Testing temperature and pH stability of the catalase enzyme in the presence of inhibitors

The catalase enzyme is analyzed under different conditions in order to get a better understanding of its function, purpose and benefit to organisms. This enzyme resides in all living organisms that have exposure to oxygen. It uses hydrogen peroxide (H2O2) as its substrate, and is responsible for breaking down H2O2 into oxygen and water, therefore neutralizing oxidative stress in the cells. Maintaining the levels of oxidative stress is highly important because of the impact that the reactive oxidative species (ROS) have on the cells. ROS damage cells by targeting DNA and proteins leading to various complications and illnesses such as cancer, diabetes, neurodegenerative diseases and they even have an impact on the process of aging. To determine the activity of the catalase enzyme and test its stability, different temperatures and pH were employed, along with examining the catalase behavior under the presence of ascorbic acid as an inhibitor. Three samples were used for this experiment: animal sample, plant sample and microorganisms. The indicator of the reaction which aided in the process of determining whether catalase is performing its function was the formation of gas bubbles in the test tubes, and the quantity of that indicator assisted in drawing conclusions about the enzyme activity. This study revealed that the optimum conditions for catalase enzyme activity tend to be 37 °C at a pH of 7, especially present in liver and yeast samples. Ascorbic acid has proven to be a valuable inhibitor of the catalase enzyme. Extremely high or low temperature, along with highly acidic or basic environments tend to alter the enzyme activity disallowing it to perform its key role

Determination of C-reactive protein concentration by the turbidimetric method in diagnostics of viral and bacterial infections

During the flu season that can impair human health, it is difficult to determine whether a patient has a bacterial or viral infection. The C-Reactive Protein marker is one of the markers in laboratory diagnostics that shows inflammatory processes in the body. The purpose of this study was to prove that the CRP marker is an indicator of bacterial infections and that it is presented in higher concentration in patients with bacterial infection than in patients with a viral infection. Therefore it is helpful in the differentiating bacterial infections from viral infections

Adherence of prebiotic fibers, antioxidants and B complex vitamins on the survival of probiotics

The studies on the relationship between probiotics and prebiotics as well as probiotics and antioxidants have gained significant increase in popularity over the past decade. However, exposure to antibiotics can result in disrupting the human microbiome, causing several related diseases. Additionally, probiotics can lose some of their beneficial effects when in contact with stomach acid which has a pH between 2 and 3. The purpose of this study was to examine the effects of prebiotic fibres and antioxidants and B complex vitamins on the survival of probiotics. When combined with those, a sufficient amount of probiotics survives and carries out their beneficial functions. This study points out the importance of mentioned compounds in maintaining a healthy organism as well as their role in preserving the life of probiotics in our gut

Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction

The ability of flavoenzymes to reduce dioxygen varies greatly and is controlled by the protein environment that can cause either a rapid (oxidases) or sluggish (dehydrogenases) reaction. Previously, a “gatekeeper” amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternate gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily, which has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternate gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60,000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) lowered its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes have a cavity binding an anion and resembling the “oxyanion hole” in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features suggesting that dioxygen reactivity might be governed by a common mechanistic principle.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by FEBS (Federation of European Biochemical Societies) and published by John Wiley & Sons, Inc. It can be found at: http://febs.onlinelibrary.wiley.com/hub/journal/10.1111/%28ISSN%291742-4658

Investigation Of 16S rRNA Gene And Gene Segments For The Determination Of Probiotics

The 16s-rRNA consists of hypervariable regions (V1 – V9) that demonstrate considerable sequence diversity among different bacteria. Species-specific sequences within a given hypervariable region constitute useful targets for diagnostic assays and other scientific investigations. Usually the size of the gene region is 1500 bp, which is large enough to be analyzed using bioinformatic tools and applied for detection. The need to advance the knowledge of the 16s-rRNA gene segments in bacterial strains would allow better understanding and better diagnostic possibilities when dealing with them. This could also be the basis for investigation of pathogenic microorganisms

Viral Vector-Based Melanoma Gene Therapy

Gene therapy applications of oncolytic viruses represent an attractive alternative for cancer treatment. A broad range of oncolytic viruses, including adenoviruses, adeno-associated viruses, alphaviruses, herpes simplex viruses, retroviruses, lentiviruses, rhabdoviruses, reoviruses, measles virus, Newcastle disease virus, picornaviruses and poxviruses, have been used in diverse preclinical and clinical studies for the treatment of various diseases, including colon, head-and-neck, prostate and breast cancer as well as squamous cell carcinoma and glioma. The majority of studies have focused on immunotherapy and several drugs based on viral vectors have been approved. However, gene therapy for malignant melanoma based on viral vectors has not been utilized to its full potential yet. This review represents a summary of the achievements of preclinical and clinical studies using viral vectors, with the focus on malignant melanoma

Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction

The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a ‘gatekeeper’ amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60 000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the ‘oxyanion hole’ in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle

Characterization of a poly(butylene adipate-co-terephthalate)- hydrolyzing lipase from Pelosinus fermentans

Certain alpha/beta hydrolases have the ability to hydrolyze synthetic polyesters. While their partial hydrolysis has a potential for surface functionalization, complete hydrolysis allows recycling of valuable building blocks. Although knowledge about biodegradation of these materials is important regarding their fate in the environment, it is currently limited to aerobic organisms. A lipase from the anaerobic groundwater organism Pelosinus fermentans DSM 17108 (PfL1) was cloned and expressed in Escherichia coli BL21-Gold(DE3) and purified from the cell extract. Biochemical characterization with small substrates showed thermoalkalophilic properties (T (opt) = 50 A degrees C, pH(opt) = 7.5) and higher activity towards para-nitrophenyl octanoate (12.7 U mg(-1)) compared to longer and shorter chain lengths (C14 0.7 U mg(-1) and C2 4.3 U mg(-1), respectively). Crystallization and determination of the 3-D structure displayed the presence of a lid structure and a zinc ion surrounded by an extra domain. These properties classify the enzyme into the I.5 lipase family. PfL1 is able to hydrolyze poly(1,4-butylene adipate-co-terephthalate) (PBAT) polymeric substrates. The hydrolysis of PBAT showed the release of small building blocks as detected by liquid chromatography-mass spectrometry (LC-MS). Protein dynamics seem to be involved with lid opening for the hydrolysis of PBAT by PfL1

Characterization of a poly(butylene adipate-co-terephthalate)-hydrolyzing lipase from Pelosinus fermentans

Certain α/β hydrolases have the ability to hydrolyze synthetic polyesters. While their partial hydrolysis has a potential for surface functionalization, complete hydrolysis allows recycling of valuable building blocks. Although knowledge about biodegradation of these materials is important regarding their fate in the environment, it is currently limited to aerobic organisms. A lipase from the anaerobic groundwater organism Pelosinus fermentans DSM 17108 (PfL1) was cloned and expressed in Escherichia coli BL21-Gold(DE3) and purified from the cell extract. Biochemical characterization with small substrates showed thermoalkalophilic properties (T opt = 50 °C, pHopt = 7.5) and higher activity towards para-nitrophenyl octanoate (12.7 U mg−1) compared to longer and shorter chain lengths (C14 0.7 U mg−1 and C2 4.3 U mg−1, respectively). Crystallization and determination of the 3-D structure displayed the presence of a lid structure and a zinc ion surrounded by an extra domain. These properties classify the enzyme into the I.5 lipase family. PfL1 is able to hydrolyze poly(1,4-butylene adipate-co-terephthalate) (PBAT) polymeric substrates. The hydrolysis of PBAT showed the release of small building blocks as detected by liquid chromatography-mass spectrometry (LC-MS). Protein dynamics seem to be involved with lid opening for the hydrolysis of PBAT by PfL1

Small cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilytica

We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities