Assignment: Some Studies of α-Amylase Production Using Aspergillus Oryzae PDF

Roxana Olteanu [email protected] TU751/3 Assignment: “Some Studies of α-Amylase Production Using Aspergillus Oryzae” By Z. Esfahanibolandbala...

Roxana Olteanu [email protected] TU751/3 Assignment: “Some Studies of α-Amylase Production Using Aspergillus Oryzae” By Z. Esfahanibolandbalaire et al., 2008 Biotechnology 1 – Down-stream Bioprocessing Dr Gwillym Williams BIOL3001 Due: Monday 8th April 2024 Assignment presented by: Roxana Olteanu C20700039 TU751/3 [email protected] Roxana Olteanu [email protected] TU751/3 1. You are provided with a standard BSA solution made up in distilled water at a concentration of 155μg/mL. What volume of this would you take (and what volume of diluent) to make up a 5 mL of the standard dilutions given below? Provide your answer to the nearest microliter. (6 marks). Concentration Volume of BSA Volume of (μg/mL) stock water (mL) (mL) 0.0 0 5 10.0 0.32 4.68 20.0 0.65 4.35 30.0 0.97 4.03 40.0 1.3 3.71 50.0 1.6 3.39 The equation C1V1 = C2V2 is used in order to calculate the volume of BSA stock. All units must first be converted from mg/mL to µg/mL, hence 5 mL = 5,000 µg/mL. In order to obtain the appropriate result, a division by 1000 (i.e.1 ml) is done which gives the result for the BSA concentration. A total concentration of 5 mL is wanted, so 5 ml is taken away from the volume of the BSA stock. This new result is the amount of water needed to make up a final solution. Workings: Roxana Olteanu [email protected] TU751/3 2. The protein concentration of a bacterial supernatant is estimated at 0.63 mg/mL. You have 1.6 L of this supernatant, and you decide to concentrate the protein by using ultrafiltration (with a 10,000 Da cut-off membrane). The final volume of the supernatant retentate after ultrafiltration is 10 mL. From the change in volume of the solution, estimate the new protein concentration (and assume that no losses of protein occur in the process.) (2 marks). The initial protein concentration is given as 0.63 mg/mL, and we have 1.6 L of this supernatant. The final volume, after ultrafiltration leaves us with 10 mL. All units must be converted to mg/mL, i.e., 1.6 L = 1600 mL. The formula to find out the new protein concentration is C1V1 = C2V2 where: o C1 = Initial protein concentration o C1 = 0.63 ml o V1 = Initial volume o V1 = 1600 ml o C2 = Final protein concentration o C2 = Unknown o V2 = Final volume o V2 = 10 ml The estimated new protein concentration after ultrafiltration is therefore 100.8 mg/mL. 3. A protein is precipitated from a bacterial supernatant by ammonium sulphate with an efficiency of 53%. The initial protein concentration was 11 mg/mL, and the volume of the supernatant that was treated with ammonium sulphate was 170 mL. The precipitated pellet is resuspended in 6 mL of buffer. What is the new protein concentration in mg/mL? (2 marks). The initial protein concentration was 11 mg/mL, and the supernatant treated with ammonium sulphate was 170 mL. The precipitated pellet is in a total volume of 6 mL of buffer and the new protein concentration is unknown. Due to all measurements being performed in mg/mL, there is no need to convert the units. The formula to find out the new protein concentration is C1V1 = C2V2 where: o C1 = Initial protein concentration o C1 = 11 mg/ml o V1 = Initial volume o V1 = 170 ml o C2 = Final protein concentration o C2 = Unknown o V2 = Final volume o V2 = 6 ml The new protein concentration is therefore 311.6 mg/mL. Roxana Olteanu [email protected] TU751/3 4. The molar extinction coefficient (ЄM) of a protein (at 280 nm) is 5 x 105 M-1cm-1. You are provided with a solution of this protein of unknown concentration and measure its absorbance at 280 nm to be 0.5 absorbance units. Use A = ЄMcl to calculate the molarity of the protein solution (assume a 1 cm path length). Express your answer in terms of mol/L and μmol/L. (2 marks). The molar extinction coefficient is 5 x 105 M-1 cm-1. The absorbance at 280 nm is 0.5 absorbance units. The concentration of the protein is unknown. If the assumption is made that the path length is 1cm, we use the Beer-Lambert law to find the concentration of the unknown protein, which is as follows: The unknown protein concentration is therefore 1 x 10-6 mol/L. 5. Read the research paper entitled ‘Some studies of α-amylase production using Aspergillus oryzae’(Esfahanibolandbalaire et al, 2008), and answer the following questions: a) What advantages do the authors cite for the production of extracellular amylase by microbes using submerged fermentation? (2 marks) The paper states that there are many advantages for the production of extracellular amylase by microbes, suing submerged fermentation. A direct passage from the paper states that “reliable scale up, simple process control, reasonably high yield and easily recoverable by a series of simple fractions and purification steps” are the main advantages. The authors continue to elaborate that, although these are some advantages, it’s important to continue to strive forward in discovering new strains in order to “remain in competitive market which can utilize low priced agrochemical side products, like molasses as a carbon sources to produce enzyme.” It’s wildly known that biopharmaceutical companies and other such large industries are, for the most part, are more likely to utilize products that can lead to a biproduct of use. Another advantage of the production of extracellular amylase by microbes is discussed when the authors explain how “microorganisms used for production of industrial scale of enzymes, such as amylase and protease, consume complex agro-carbon sources such as molasses, water-waste of potato, glucose, starch and pulp and paper” which are relatively cheap and easily accessible products. Roxana Olteanu [email protected] TU751/3 The paper persists in explaining that the “production of various enzymes, especially amylase, amyloglucosidase and related glucanases by fungi, appears to be promising.” b) What was the percentage working volume of shake flask used in the study? (2 marks) The paper details the following details: o All culture experiments were performed in 500 mL Erlenmeyer flasks containing 100 mL of production medium. o The Erlenmeyer flasks containing required amount of medium was inoculated under sterile conditions and were stopped with cotton bungs and covered with aluminium seals. In order to find out the percentage working volume of shake flask used in the study; we use the following formula: Where the known information is as stated above. When subbing the values into the formula; an answer of 20% working volume is found to be used in the study. c) Why were the glucose, lactose, maltose, starch and corn steep liquor sterilized separately from the basal medium? (2 marks) Glucose, lactose, maltose, starch, and corn steep liquor were first sterilized separately before being introduced to the basal medium because each carbon source had proven to have a variety of different effects on the enzyme activity, depending on its concentration. The two graphs below were taken directly from the studied paper, which show (in figure 2) the effects of different carbon on enzyme production, and (in figure 3) the effects of soluble starch and maltose on enzyme production. Roxana Olteanu [email protected] TU751/3 The paper starts with a focus on glucose and lactose to have shown a “narrow effect on enzyme activity”. This is important, because according to the graph, in comparison with the other carbon sources, both glucose and lactose have a very small effect on the overall enzyme production. The effect of glucose concentration was confirmed to have an effect at 1 to 10 gL-1, just as lactose was also confirmed to have an effect at a concentration of 5 to 15 gL-1 in terms of α-amylase production. The paper investigated, with respect to both values in each case, that “biomass growth and enzyme production was negligible.” Due to these negligible results, they would not have been of much use to the overall product, and hence, a separate investigation under sterile conditions was undertaken do identify which carbon source would be best for the product. Furthermore, there are three other carbon sources which show a much more promising result when compared to glucose and lactose. Figure 2 and figure 3 both show that the concentrations of soluble starch, maltose and corn-starch to yield a much higher enzyme activity with respect to the concentration used. The soluble starch, maltose and corn-starch were “investigated from 5 to 30 gL-1, where α-amylase production has increased.” However, when looking at figure 3, it is clear that when the concentration is increased, the enzymatic activity is at a loss. This proves that there is a “goldilocks zone” where the enzymatic activity is at its best. The reason these carbon sources had to have been sterilizes separately from the basal medium, for starters, is to get rid of any unnecessary impurities, but also to discover which carbon source would work best for the final product. d) Which sugar was used to form a standard curve in the DNS assay? (2 marks) According to the graph depicted in figure 6 below, corn-steep liquor was used to form a standard curve in the DNS assay. Corn-steep liquor is generally used due to its high level of sugars and other useful proteins. In the case of this study, the authors elaborate how “as the corn-steep powder was increased from 0.5 to about 1.0 gL-1, it had meagre effect on the α-amylase production comparing with other three types of nitrogen sources.” This means that corn-steep liquor was used as both a sugar and nitrogen source. Roxana Olteanu [email protected] TU751/3 e) What pH value was optimal for production of α-amylase? (2 marks) The study conducted tests which varied the pH level with respect to the enzymatic activity, as shown in figure 1 below. When studying this graph, it is clear that the optimum pH lies in the middle somewhere between 6 and 7, i.e., a near neutral state. The authors explain how the pH is one factor which governs the size of the pellets produced throughout the experiment, alongside fungal growth, production and stability of amylolytic enzyme. They continue by expressing that: o At initial pH of 6 and 7± 0.1, pellets were more approximately spherical in the medium (visual observation). o At a pH of about 6.2 ± 0.1, more enzymes production has been detected. o Therefore, it can be concluded that the initial pH of 6.2 ± 0.1 has considerable influence on the enzyme production. o This shows coherence with optimum activity of α-amylase by 1 value. f) Which carbon source caused catabolite repression? The carbon source that caused catabolite repression was found to be glucose. The graph depicted in Figure 2, (question 5. c) ) shows how glucose was the one carbon source with the lowest value for enzymatic activity, even in comparison with another low value such as lactose. Initially, the authors of this investigation had planned on utilising molasses as their carbon source, however due to too may impurities such as “ mud, sodium chloride, calcium carbonate, gum material, heavy metals, pesticide and undesirable microbial contaminations”, they’ve opted for other sources. As glucose is easily obtainable and an inexpensive carbon source, the authors performed tests to confirm the enzymatic activity is of a favourable yield. Roxana Olteanu [email protected] TU751/3 However, they’ve discovered when “effects of glucose concentration on the enzyme production was studied, and on the contrary to remarkable biomass production, negligible enzyme activity has been detected. It can therefore be concluded that “glucose has acted as catabolite repressor for the production using the present Aspergillus Oryzae strain.” g) What was the best nitrogen source for α-amylase production? (2 marks) The best nitrogen source for this experiment had been found to be a combination of yeast and specific amount of sodium nitrate, while maintaining certain carbon/nitrogen ratio, which resulted in a considerable amount of α-amylase production. The authors explained how they studied multiple strong candidates for a good nitrogen source for this experiment, however the “production of α-amylase is affected by selected and low nitrogen concentration; therefore, it’s required to pin point proper type percentage of necessary nitrogen compound.” An example of a nitrogen sources used in the study are ammonium salts such as ammonium chloride, nitrate, and sulphate on α-amylase production. It has then been discovered that ammonium chloride and sulphate had a meagrely affect on α-amylase production, which may have been due to changing the production environment to be more acidic, which was not ideal. Hence, the combination of yeast and sodium nitrate under very specific conditions, were found to be the best nitrogen sources for this experiment. h) The authors indicate that fungal pellets of compact and spherical size are preferred for enzyme production. Why is this normally the case? (2 marks) The authors found that fungal pellets of compact and spherical size are preferred for enzyme production, by discovering what the alternative may lead to. They state that the effect on inoculum size on fungi growth form in submerged culture, is of considerable importance in fermentation technology as it influences growth rate, aeration, power consumption for stirring and the ease with which biomass ca be separated from the broth. They concluded that “in lower inoculum, fungi have to undergo extensive growth and branching before nutrient utilization, resulting in conservative production of α-amylase. The irregular shape, big size pellets might have consumed major amounts of substrate at initial stages of fermentation.” Hence, the preferred smaller and spherical sizes of the pellets.