Aspergillus Oryzae, Used to Produce ?-Amylase - Science Assignment Help

Assignment Task:

Task:

1. Aspergillus oryzae, used to produce α-amylase, demonstrates pleomorphism. Briefly, explain what is meant by pleomorphism for a filamentous microorganism 

2. You conduct a count of Aspergillus oryzae spores using the haemocytometer shown below:

You obtain a count of 72 spores over the area of the square surrounded by the small circle in the diagram (see red arrow; note that the square surrounded by the large circle is 1 mm 2 ). The spore preparation was diluted 1-in-25 before measurement. The depth of the graduated area of the slide is 0.1 mm. What is the spore concentration expressed as spore/mL? 
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3. The α-amylase produced by Aspergillus oryzae has an pI value of 3.5. If you wished to purify this by ion exchange chromatography with a mobile phase of pH 7, which type of ion exchange resin would you use? 

4. The α-amylase produced by Aspergillus oryzae has an extinction coefficient of Ε 280nm 1mg/mL = 2.07. You are provided with a solution of this enzyme of unknown concentration and measure its absorbance at 280 nm to be 0.58 absorbance units. What is the concentration of this solution in mg/mL and mol/L (molecular weight of α-amylase is 50,000 Da)

5. The medium compostion used to grow Aspergillus oryzae to produce α-amylase is as follows (in g/L): citric acid, 2.0; MgSO 4 .7H 2 O, 2.0; KH 2 PO 4 , 2.0; K 2 SO 4 , 2.0; (NH 4 ) 2 SO 4 , 3.0; CaCl 2 , 0.8; yeast extract, 5.0; trace metal solution, 0.5 mL/L (citric acid, 3.0; ZnSO 4 , 0.29; FeSO 4 , 0.28; CuSO 4 , 0.25; MnSO 4 , 0.25, NiCl, 0.05). Which components of this medium will act as the nitrogen source for the fermentation? (2 marks)

6. The solids-liquid separation step may be carried out by either filtration or centrifugation. From your knowledge of the performance charactistics of these methods, provide any two ‘rules-of-thumb’ for recommending one method over the other for cell separation 

7. Provide three functions of the dewatering step of downstream processing 

8. In the development stage of a molecule purification strategy you wish to test the ability of 30% ammonium sulphate saturation to precipitate your protein. You have 378 mL of culture supernatant. What weight of solid ammonium sulphate must you add to this volume to achieve 30% saturation? (table overleaf)

9. After conducting the procedure in question 8 you wish to use the 30% ‘cut’ of ammonium sulphate to test the effectiveness of 50% saturation. How much more ammonium sulphate must to added to 75 mL of the 30% ammonium sulphate sample to achieve 50% saturation? 

10. Different proteins require different percent ammonium sulphate treatments to yield effective precipitation. Explain the reason for this

11. What is ‘membrane polarization’ in ultrafiltration and how can it be lessened? 

12. Briefly, explain the operational principle of dialysis to desalt a protein solution 

13. ‘Dialysis is an equilibrium-based process’. Explain the meaning of this statement 

14. Desalting may be performed by ultrafiltration, dialysis or gel filtration chromatography. If you had all three techniques available to you, which one would you recommend to a novice biotechnologist for desalting and why?

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