Section: 807A (Wednesday)
Group: 1
Names: John Gibson, Alex Haney
Instructor: Hui Yang, PhD, George Gikas TA
Date: Oct 21, 2024
University Of Massachusetts,
Lowell, Fall 2024
Beta-Galactosidase Reaction Conditions Versus Reaction Intensities By Spectrophotometry
Abstract
β-galactosidase enzyme is a biological enzyme naturally occurring in prokaryotes, such as E. coli bacteria, to break down lactose disaccharides for glucose metabolism, such as glycolysis, to generate ATP, and to sustain cellular life. Mammals, including humans, produce analogs of β-galactosidase, such as lactase, in their digestive track. Beyond naturally occurring processes, this enzyme can be used as an expression reporter marker of the LacZ gene engineered into transgenic organisms for engineering, medical investigation, and agricultural purposes.
The big question of this enzyme is whether its performance characteristics are suitable for experimental settings and what reaction conditions and substrates this enzyme can tolerate. This study hypotheized the lactose analog ortho-nitro-beta-D-galactopyranoside (ONPG) as the reaction substrate as the product of ONPG sugar cleavage reaction with β-galactosidase enzyme was ortho-nitro-pyranoside (ONP), yellow in color, according to literature (1).
In this experiment study, the ONPG sugar cleavage reaction with β-galactosidase enzyme was performed under a pH range between 2 and 12 with spectrophotometer readings to compare reaction speeds, and the enzyme concentration effect was characterized with a parabola equation. The expected color change was observed during the experiment. Reaction curves with time progression were plotted. The enzyme concentration tested was between 0.05 mg/mL and 0.40 mg/mL β-galactosidase, with a negative control test of 0.0 mg/mL β-galactosidase. The substrate ONPG concentration was 0.3 mM. The optimal pH value for the β-galactosidase enzyme was estimated to be between 6 and 8, likely close to pH 8. However, this was not precisely determined due to a small R-square statistical correlation. When the pH condition was unfavorable for the reaction, a high enzyme concentration was hypothesized to accelerate the reaction and “rescue” the reaction, and this hypothesis was verified successfully as the experiment produced a comparable level of product as in a favorable pH condition. ONPG sugar cleavage reaction with β-galactosidase enzyme was thus deemed effective as a biological marker of protein translation.
The spectrometer Spec20 was used in this experiment.
Introduction
β-galactosidase enzyme is a bacterial protein found in E. coli and other species to digest disaccharide lactose, cleaving the disaccharide into its constituent monosaccharides, namely glucose and galactose. Glucose, in turn, feeds into glycolysis of cellular respiration to produce ATP as the energy for cellular processes, both in eukaryotes and prokaryotes. The knowledge of β-galactosidase cleaving lactose provided an understanding of Lac operon gene expression regulation, first fully investigated by Francois Jacob and Jacques Monod (2). The understanding of Lac operon facilitated the modern conceptions of gene promotors, transcription factors, repressors, enhancers, and small signaling molecules and is vital in modern genetic engineering, molecular manipulations in medicine, agriculture, industrial, and other applications.
E. coli and other species of bacteria with functional β-galactosidase coding and regulation convey evolutionary advantages; when exposed to a lactose-rich environment, they can outgrow and out-compete other bacteria that lack the enzyme gene or proper regulation of the gene. Homologue β-galactosidase enzymes, such as lactase, are also naturally occurring and synthesized in mammals, including humans, digestive glands, namely, the small intestines, to facilitate milk digestion. People with down-regulated, insufficient lactase synthesis develop lactose intolerance. In contrast, groups of people in Europe and West Africa with up-regulated lactase inheritance can take advantage of livestock milk nutrients to improve their health outcomes (3).
Enzymes are biological catalysts, allowing reactions to occur at lower chemical energy and accelerated reactions. Enzymes are specific to reaction substrates; the β-galactosidase enzyme cleaves the ether bond between 2 monosaccharides in the β configuration bond angle. ONPG is a lactose analog with an ether bond between a monosaccharide and a nitrogenous ring, also with the β configuration bond angle, and hence, β-galactosidase can cleave the bond. When the nitrogenous ring is released from the ether bond, its light-absorbing energy range shifts, enhancing absorption at 420 nm light according to literature (1). This study hypothesized this reaction correct and suitable reaction concentration at around 0.3 mM ONPG, 0.20 mg/mL β-galactosidase, pH 7.
A first assay should measure the reaction speed under a wide pH range to fully characterize an enzyme's activity with reaction condition perturbations. The pH range was set up in this particular experiment with buffer solutions. Different buffer chemicals have different buffering ranges and capacities, so each pH setting requires a separate reaction tube. Furthermore, an enzyme’s binding and catalyzing speeds are a complex interplay between substrate and product concentration in relation to the enzyme concentration. It is often best to introduce perturbates of many varying substrate and enzyme concentrations and obtain the reaction intensity curves for research and industry productions.
Due to time constraints, this experiment only tested varying enzyme concentrations of β-galactosidase. However, this experiment hypothesizes that enzyme concentration can directly enhance the reaction intensity, and when a reaction is in an unfavorable pH condition, such as pH 10, the high enzyme concentration can accelerate a reaction to be comparable to an optional pH condition. The experiment first used 7 reaction tubes with buffers at pH 2 to 12 to estimate the optimal pH level of the ONPG cleavage reaction by β-galactosidase. Then, 8 reaction tubes were given varied enzyme concentrations, producing reaction intensity curves, following an instructor experiment manual (1). Finally, a reaction at an unfavorable pH condition was given a high concentration of enzyme to observe an accelerated reaction.
Material And Method
Testing Enzyme pH Tolerance Range
Spectrophotometer Spec20 was turned on and warmed up for 20 minutes before any measurements were taken.
For varying pH tests, 7 reaction tubes were set up with 0.3 mM ONPG, 0.1 mg/mL β-galactosidase (from crushed Lactaid pills), and 0.02 M Na2PO4 at pH 2, 4, 6, 7, 8, 10, and 12, one for each. Each reaction was given 3 minutes reaction time and immediately had their absorbance of light at 420 nm (A420) measured. Timing started when ONPG was mixed in. The spectrophotometer was calibrated before the first reaction started.
Recording Time Progression With Varying Enzyme Concentration
For varying enzyme concentration tests, 8 reaction tubes were set up with 0.3 mM ONPG, 0.00 mg/mL, 0.05 mg/mL, 0.10 mg/mL, 0.15 mg/mL, 0.20 mg/mL, 0.25 mg/mL, 0.30 mg/mL, 0.40 mg/mL β-galactosidase, and 0.02 M Na2PO4 at a constant pH 7. Each reaction was given 3 minutes reaction time in the spectrophotometer, and as their A420 were measured. Timing started when ONPG was mixed in. The spectrophotometer was calibrated again before the first reaction started.
Testing Hypothesized High Enzyme Concentration
For the final test with unfavorable pH but high enzyme concentration, 1 reaction tube was set up with 0.3 mM ONPG, 0.40 mg/mL β-galactosidase, and 0.02 M Na2PO4 at pH 10. At the end of the 3-minute reaction, A420 was recorded. The spectrophotometer was calibrated for the third time before the reaction started.
Result
The three experiment sections, namely testing enzyme pH tolerance range, recording time progression with varying enzyme concentration, and testing hypothesized high enzyme concentration reaction, were performed. All reactions produced a clear yellow product except negative controls with zero enzyme contractions. For varying pH tests, as shown in Table 1 below, the varying pH reactions produced A420 between 0.01 and 0.20. The yellow color in the reaction tubes were lightest at pH 2 and 12, and strongest between pH 6 and 8.
Table 1. A420 Of ONPG Cleavage With Beta-Galactosidase
Legend: A420, light absorbance at 420 nm wavelength after 3 minutes reaction
As shown in Figure 1 below, the A420 had an upward trend between pH 2 and 6, and a downward trend was observed between pH 8 and 12.
Figure 1. A420 Of ONPG Cleavage With Beta-Galactosidase
Caption: A420, light absorbance at 420 nm wavelength after 3 minutes reaction
For the varying enzyme concentration tests, as shown in Table 2, the A420 ranged from 0 to 0.15 at the highest enzyme concentration of 0.40 mg/mL. The yellow color in the reaction tubes appeared visible the slowest with the lowest enzyme concentration of 0.15 mg/mL and progressively became quicker and quicker as the enzyme concentration increased toward 0.40 mg/mL.
Table 2. Various Enzyme Concentration Reaction Intensity
Legend: The reactions occurred at pH 7, enzyme concentration varied from 0.00 mg/mL to 0.35 mg/mL , reaction time between 0 and 3 minutes, reaction absorbances were the recorded cells
As shown in Figure 2, the A420 in Table 2’s recording cells were plotted with time as the X-axis variable.
Figure 2. Enzyme Beta-Galactosidase Concentration Reaction Curves
Caption: The enzyme concentration ranged between 0.00 mg/mL and 0.40 mg/mL. Each curve color corresponds to a particular enzyme concentration.
For the last reaction with, 0.40 mg/mL β-galactosidase, and 0.02 M Na2PO4 at pH 10. The A420 was 0.06 at the end of the 3-minute reaction.
Discussion
The yellow color of reactions’ products matches literature descriptions of ortho-nitro-pyranoside (ONP), implicating hypothesized reactions occurring (1). Figure 1’s upward curve at pH 6 and downward curve at pH 8 makes sense since enzymes work in biological systems at biological pH levels around 7. The high A420 plateau between pH 6 and 8 means that β-galactosidase cleaves ONPG’s sugar subunit fastest in that pH range. The implication is that bacteria species that utilize β-galactosidase prefer the environment near the neutral pH 7 point. However, the trend curve could not give a definitive optimal pH number as the statistical correlation R-square value is only 0.613. This can be attributed to the stock phosphate pH buffer solution for the pH 7 reaction, such as production error. This error did not affect other phosphate pH buffer solutions as the other data points did not deviate from the trend curve.
As shown in Figure 2, the higher the enzyme concentration, the faster the curve reaches the optimal level plateau for that curve. This also means that the higher the enzyme concentration, the quicker the substrate ONPG is consumed. After ONPG is consumed, the A420 fluctuates without a definitive upward or downward trend.
As shown in Figure 3 below, Figure 2’s reaction complete (peak) time is outlined in a dashed curve. Reaction complete (peak) time was between 0.5 minutes with 0.4 mg/mL enzyme and 3 minutes with 0.05 mg/mL enzyme. This was expected as the higher concentration enzyme accelerated the reactions.
Figure 3: Enzyme Beta-Galactosidase Concentration Reaction Complete time
Caption: The enzyme concentration ranged between 0.00 mg/mL and 0.40 mg/mL. Each curve color corresponds to a particular enzyme concentration.
For the last reaction with 0.3 mM ONPG, 0.40 mg/mL β-galactosidase, and 0.02 M Na2PO4 at pH 10, the A420 at 0.06 at the end of the 3-minute reaction is comparable to the reaction at pH 7 in first 7-tube reactions, which had A420 of 0.09. The high enzyme concentration of 0.40 mg/mL β-galactosidase compensated for the unfavorable pH 10 condition. In contrast, without such enhanced enzyme concentration, the pH 10 test with the same substrate concentration reached only 0.01 of A420 (Table 1’s second last row).
Conclusion
This experiment demonstrated that the ONPG substrate is a suitable reaction marker for gauging enzyme β-galactosidase concentration. The pH level that this enzyme can tolerate is large, between 2 and 12. The concentrations suitable for the reaction are centered at 0.3 mM ONPG and 0.2 mg/mL β-galactosidase. As hypothesized, the unfavorable pH 10 condition was rescued and had accelerated reaction speed when enzyme concentration was enhanced. This experiment successfully demonstrated the optimal pH for β-galactosidase activity between pH 6 and 8. The reaction curves of various enzyme concentrations made sense; the higher the enzyme concentration, the faster the reaction completely consumes the reactant substrate.
References
Yang, Hui. (2024). Principles Of Biology I Laboratory Manual Fall 2024. [Unpublished Immunology Laboratory Manual]. Department of Biological Sciences, University of Massachusetts, Lowell.
Nickle, T., & Barrette-Ng, I. (2022, April 9). 12.1: The Lac Operon. Biology LibreTexts. https://bio.libretexts.org/Bookshelves/Genetics/Online_Open_Genetics_(Nickle_and_Barrette-Ng)/12%3A_Regulation_of_Gene_Expression/12.01%3A_The_lac_Operon
Malik TF, Panuganti KK. Lactose Intolerance. 2023 Apr 17. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan–. PMID: 30335318.
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