BIOL1004 practical 2 2020 PDF

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Practical 2 (aka. Lab 2) from BIOL1004, year 2020...

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Effects of temperature and pH on Bromelain activity Bartholomew Gomes Introduction Enzymes are biological macromolecules, typically proteins, that accelerate intercellular chemical reactions (Stryer, Berg & Tymoczko 2002). There are numerous types of enzymes, one of them being protease – the enzyme responsible for catalyzing the breakdown of proteins through peptide bonds cleavage (King et al. 2014). One natural source of proteolytic enzymes is plants, specifically in fresh fruits. Pineapples (Ananas comosus) and kiwifruits (Actinidia chinensis) contain bromelain (Rowan et al. 1990) and actinidin (Baker et al. 1980), respectively. On the other hand, fruits like apples and oranges are yet to be associated with any established proteases. Several factors such as temperature, pH, or substrate concentration have a profound impact on enzyme activity (Newsholme 1965). Extreme temperatures, usually high, breaks hydrogen bonds and hydrophobic interactions resulting in a denatured enzyme that cannot function properly due to loss of structure (Daniel et al. 1996). Similarly, due to the tendency of proteins to denature, a proteolytic enzyme can only function effectively in certain pH ranges (Hoffman & Teicher 1961). One of the most common enzyme activity assays and the technique that will be used in this experiment will be a gelatin hydrolysis: a test that detects the presence of a proteolytic enzyme that liquifies gelatin (Kohn 1953; Greene & Larks 1955). The relevance of this paper lends itself to the potential clinal and therapeutic applications of the protease, bromelain (Rathnavelu et al., 2016). It is already administered for its well-established anti-inflammatory, antithrombotic and fibrinolytic properties (Pavan et al. 2012; Rathnavelu et al., 2016). The focus of this paper is controlling bromelain enzyme activity to enhance its potency as a skin debridement agent (Rosenberg et al. 2004). Aim In this study, gelatin samples were mixed with fresh fruit extract (containing different proteases) and subjected to various temperatures and pH levels to determine the optimal conditions for enzyme activity. Materials and Methods The gelatin hydrolysis test was carried out according to the description in the lab manual (RSB 2020). In brief, all test tubes were labelled in four series: A1–A5, B1–B5, C1–C6, and HCl1– HCl5. The A series tubes were filled with different fruit extracts (i.e. water, apple, orange, kiwifruit, and pineapple, respectively). The B series tubes varied the temperature conditions (i.e. 0C, room temperature, 40C, 70C, and 100C, respectively). The HCl series tubes were used to perform serial dilutions which helped vary the pH levels for the C series tubes. Here, an adjustment made to the method was the incorporation of pH paper to measure and record each tube of the serial dilution. For gelatin preparation, 50 grams of power was added per liter of water. The incubation times were 20 minutes for all the series except the C series, for which it

was only 10 minutes. The steps for C series mere mirrored with NaOH instead of HCl by a lab partner, resulting in a D series. Results Table 1. Gelatin hydrolysis of all the series (A, B, C, D).

Type of fruit

Liquefaction

Temperature Liquefaction [C] Water 0 + Apple ~20 + Orange 40 + Kiwifruit + 70 Pineapple + 100 *four different measurements, **four different measurements

pH

Liquefaction

1 2 3* 4** 8

+ + + +

In the A series, water, apple, and orange solutions set while the kiwifruit and pineapple extracts failed to set. In the B series, temperatures between 0C and 40C did not set while the 70C and 100C conditions did set. In the C and D series, only the solution with a pH 1 set. Discussion Based on the results, we can assume both apple and orange extracts had relatively low levels of protease activity. Following this logic, kiwifruit and pineapple can be assumed to have higher levels of protease activity which is supported by prior research highlighted in the introduction. Although the results did not present an optimum temperature or pH, we were still able to figure out a broader range. In terms of temperature, enzyme activity seemed to deteriorate from 70C and up; we can claim that bromelain is efficient from 0C to 40C. Speaking of pH, enzyme activity seemed to only be hindered by extremely acidic environments (i.e. pH 1). Further research must be conducted to determine whether extremely basic environments would have a similar effect, since our experiment peaks at a mere pH 8. By observing that majority of the pH measurements of both acidic and basic dilution series lied in the 3-4 range, we can claim that bromelain is a powerful buffer. Current research regarding its buffering capacities highlight its potency in the field of winemaking (Benucci et al. 2011). Existing studies exploring bromelain cites its effective pH range as 2-10 and its optimum temperature as 30C (Khan et al. 2003) which is well reflected by the results of this paper. The primary limitation of this experiment is the inability to accurately determine the optimum pH and temperature values, instead only proving us with a range. Enzyme assays that would be more effective in this regard the use of fluorogenic substrates (Freeman et al. 1995), magnetic resonance, or mass spectroscopy (Ou et al. 2018). Acknowledgements I would like to acknowledge and thank Isha Singhal for their contribution in data collection for the NaOH component of this experiment. References

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Stryer L., Berg J.M., Tymoczko J.L. (2002). Biochemistry (5th ed.). San Francisco: W.H. Freeman. ISBN 0-7167-4955-6. Ou, Yangguang et al. “Methods of Measuring Enzyme Activity Ex Vivo and In Vivo.” Annual review of analytical chemistry 11(1): 509-533. doi:10.1146/annurev-anchem-061417-125619....