An Enzyme With Wide Distribution is a Potential Surrogate Parameter for the Estimation of Fecal Contamination in Urban Waters
Enzyme Activity Slyd Contamination | An enzyme with widespread distribution has been identified as a potential surrogate parameter for estimating fecal contamination in urban waters. This enzyme, bd-galactosidase (GAL), is susceptible to heavy metal stress and has shown great promise as a rapid “early warning” indicator of fecal contamination. It has been used to measure the amount of bacterial nitrate in human urine.
Enzyme activity bd-galactosidase (GAL) is a potential surrogate parameter for estimating the level of fecal contamination in urban waters.
Inducible bacterial enzymes, bd-galactose, and bd-glucuronidase were studied in Denmark to detect waterborne Campylobacter jejuni. The enzyme activity of the target bacteria was detected more accurately when the induced bacterial enzymes were present than when the induced bacteria were not. Despite the differences between the target and non-target bacteria, the results from this study are consistent with the use of bd-galactosidase as a potential surrogate parameter for the assessment of fecal contamination in urban waters.
This method may not be susceptible to detecting fecal contaminants in drinking water. However, this test is sensitive enough to detect E. coli in water after ultraviolet light disinfection. In the study, the authors found that this method effectively detected both Salmonella and Escherichia coli.
To assess the potential of a bacterial enzyme as a surrogate for determining the level of fecal contamination, isolated pure cultures were grown in 10% tryptic soy broth without dextrose. IPTG, a product of Sigma Chemical Co., was used to induce bd-galactosidase in E. coli and other bacterial strains. Noninduced cells were grown in TSB or MetGlu without IPTG. After one to four days, the culture reached a stationary phase.
Various factors can cause the presence of fecal contamination in urban waters. One example is fecal contamination caused by sewage treatment plants. This contaminant can harm receiving waters. Whether sewage treatment processes cause fecal contamination, the presence of E. coli bacteria will require further action.
GAL activity is a rapid “early warning” indicator of gross sewage contamination
Indicators of fecal pollution are organisms that can be detected in many sewage. The organisms used as indicators are typically E. coli, total coliforms, and sulfite-reducing bacteria. They have several essential functions, including indicating whether the sewage treatment process is working. A perfect indicator must be able to survive as long as the worst potential pathogens.
GAL activity is a ubiquitous enzyme
The sled gene encodes an enzyme of the FKBP family. It is present in nearly every cell in the body and is widely distributed throughout the environment. The enzyme causes syphilis, or inflammation, in the body. The gene is ubiquitous, and contamination by sly is highly prevalent. The study demonstrates the importance of identifying GAL in contaminated water.
Endo-b-gal is an essential component of whey proteins. It may function in the hydrolysis of lactose. Endo-b-gal may also function in the hydrolysis of lactose, but these are untested. Moreover, this enzyme is not yet classified as a slyd contamination agent. The enzyme activity in the milk of lactose intolerant mice has been linked to food-borne diseases.
GAL activity is sensitive to heavy metal stress
Heavy metals affect many organisms through a variety of physiological mechanisms. Fish, for example, may become more vulnerable to predation, suffer reduced reproductive success, or even die. While these effects are often not observable, survey responses may be limited to changes in the relative abundance of metal-tolerant species or decreases in the abundance of metal-sensitive species. Furthermore, gill irritation or collapse of chromophores may result in various physical anomalies in fish. This condition may be exacerbated by a reduction in ion regulation in fish.
The exposure of humans to heavy metals has skyrocketed due to industrialization and anthropogenic activities. Several metals have been highly toxic to humans and cause significant health problems, including mercury, lead, chromium, cadmium, and arsenic. Exposure to these metals through air and water can lead to acute or chronic poisoning. Toxic metals can also cause damage to human tissue through bioaccumulation. Ultimately, heavy metals disrupt cellular processes, leading to harmful effects on human health.
As metals enter the atmosphere, they become precipitated onto land and water. Significant runoff from storms causes episodic exposure to metals. Smaller runoff events are often caused by activities like watering lawns or washing cars. The magnitude of the episodes of exposure is dependent on how much dry deposition occurs between events and how much water is discharged. The highest levels are experienced early in the first flush. Acid rain and soils with acid-forming parent material enhance metal mobility.
Recombinant His-tagged proteins expressed in Escherichia coli
Several methods are available for the purification of recombinant His-tagged proteins. One of the most widely used methods is affinity purification, which uses a small His-tag (typically six or ten histidines) fused to the N or C terminus of the target protein. This method is inexpensive and easy to implement, and the E. coli strain is easy to grow and yields high-quality proteins.
Another method of improving the expression of recombinant proteins in bacteria is strain engineering. Researchers have developed strains that can express histidine-tagged proteins more efficiently than other types of bacteria. The method was initially used in a study by Makino, T. et al. in Applied Microbiology and Biotechnology.
A genetically engineered fusion protein (GE-A-Tag) is a highly efficient tool for producing recombinant eukaryotic proteins. This technique was used to produce a prion-like protein, Shadow. The recombinant Shadow was expressed with a (His)6-tag and solubilized in 8 M urea. It was then bound to a Ni(2+) column and eluted using an imidazole gradient. The purified Shadow was then subjected to size exclusion chromatography. The purified protein was desalted to remove salts.
Another method used to purify recombinant His-tagged proteins is affinity-purified peptide-based tags. The immobilized Ni(II) ion is effective for milligram-scale protein production, and it is a natural partner for 6xHis-tagged proteins. However, the Ni(II) ion is considered the best compromise for purifying recombinant His-tagged proteins.
CBD tag is a chitin tag
CBD-tagged proteins are helpful as a purification tool for enzyme activity. These proteins are commonly detected in food samples. CBD-tagged proteins can be removed by incubation with chitin beads. Using CBD-tagged proteins reduces contamination of His-tagged protein. CBD-tagged proteins also have a high affinity for chitin resin and effectively remove significant contaminant Ni-NTA in food samples.
This method has several advantages. It is compatible with diverse buffer conditions, including 50 mM to 2 M NaCl, 6 M urea, and high imidazole concentrations. Chitin beads are handy for direct loading of elution fractions after Ni-NTA and chitin bead treatment. CBD-tagged proteins are removed from the flowthrough using Ni-NTA-Chitin beads purification.
A novel biotechnological approach to detect contaminants with SlyD uses the CBD tag to identify chitin proteins. The CBD tag is a chitin molecule that affects the enzyme activity of aceE protein and the pyruvate dehydrogenase complex. The method is susceptible and reproducible, and the resultant tags will enable you to detect contaminants in your products without any difficulty.
The fusion tags made of CBMs are reliable and practical. They are precise and low-cost. The CBM’s ability to bind substrates is unique and helps avoid non-specific binding. It is also compatible with enzyme activity slyd contamination. CBMs are the ideal choice for your application when it comes to enzyme activity slid.