose were attained in shake flask assays. In this work, cellulose-rich seaweed waste was shown to be an upgradable, sustainable source of carbohydrates.

Low-density polyethylene films incorporated with silver nanoparticles to promote antimicrobial efficiency in food packaging.

Technological innovations in packaging are intended to prevent microbiological contaminations for ensuring food safety and preservation. In this context, researchers have investigated the antimicrobial effect of low-density polyethylene films incorporated with the following concentrations of silver nanoparticles: 1.50, 3.75, 7.50, 15.00, 30.00, 60.00, and 75.00 µg/ml. The films were characterized using field emission gun scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetry, and differential scanning calorimetry. From the results of these techniques, it could be concluded that the silver nanoparticles incorporated in the low-density polyethylene films did not influence their physical, chemical, and thermal properties. The direct contact assays, shake-flask assays, and bacterial images obtained using scanning electron microscopy were used to analyze the antimicrobial activity of the films. In the microbial analyses, it was verified that the nanostructured films exhibited antimicrobial properties against all the microorganisms studied, although more notably for fungi and Gram-negative bacteria than the Gram-positive bacteria. Moreover, it was discovered that the packages, in which silver nanoparticles were incorporated, inhibited the growth and reproduction of bacterial cells during the early stages. These results suggest that the extruded low-density polyethylene films incorporated with silver nanoparticles may be an essential tool for improving food quality and safety.

Factorial design in fermentation medium development for hyaluronic acid production by Streptococcus zooepidemicus

Hyaluronic acid (HA) has biological functions of interest to medical and cosmetic industries. The optimization of a fermentation medium contributes to improve the microbial production of the polymer on a large scale and make the product more accessible to the market. Factorial design and response surface were used to evaluate the concentration of sucrose, yeast extract, glutamine, glutamate and oxalic acid on the HA production by Streptococcus zooepidemicus in shake flasks. Biomass, lactate and acetate production were also studied. Yeast extract showed to be the main significant variable for all the studied responses, showing positive effect. For the HA production, glutamine was also significant and presented a positive effect. The best condition for the polymer production was 50 g L-1 of yeast extract and sucrose and 0.6 g L-1 of glutamine, glutamate and oxalic acid. In this condition, the fermentation was carried out in bioreactor with pH controlled at 8 and 0.5 vvm. HA production in bioreactor was explained by logistic regression model and the maximum concentration asymptotic of HA was 0.860 g L-1. An increase of 34% on polymer production was observed when compared to shake flask assay. Thus, it was possible to optimize the polymer production using statistical techniques.

Preparation, Biodistribution, and Scintigraphic Evaluation of 99mTc-Clindamycin: an Infection Imaging Agent

Bacterial infection is found to be the cause of death throughout the world. Nuclear medicine imaging with the help of radiopharmaceuticals has great potential for treating infections. In the present work, clindamycin, a lincosamide antibiotic, was labeled with technetium-99m (~380MBq). Clindamycin has been proven to be efficient for treating serious infections caused by bacteria such as Staphylococcus aureus. Quality control, characterization, biodistribution, and scintigraphy of radiolabeled clindamycin were done, and labeling efficiency was determined by ascending paper chromatography. More than 95% labeling efficiency with technetium-99m ((99m)Tc) was achieved at pH6-7 while using 2.5-3μg SnCl2 · H2O as a reducing agent and 100μg of ligand at room temperature. The characterization of the compound was performed by using electrophoresis, HPLC and shake flask assay. Electrophoresis indicates the neutral behavior of (99m)Tc-clindamycin. HPLC analysis confirms the single specie of the labeled compound, while shake flask assay confirms high lipophilicity. The biodistribution studies of (99m)Tc-clindamycin were performed Sprague Dawley rats bearing bacterial infection. Scintigraphy and biodistribution studies showed a high uptake of (99m)Tc-clindamycin in the liver, heart, lung, and stomach as well as at S. aureus-infected sites in rabbits.

Improvement on the yield of polyhydroxyalkanotes production from cheese whey by a recombinant Escherichia coli strain using the proton suicide methodology

In this work Escherichia coli strain CML3-1 was engineered through the insertion of Cupriavidus necator P(3HB)-synthesis genes, fused to a lactose-inducible promoter, into the chromosome, via transposition-mediated mechanism. It was shown that polyhydroxyalkanotes (PHAs) production by this strain, using cheese whey, was low due to a significant organic acids (OA) synthesis. The proton suicide method was used as a strategy to obtain an E. coli mutant strain with a reduced OA-producing capacity, aiming at driving bacterial metabolism toward PHAs synthesis.Thirteen E. coli mutant strains were obtained and tested in shake flask assays, using either rich or defined media supplemented with lactose. P8-X8 was selected as the best candidate strain for bioreactor fed-batch tests using cheese whey as the sole carbon source. Although cell growth was considerably slower for this mutant strain, a lower yield of OA on substrate (0.04CmolOA/Cmollac) and a higher P(3HB) production (18.88gP(3HB)/L) were achieved, comparing to the original recombinant strain (0.11CmolOA/Cmollac and 7.8gP(3HB)/L, respectively). This methodology showed to be effective on the reduction of OA yield by consequently improving the P(3HB) yield on lactose (0.28CmolP(3HB)/Cmollac vs 0.10CmolP(3HB)/Cmollac of the original strain).

99mTc-Daunorubicin a potential brain imaging and theranostic agent: synthesis, quality control, characterization, biodistribution and scintigraphy

Daunorubicin is a chemotherapeutic antibiotic of the anthracycline family used for the treatment of many type of cancers when doxorubicin or other less effective drugs cannot be used. The aim of the present study was labeling of Daunorubicin with (99m)Tc, quality control, characterization, and biodistribution of radiolabeled Daunorubicin. Labeling efficiency was determined by ascending paper chromatography. All the experiments were performed at room temperature (25°C±2°C). More than 96% labeling efficiency with (99m)Tc was achieved at pH 5-6, 2-4 μg stannous chloride and 300 μg of ligand in few minutes. The characterization of the compound was performed by using HPLC, electrophoresis and shake flask assay. Electrophoresis indicates that Tc-99m-Daunorubicin is neutral, HPLC confirms the single specie of the labeled compound, while shake flask assay confirms high lipophilicity. The biodistribution studies of (99m)Tc-Daunorubicin were performed in rats. Significantly higher accumulation of (99m)Tc-Daunorubicin was seen in brain of normal rats. Scintigraphy was also indicating higher accumulation of (99m)Tc-Daunorubicin in brain of normal rabbits.

Detection of antibiotic-producing Streptomyces inhabiting forest soils.

A modified agar overlay technique was developed for detecting antibiotic production by Streptomyces isolates and compared with two routinely used overlay procedures and the shake flask-disk assay method. Two types of inhibitory patterns could be discerned in the modified overlay method, and one of these was always exhibited by Streptomyces isolates which produced extractable antimicrobial substances. In a preliminary screening of 600 isolates, higher numbers of antibiotic-producing streptomyces were detected with the modified overlay procedure than with the streak or pour overlay method and the shake flask assay. The modified overlay distinguished between antibiotic production and other antagonistic effects. The inhibitory agents extracted after 6 days of culture growth on the modified overlay agar were of a higher concentration and more stable over longer periods of time than those recovered from shake flasks. It was observed that acidoduric streptomyces produced a greater proportion of antibiotics against gram-negative bacteria than did neutrophilic cultures, and this effect was greatest with the isolates from the most acidic soils. The neutrophiles were more active against gram-positive bacteria and, upon antibiotic classification, it appeared that the neutrophilic and acidoduric cultures from various soils produced different antimicrobial compounds.