Extremophile enzyme activity lab: using catalase from Pyrobaculum calidifontis to highlight temperature sensitivity and thermostable enzyme activity

Robinson N.P.

This issue of Emerging Topics in the Life Sciences highlights current areas of research in the field of archaeal biology and the following introductory editorial sets the stage by considering some of the key developments over the last four decades since the initial identification of the archaea as a unique form of life. Emerging topics from this vibrant and rapidly expanding field of research are considered and detailed further in the articles within this issue.

Bryer P.J.

Finding the right enzyme experiment can be problematic, depending what one is trying to show, what supplies and equipment are available, and the time one can devote to the topic. I’ve developed simple and inexpensive labs for looking at catalase and invertase activity using yeast encapsulated in sodium alginate. Single-celled yeast, Saccharomyces cerevisiae, are encapsulated in sodium alginate, a readily available extract from brown algae that, when it comes in contact with calcium chloride (CaCl2), forms a sphere or “bead.” These spheres may then be put into a solution containing substrate to test for enzyme activity. The spheres are easy to manipulate, one doesn’t have the variability and mess of a yeast solution, and since there are no cells in solution, there is nothing to interfere with the various assay methods one might want to use to test for product. The graduated cylinder method for testing catalase activity introduced here is especially good for collecting large amounts of data that enable students to use statistics and, unlike similar yeast catalase experiments using paper disks and a yeast solution, the yeast spheres are easy to manipulate and there is very little variability. I have used this procedure with students in class and with teachers in workshops with positive results and comments.

Dolan E.L., Collins J.P.

Research on how people learn shows that teaching using active learning is more effective than just lecturing. We outline four concrete ways instructors can begin to apply active learning in their teaching: backward instruction design; expecting students to learn more than facts; posing “messy” problems for students to solve; and expecting students to talk, write, and collaborate. Each tactic is supported with references demonstrating its efficacy and advice and links to resources for getting started with active learning.

Furlong M., McFarlane R.

Standard "cookbook" laboratory activities that are used to teach students the optimal physical growth conditions of microorganisms should be modified so that they more effectively foster student's higher order cognitive skills and attract student interest. This paper describes a laboratory activity that engages students in an inquiry-based approach to studying the physical growth requirements of microorganisms. In this activity, students design and implement an experiment to obtain pure cultures of specific microorganisms, with distinct growth properties, that are provided to them in a mixed culture.

Freeman S., Eddy S.L., McDonough M., Smith M.K., Okoroafor N., Jordt H., Wenderoth M.P.

Significance The President’s Council of Advisors on Science and Technology has called for a 33% increase in the number of science, technology, engineering, and mathematics (STEM) bachelor’s degrees completed per year and recommended adoption of empirically validated teaching practices as critical to achieving that goal. The studies analyzed here document that active learning leads to increases in examination performance that would raise average grades by a half a letter, and that failure rates under traditional lecturing increase by 55% over the rates observed under active learning. The analysis supports theory claiming that calls to increase the number of students receiving STEM degrees could be answered, at least in part, by abandoning traditional lecturing in favor of active learning.

Iwase T., Tajima A., Sugimoto S., Okuda K., Hironaka I., Kamata Y., Takada K., Mizunoe Y.

In this study, an assay that combines the ease and simplicity of the qualitative approach for measuring catalase activity was developed. The assay reagents comprised only hydrogen peroxide and Triton X-100. The enzyme-generated oxygen bubbles trapped by Triton X-100 were visualized as foam, whose height was estimated. A calibration plot using the defined unit of catalase activity yielded the best linear fit over a range of 20–300 units (U) (y = 0.3794x − 2.0909, r2 = 0.993). The assay precision and reproducibility at 100 U were 4.6% and 4.8%, respectively. The applicability of the assay for measuring the catalase activity of various samples was assessed using laboratory strains of Escherichia coli, catalase-deficient isogenic mutants, clinically isolated Shiga toxin-producing E. coli, and human cells. The assay generated reproducible results. In conclusion, this new assay can be used to measure the catalase activity of bacterial isolates and human cells.

Emmert E.A.

The safe handling of microorganisms in the teaching laboratory is a top priority. However, in the absence of a standard set of biosafety guidelines tailored to the teaching laboratory, individual educators and institutions have been left to develop their own plans. This has resulted in a lack of consistency, and differing levels of biosafety practices across institutions. Influenced by the lack of clear guidelines and a recent outbreak of Salmonella infections that was traced back to teaching laboratory exposures, the Education Board of the American Society for Microbiology charged a task force to develop a uniform set of biosafety guidelines for working with microorganisms in the teaching laboratory. These guidelines represent best practices for safely handling microbes, based on the safety requirements found in the Centers for Disease Control and Prevention’s (CDC’s) Biosafety in Microbiological and Biomedical Laboratories (BMBL). Guidelines for safely handling microbes at both biosafety level 1 (BSL1) and biosafety level 2 (BSL2) were developed. The guidelines are brief by design for ease of use and are accompanied by an extensive appendix containing explanatory notes, sample documents, and additional resources. These guidelines provide educators with a clear and consistent way to safely work with microorganisms in the teaching laboratory.

Amo T., Paje M.L., Inagaki A., Ezaki S., Atomi H., Imanaka T.

A novel, facultatively aerobic, heterotrophic hyperthermophilic archaeon was isolated from a terrestrial hot spring in the Philippines. Cells of the new isolate, strain VA1, were rod-shaped with a length of 1.5 to 10 μm and a width of 0.5 to 1.0 μm. Isolate VA1 grew optimally at 90 to 95 °C and pH 7.0 under atmospheric air. Oxygen served as a final electron acceptor under aerobic growth conditions, and vigorous shaking of the medium significantly enhanced growth. Elemental sulfur inhibited cell growth under aerobic growth conditions, whereas thiosulfate stimulated cell growth. Under anaerobic growth conditions, nitrate served as a final electron acceptor, but nitrite or sulfur-containing compounds such as elemental sulfur, thiosulfate, sulfate and sulfite could not act as final electron acceptors. The G+C content of the genomic DNA was 51 mol%. Phylogenetic analysis based on 16S rRNA sequences indicated that strain VA1 exhibited close relationships to species of the genusPyrobaculum. A DNA–DNA hybridization study revealed a low level of similarity (≤ 18%) between strain VA1 and previously described members of the genusPyrobaculum. Physiological characteristics also indicated that strain VA1 was distinct from thesePyrobaculumspecies. Our results indicate that isolate VA1 represents a novel species, namedPyrobaculum calidifontis.

Amo T., Atomi H., Imanaka T.

ABSTRACT We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70°C. The enzyme exhibited a K m value of 170 mM toward H 2 O 2 and a k cat value of 2.9 × 10 4 s −1 ·subunit −1 at 25°C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene ( kat Pc ). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of kat Pc revealed that no orthologue genes were present on the archaeal genomes, including those from the “aerobic” (hyper)thermophilic archaea Aeropyrum pernix , Sulfolobus solfataricus , and Sulfolobus tokodaii . Therefore, Kat Pc can be considered a rare example of a manganese catalase from archaea.

Kimbrough D.R., Magoun M.A., Langfur M.

The action of the enzyme catalase on aqueous hydrogen peroxide to generate oxygen gas is a well-established demonstration. Catalase is typically obtained by aqueous extraction of a potato, and the potato extract is mixed together with 3% hydrogen peroxide. The oxygen that is produced can be collected over water. Variations on the procedure can demonstrate the dependence of catalytic activity on temperature or the presence of inhibitors.

Woese C.R., Fox G.E.

A phylogenetic analysis based upon ribosomal RNA sequence characterization reveals that living systems represent one of three aboriginal lines of descent: ( i ) the eubacteria, comprising all typical bacteria; ( ii ) the archaebacteria, containing methanogenic bacteria; and ( iii ) the urkaryotes, now represented in the cytoplasmic component of eukaryotic cells.

Williams J.

1. The velocity of decomposition of hydrogen peroxide by catalase as a function of (a) concentration of catalase, (b) concentration of hydrogen peroxide, (c) hydrogen ion concentration, (d) temperature has been studied in an attempt to correlate these variables as far as possible. It is concluded that the reaction involves primarily adsorption of hydrogen peroxide at the catalase surface. 2. The decomposition of hydrogen peroxide by catalase is regarded as involving two reactions, namely, the catalytic decomposition of hydrogen peroxide, which is a maximum at the optimum pH 6.8 to 7.0, and the "induced inactivation" of catalase by the "nascent" oxygen produced by the hydrogen peroxide and still adhering to the catalase surface. This differs from the more generally accepted view, namely that the induced inactivation is due to the H2O2 itself. On the basis of the above view, a new interpretation is given to the equation of Yamasaki and the connection between the equations of Yamasaki and of Northrop is pointed out. It is shown that the velocity of induced inactivation is a minimum at the pH which is optimal for the decomposition of hydrogen peroxide. 3. The critical increment of the catalytic decomposition of hydrogen peroxide by catalase is of the order 3000 calories. The critical increment of induced inactivation is low in dilute hydrogen peroxide solutions but increases to a value of 30,000 calories in concentrated solutions of peroxide.

Rahmah W., Khoiruddin K., Wenten I.G., Kawi S.

Gruznov D.V., Gruznova O.A., Sokhlikov A.B., Lobanov A.V., Chesnokova I.P.

Natural bee honey can change its physicochemical and biological properties during storage. Literature data on long-term storage of honey at low temperatures (from 0 to -20 °C) indicate that this ensures the stability of some physicochemical parameters. Despite the potential benefits of these temperature regimes for practical use, it is important to consider their potential negative effects on honey quality. The aim of this study was to investigate the influence of various temperature conditions on the physicochemical and biochemical parameters of linden, buckwheat, and sunflower honeys during storage for 12 months. For the first time, a wide range of physicochemical parameters of honey samples was analyzed before and during storage for 12 months at temperatures of 18, 10, 5, 0, -5, -10 and -18 (±2) °C. The evaluation of the physicochemical parameters before storage demonstrated that the samples fully complied with the Interstate Standards. Throughout the storage period, the HMF level remained stable at -18 °C, whereas it significantly increased at higher temperatures; for example, after 12 months at 18 °C, the increase ranged from 472.5% to 488.1%, depending on the botanical origin of the honey. However, maximum permissible concentration – MPC (25 mg/kg) was not exceeded. A decrease in the activity of diastase, D-glucose-1-oxidase and catalase was observed at all temperature conditions already during the first month of storage. Minimal changes were observed at 0 and 5 °C. Hydrogen peroxide (H2O2) remained stable in this temperature range. Moisture content, total mass fraction of reducing sugars, and acidity did not change significantly in all samples. An antimicrobial study using test cultures found that honey stored at 5 and 0 °C had the greatest inhibitory effect. The data obtained demonstrates that the optimal temperature range for 12-month storage of honey is between 5 and 0 °C. These findings can be used as supplementary guidance when making amendments to regulatory documents governing storage requirements for this product.