MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY – HO CHI MINH CITY FACULTY CHEMICAL ENGINEERING AND FOOD TECHNOLOGY PRACTICAL REPORT ENZYME TECHNOLOGY Instructor Nguyen Minh Xuan Hong GRO.
MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY – HO CHI MINH CITY FACULTY: CHEMICAL ENGINEERING AND FOOD TECHNOLOGY PRACTICAL REPORT ENZYME TECHNOLOGY Instructor: Nguyen Minh Xuan Hong GROUP - MEMBERS Đỗ Hồng Ánh Mai Đỗ Thị Yến Ly Nguyễn Thu Hiền Võ Nguyễn Thục Trinh Bùi Thiên Lộc 14th March 2022 - Ho Chi Minh city TABLE OF CONTENT TABLE OF FIGURES iii Lab 1: ENZYME KINETICS Experiment 1.1 Effect of enzyme concentration on enzyme activity 1 Abstract Background Materials and Methods Results Discussion Experiment 1.2 Effect of substrate concentration on enzyme activity Abstract Background Materials and Methods 4 Results 5 Discussion Experiment 1.3 Specificity of enzyme Abstract Background Materials and Methods Results Discussion Experiment 1.4 Effect of temperature on enzyme activity 11 Abstract 11 Background 11 Materials and Methods 11 Results 13 Discussion 15 Experiment 1.5: Effect of pH on enzyme activity 16 Abstract 16 Background 16 i Materials and Methods 16 Results 18 Discussion 19 Experiment 1.6: Effect of Inhibitor and Activator on enzyme activity 20 Abstract 20 Background 20 Materials and Methods 20 Results 20 Discussion 21 Lab 3: Enzyme protease 23 Experiment 3.3: Effect of enzyme bromelain on meat tenderizing 23 Abstract 23 Background 23 Materials and Methods 23 Results 24 Discussion 26 Lab 4: Enzyme pectinase 27 Experiment 4.1: Determination of pectinase activity using color-metric method 27 Abstract 27 Background 27 Materials and Methods 27 Results 28 Discussion 30 Experiment 4.2 Application of pectinase in clarification of apple juice 31 Abstract 31 Background 31 Materials and methods 31 Results 32 Discussion 32 References 33 ii TABLE OF FIGURES Figure The effect of enzyme amylase concentration on enzyme activity Figure The relationship between enzyme concentration and reaction time Figure The effect of substrate concentration on enzyme activity Figure The relationship between starch concentration and reaction time Figure Fehling solution (mixed) and three prepared test tubes Figure Results of three test tubes after keeping in hot water for 1-3 and cool down Figure Test tubes were prepared at three different assay temperatures 13 Figure Drops of each solution were disappeared in black blue color 14 Figure Relationship between temperature and reaction time 14 Figure 10 Test tubes prepared at three different pH with the enzyme solution 17 Figure 11 Test tubes prepared at three different pH with buffer instead of enzyme solution 17 Figure 12 Drops of each solution with Lugol solution until all reactions have been completed 18 Figure 13 The chart shows the relationship between pH and reaction time 19 Figure 14 The results of three tubes with Lugol solution 21 Figure 15 Meat samples before the experiment 24 Figure 16 Meat samples before the experiment 24 Figure 17 Meat samples after the experiment 25 Figure 18 Meat samples after the experiment 25 Figure 19 Meat samples after the experiment 26 Figure 20 Samples of two test tubes which 5ml Anthrone in 2.5 ml diluted filtrate 28 Figure 21 The initial value of the spectrophotometer 29 Figure 22 The value of test tube is measured 29 Figure 23 The value of test tube is measured 30 Figure 24 Diagrammatic representation of different steps involves in apple fruit juice 31 Figure 25 Results of four test tubes for observation and discussion 32 iii Lab 1: ENZYME KINETICS RATE OF REACTION Experiment 1.1 Effect of enzyme concentration on enzyme activity Abstract Enzymes are catalysts which lower the activation of chemical reactions, thus making them happen more rapidly In this lab, enzyme kinetics are examined utilizing various experimental techniques, including measurements of temperature The purpose of this lab is to investigate the impact of enzyme concentration on enzyme activity If the concentration of the enzyme is increased, the rate of the reaction will be increased as well The dependent variable is the rate of the reaction Background Enzymes are catalysts that lower the activation of chemical reactions, thus making them happen more rapidly Enzyme kinetics is the study of catalytic reactions, or reaction rate, which occurs in the presence of enzymes under varying conditions, specificities, and mechanisms such as the proximity effect, orientation effect, catalytic effect, and energy effect; the studies are conducted under assorted circumstances, such as temperature, pH, and component concentrations in correlation to reaction rates For example, enzyme concentration directly relates to reaction rates whereby an increase in enzyme concentration will also increase the rate of the reaction in a linear ENZYME CONCENTRATION relationship (as seen in the above graph on the left) In experiment 1, different amounts of enzyme and the same amounts of substrate were put in a test tube Then those were observed using Lugol solution to see how fast they reacted to produce the product Materials and Methods Materials - 1% starch solution - Buffer solution (distilled water) - Lugol solution - Amylase enzyme solution Laboratory tools - Test tubes - Pipette - Test tube stands - Sticker notes *Methods: - Assemble and label clean test tubes to prevent contamination or premature reaction from occurring - Measure enzyme amylase and starch then distribute chemicals as follows: Tube Tube Tube Tube Tube Distilled water ml 0.9 ml 0.8 ml 0.5 ml ml 1% Enzyme ml 0.1 ml 0.2 ml 0.5 ml ml 1% Starch ml ml ml ml ml [ Enzyme] 0% 0.05% 0.1% 0.25% 0.5% - Check the reaction with Lugol solution after each 30 seconds until all reactions have completed Record the time Results [ Enzyme] 0% 0.05% 0.1% 0.25% 0.5% Reaction No react 5.36 mins 4.11 mins 3.18 mins 2.32 mins time (min) Figure The effect of enzyme amylase concentration on enzyme activity Effect of enzyme concentration 0.60% [ Enzyme] 0.50% 0.40% 0.30% 0.20% 0.10% 0.00% [ Reaction time] Figure The relationship between enzyme concentration and reaction time Discussion It was found that as the concentration of enzyme increased, the speed of reaction increased and the reaction time decreased As there was a higher amount of enzyme, the substrate had more chances to bind to the amylase active sites and reacted more at once, therefore increasing the rate of reaction Thus, the higher amount of enzyme is, the more effective its activity is Higher levels of enzyme mean there will be a higher turnover rate of the product Experiment 1.2 Effect of substrate concentration on enzyme activity Abstract As the concentration of the substrate was increased, the rate of the reaction also increased for reasons similar to the concentration of the enzyme The amount of substrate increased there were more substrate molecules to bond with the active site of the amylase enzyme Different amounts of starch and the same amount of enzyme amylase were put in a test tube, then were observed using Lugol solution to show the goal of the experiment is how the effectiveness of amylase activity under effect of substrate concentration according to the speed of the reaction Background This lab provides changing of starch concentrations affect the rate of reaction of enzyme amylase reaction Controlling this factor in a cell is one way that an organism regulates its enzyme activity and so its metabolism As we know, enzymes will work best if there is plenty of substrates As the concentration of the substrate increases, so does the rate of enzyme activity However, when the enzymes become saturated and no more substrates can fit at any one time, the rate of enzyme activity does not continue to increase indefinitely In experiment 2, different amounts of enzyme and substrate were put in a test tube Then those were observed using Lugol solution to see how fast they reacted to produce the product Materials and Methods Materials - 1% starch solution - Buffer solution (distilled water) - Lugol solution - Amylase enzyme solution Laboratory tools - Test tubes - Test tube stands - Pipette - Sticker notes Method - Assemble and label clean test tubes to prevent contamination or premature reaction from occurring - Measure enzyme amylase and starch then distribute chemicals as following: Distilled water 1% Starch 1% Enzyme [ Enzyme] Tube 1.8 ml Tube 1.6 ml Tube 1.3 ml Tube 0.8 ml Tube 0.3 ml ml 0.2 ml 0% 0.2 ml 0.2 ml 0.1% 0.5 ml 0.2 ml 0.25% 1.0 ml 0.2 ml 0.5% 1.5 ml 0.2 ml 0.75% - Check the reaction with Lugol solution after each 30 seconds until all reactions have completed Record the time Results [ Starch] 0% Reaction No react time (min) 0.1% 1.04 mins 0.25% 2.38 mins 0.5% 4.08 mins Figure The effect of substrate concentration on enzyme activity 0.75% 7.34 mins Effect of substrate concentration 0.80% 0.70% [ Starch] 0.60% 0.50% 0.40% 0.30% 0.20% 0.10% 0.00% [ Reaction time] Figure The relationship between starch concentration and reaction time Discussion Likewise, it was found that as the concentration of starch was increased, the speed of reaction increased When there is more substrate, there will be just as much enzyme, also making the rate of the reaction increase But in the case of experiment 1.2, the amount of enzyme is kept the same for each tube, a higher amount of starch does not have enough enzyme to react by the maximum velocity, so the rate of reaction was decreasing and the reaction time was longer Experiment 1.6: Effect of Inhibitor and Activator on enzyme activity Abstract The goal of this experiment is to observe how inhibitors and activators affect enzyme amylase activity To observe the activity of enzyme amylase during the starch hydrolysis, it is affected by the activator substances or inhibitors, we use NaCl, CuSO4, and distilled water in this experiment From that, we can determine which substance is an inhibitor, which is an activator substance Background Just as enzymes act as a catalyst to biochemical reactions, enzyme activity can be affected by other molecules: inhibitors are molecules that decrease enzyme activity and activators are molecules that increase activity Enzyme inhibitors are compounds that modify the catalytic properties of the enzyme and therefore, slow down the reaction rate, or in some cases, even stop the catalysis Such inhibitors work by blocking or distorting the active site Enzyme activators are chemical compounds that increase the velocity of enzymatic reactions The binding of an activator to a regulatory site stabilizes the shape that has the functional active site Amylase will increase in the environment with NaCl, vice versa CuSO4 will inhibit the effect of this enzyme Materials and Methods * Materials: - 1% enzyme amylase solution - 1% NaCl - 1% CuSO4 - Distilled water - 1% starch solution - Lugol solution * Methods: Prepare tubes as follows: Tube 1: 0.5 ml of 1% amylase solution and 0.5 ml of 1% NaCl Tube 2: 0.5 ml of 1% amylase solution and 0.5 ml of 1% CuSO4 Tube 3: 0.5 ml of 1% amylase solution and 0.5 ml of distilled water Add ml of 1% starch solution to each tube Keep it at room temperature for 1-3 minutes Then check the reaction with Lugol solution Observe and compare the results Results Tube 1: Only appear the color of Lugol solution, Tube 2: Purple-blue color 20 Tube 3: Color of Lugol solution, Figure 14 The results of three tubes with Lugol solution Discussion Enzyme inhibitors, activators can also affect amylase enzyme activity Tube 1: Do not appear purple color characteristic indicates that the starch has been completely hydrolyzed by amylase enzyme so when to add Lugol solution, the tube only appears the color of Lugol solution Thus, the appearance of NaCl as a catalyst increases starch degradation of the enzyme amylase because the chloride ions activate the reaction NaCl is an activator If the concentration of sodium chloride increases, the rate of enzyme activity will gradually slow down because high amounts of sodium chloride disrupt the bonds and structure of the active site This then leads to denaturation and the starch is no longer able to bind to some of the active sites Enzyme activity will gradually slow down as more and more enzymes become denatured and finally stop Thus, such a low concentration is selected as the most suitable, to investigate the activating property of Cl- ions Tube 2: At room temperature, starch has torsion-type amylose molecules with perforated springs, the starch absorbs Iodine in Lugol solution for special purple-blue The amylase was used to hydrolyze the alternate glycosidic bonds in starch By doing this it meant that the starch, a polysaccharide was broken down into maltose, a disaccharide However, copper sulfate would interfere with starch for the active sites and so prevent the 21 formation of enzyme-substrate complexes Fewer complexes formed means that the time taken to hydrolyze the starch would be greater and the rate of the reaction would be slower Starch has not been hydrolyzed as the copper ion is a non-specific inhibitor of the enzyme So, CuSO4 is an inhibitor Tube 3: The starch is hydrolyzed by a part of dextrin Enzyme amylase takes time to hydrolyze the starch, so the purple-blue is lost after appearing The time took place longer than tube but still happened Thus, distilled water is not an activator is also an inhibitor 22 Lab 3: Enzyme protease Experiment 3.3: Effect of enzyme bromelain on meat tenderizing Abstract The purpose is to investigate the meat tenderizing properties of bromelain, which is derived from pineapple extract Tenderization of meat is described as the breaking down of collagen in meat to make it more palatable, and bromelain, which is a combination of two proteases, achieves the same by degrading the collagen fibers of the meat This experiment uses heat treatment and enzyme catalysis of pineapple so as to make the meat softer Bromelain is an enzyme discovered in pineapple that has been used in medicine and meat tenderization Pineapple stems are used to make it commercially Background Bromelain is a plant-derived proteolytic enzyme found in the Bromeliaceae family, which includes Pineapple Cysteine proteinases are widely utilized in the food and pharmaceutical sectors, as well as in diagnostic labs, due to their catalytic activity Bromelain is also economically important because the enzyme is derived from pineapple parts such as the stem and wastes, and the technology for using it is simple and inexpensive It can be used in the home or in the workplace to tenderize tough meat, and it is a better alternative to chemical tenderizers or other plant proteases Materials and Methods *Materials - Pineapple -4 dishes (made by silver papers) -Meat *Laboratory tools: - Pot of boiling water *Methods - Pineapple juice was extracted using a juicer or filter cloth after the hard shell was removed and the pineapple was sliced into small pieces - Meat is cut into long, thin pieces so that the muscle below may be seen Divide the meat into four halves and serve in the following order: + Dish 1: no heat treatment, control + Dish 2: pour in the pineapple juice + Dish 3: heat-treated control (cook for minutes) + Dish 4: add pineapple juice, set aside for hours at room temperature, then cook for minutes - Then, comparing and contrasting the structure of the meat in four different meals 23 Results - Samples before processing as required by the experiment: Figure 15 Meat samples before the experiment Figure 16 Meat samples before the experiment 24 Figure 17 Meat samples after the experiment Figure 18 Meat samples after the experiment 25 Figure 19 Meat samples after the experiment -From the pictures above, it can be seen that the meat on the plates is initially prepared the same All are uniform in color, meat texture and original toughness are the same at figure 15 and figure 16 -In figure 17, 18 and 19 the texture has a lot of changes Details that, dish and are more dry and tough than dish and Discussion After arranging the experimental dishes as required in Figures 17 and 18 at different temperatures and requirements, it is easy to see that the structure of the meat in each dish has changed markedly Specifically, on dishes with pineapple juice, we noticed the texture of the meat was softer than the other two dishes without pineapple juice In dish 1, the meat will be dry and dark in color when left for a long time outside Regarding dish 2, the meat becomes softer when soaked in pandan leaf juice Sample heat treatment can remove more water than in dish 3, so the meat becomes tougher and drier In dish 4, because there is a combination of pineapple juice and heat treatment, the structure is harmonious, soft, and not too dry like the sample of dish and we can clearly see the texture of the meat on this plate is quite even As a result of these findings, bromelain generated from pineapple extract may be utilized to effectively tenderize meat 26 Lab 4: Enzyme pectinase Experiment 4.1: Determination of pectinase activity using color-metric method Abstract The purpose of this experiment use a colorimetric technique in order to have pectinase activity was measured Based on a defined translation, a practical approach for color comparison research has been created Other chemical processes take longer than this procedure The galacturonic acid generated by the enzyme pectinase is quantified by analyzing the enzyme pectinase 𝑍𝑛𝑆𝑂4 bonds to the pectin substrate Background The enzyme pectinase is present in plant cell walls By catalyzing the deesterification of pectin's methoxyl group, this enzyme transforms pectin into pectic acid Because they assist preserve juice quality clear, constant, and consistent in largescale manufacturing, pectinases have become an essential aspect of the fruit juice industry and other biotechnological uses They are also employed in infant nourishment, oil extraction, canned orange juice, and date fruit sugar extract The monomeric galacturonic acids produced by pectinase during catalyzing pectin breakdown were measured to determine the enzyme's activity Specific activity units were used to express the results The quantity of enzyme that hydrolyzes 1g pectin to galacturonic acids in 60 minutes under normal conditions was defined as one unit of enzyme activity (30C, pH 3.9-4.1) Materials and Methods *Materials - 1% pectin solution - 15% 𝑍𝑛𝑆𝑂4 solution - 1% enzyme pectinase solution - 0.2% Anthrone solution in sulphuric acid - Distilled water *Laboratory tools: - Test tube - Pipette - Cups - Optical density meter *Methods - In a test tube, combine 10 mL of 1% pectin solution with mL of 1% enzyme pectinase solution and bake at 30°C for 60 minutes (pH of pectin solution = 3.9-4.1) - The mixture is then filtered using filter paper after being added mL of 15% ZnSO4 solution The filtrate is times diluted 27 - In 10 minutes, mL of Anthrone solution is violently combined with 2.5 mL of diluted filtrate After that, it is placed in a water bath at 70°C for 12 minutes It is then allowed to cool to room temperature before having its OD measured at A584 - Water is used as a control sample instead of the enzyme Results Figure 20 Samples of two test tubes which 5ml Anthrone in 2.5 ml diluted filtrate 28 Figure 21 The initial value of the spectrophotometer Figure 22 The value of test tube is measured 29 Figure 23 The value of test tube is measured OD = ODT – ODCT = 1.632 – 1.226 = 0.406 Pectinase activity = (0.34 x OD – 0.0104)/m = (0.34 x 0.406 – 0.0104)/5 = 0.0255 ODT: A584 of the test sample ODCT: A584 of the control sample m: the amount of enzyme used (g or mL) Discussion When pectin is hydrolyzed in the standard state (pH = 3.9-4.1, time 60 minutes, temperature 30°C, manual pectin at 0.66 percent concentration, degree hydrolysis 30 percent), 1ml of pectin is converted to galacturonic acid By the obtained result of 0.0255, the pectinase activity can be changed to 0.0255ml galacturonic acid pectin after 60 minutes 30 Experiment 4.2 Application of pectinase in clarification of apple juice Abstract It has long been known that pectic enzymes were responsible for the clarification of cloudy fruits juices by breaking down pectic polysaccharides into simpler molecules such as galacturonic acids, galactose, etc The goal of this experiment is to discuss the reactive activity of pectinase at specific amounts with a similar volume of apple juice and understand the advantages of the enzyme in the fruit juice industry The techniques used in this experiment are weight, measurement, extraction, dilution, mix, and observation for the best results Finally, we found that various amounts of pectinase affected the clarity of apple juice after being treated Background Experiment 4.2 is conducted to explain the effectiveness of enzyme pectinase in the food industry, especially in fruit juice processing Figure 24 Diagrammatic representation of different steps involves in apple fruit juice As we know, pectin is an acidic heteropolysaccharide present in the middle lamella of the plant cell wall Pectinases are used to break down the glycosidic bonds present between monomer sugars like galacturonic acid by hydrolysis, trans-elimination, and deesterification reactions This mechanism helps to reduce the overall production cost, obtain a high yield of juice as well as enhance the clarity and quality of products leading to the visual acceptance of juice by the consumers To obtain the desired results, procedures and process parameters are needed to be optimized for example enzyme concentration (0; 0.2; 0.5; 1mL), treatment duration (30 minutes), incubation temperature (45-50°C) Materials and methods *Materials: - Apples - 1% enzyme pectinase solution - Distilled water *Laboratory tools: 31 - Knife - Electronic stove - Cupholder - Thermometer - Pipette - Filter cloth - Test tube - Blender *Methods: - Weigh and cut apples into small pieces - Use the blender to take the apple juice from apples - Dilute 20mL of the juice with distilled water to make a 100mL diluted solution - Lable four test tubes and add solution as follows: Tube Tube Tube Tube Apple juice (mL) 8 8 Pectinase (mL) 0.2 0.5 Water (mL) 1.8 1.5 1.0 - Keep all tubes at 45-50°C in 30 minutes - Do filtration each tube by the filter cloth for easier observation Results Figure 25 Results of four test tubes for observation and discussion From the results above, we can observe by eyes that, the increasing clarity in order is tube 1, tube 2, tube 3, tube Discussion Enzyme pectinase helps to cut the glycosidic bonds in pectic polysaccharides to release monomer sugars The more amount of used enzyme is, the more effective the result is In test tube with no enzyme concentration (0mL), there was no activity of pectinase and no cutting large molecules into simpler soluble ones Therefore, the solution in this test tube presented the cloudy apple juice was considered as the suspension system with 32 colloidal properties of insoluble substances such as polyphenols and pectin Test tubes 2, 3, and especially test tube showed the clarity of apple juice was improved with higher enzyme concentration The explanation for these results is more enzyme concentration leads to more active sites for the combination with substrates So higher amount of enzyme pectinase reacted, more pectic polysaccharides were solved to soluble molecules with no colloidal properties which caused the cloudy phenomenon in fruit juice In conclusion, the enzyme pectinase has a significant effect in improving the clarity of apple juice References Enzyme review https://www.khanacademy.org/science/ap-biology/cellular-energetics/environmentalimpacts-on-enzyme-function/a/hs-enzymesreview#:~:text=Enzyme%20concentration%3A%20Increasing%20enzyme%20concentrat ion,additional%20enzymes%20to%20bind%20to Effect of temperature, substrate, enzyme concentration, and pH on reaction rate https://www.bbc.co.uk/bitesize/guides/zwxv6yc/revision/2?fbclid=IwAR0HZdqha5x5vti ns6jOXRlulVc-S665FOSUucP76oGcFe7mzIH-PpUP-qY Fehling Test https://chemdemos.uoregon.edu/demos/Fehling-Test# Starch Hydrolysis of Amylase https://www.ukessays.com/essays/biology/starch-hydrolysis-of-amylase-biologyessay.php The Effects of Temperature on Enzyme Activity and Biology https://sciencing.com/effects-temperature-enzyme-activity-biology-6049.html Optimal Temperature for Enzyme Amylase https://www.ukessays.com/essays/biology/optimal-temperature-for-enzyme-amylasebiology-essay.php Starch and Iodine https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Modules_( Biological_Chemistry)/Carbohydrates/Case_Studies/Starch_and_Iodine Amylase: Thủy phân tinh bột ngành đường https://www.mdi.vn/chi-tiet-san-pham/enzyme/amylase:-thuy-phan-tinh-bot-nganhduong-p761.html Tenderisation of meat using bromelain from pineapple extract https://www.researchgate.net/publication/306159233_Tenderisation_of_meat_using_bro melain_from_pineapple_extract 33 Optimization of the Effect of Pineapple By-Products Enhanced in Bromelain by Hydrostatic Pressure on the Texture and Overall Quality of Silverside Beef Cut https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760178/ Pectic Enzymes in the Clarification of Apple Juice https://www.tandfonline.com/doi/pdf/10.1080/00021369.1964.10858304?fbclid=IwAR0 mRqeeENd3Jjri_b_nwHwMrZNvhWcG3TPiUPNJXjq94-mh71Mi3DpOXIQ Pectinase: A Useful Tool in Fruit Processing Industries https://juniperpublishers.com/nfsij/pdf/NFSIJ.MS.ID.555673.pdf?fbclid=IwAR0pgoDVz -IIHtCGeS4stmCq1wvjK9T7PXlfEfaj-Vw5QKaTNfU0Nzo6PpI 34 ... https://www.khanacademy.org/science/ap-biology/cellular-energetics/environmentalimpacts-on -enzyme- function/a/hs-enzymesreview#:~:text =Enzyme% 20concentration%3A%20Increasing%2 0enzyme% 20concentrat ion,additional%20enzymes%20to%20bind%20to Effect of temperature, substrate, enzyme. .. effect of enzyme amylase concentration on enzyme activity Effect of enzyme concentration 0.60% [ Enzyme] 0.50% 0.40% 0.30% 0.20% 0.10% 0.00% [ Reaction time] Figure The relationship between enzyme. .. effect of enzyme amylase concentration on enzyme activity Figure The relationship between enzyme concentration and reaction time Figure The effect of substrate concentration on enzyme activity