Short Q's Flashcards
(100 cards)
1
Q
What is catabolite repression? Briefly describe one method of overcoming this to ensure optimal enzyme production.
A
repression at the level of transcription of an enzyme (usually involved in degradation) due to the presence of an easily utilizable carbon source in the medium (usually glucose). Avoid by (i) mutation in glucose catabolic pathway which causes slower utilization; (ii) avoid repressing C source in medium; (iii) slow feeding of repressive substrate.
2
Q
What is the relationship between product concentration at the end of upstream processing (at the start of downstream processing) and the final selling price of a biomolecule?
A
the more dilute a molecule, then the higher its value.
3
Q
Name the four steps involved in the downstream processing of a
biopharmaceutical.
A
Solids liquid separation; dewatering; purification; formulation
4
Q
Identify and briefly explain the four major steps in the downstream processing of
a biopharmaceutical.
A
: Solid-liquid separation (removal of cells), dewatering (concentration by precipitation or ultrafiltration), purification (chromatography) and formulation (addition of stabilizers, etc.).
5
Q
‘A consideration of downstream processing must begin at the upstream processing stage’. Identify one upstream processing factor that might either complicate or simplify the purification of a target protein, and briefly explain its relevance.
A
Inclusion of growth medium components that complicate subsequent purification (contaminant IgG in serum and monoclonal antibody purification; certain antifoams may affect ultrafiltration or IEX; choice of medium ingredients may complicate purification scheme). Other factors include selection of organism that actively secretes protein, selection of expression host with low level of endogenous extracellular proteases
6
Q
- Identify and briefly explain the four major steps in the downstream processing of a biopharmaceutical.
A
Solid-liquid separation (removal of cells), dewatering (concentration by precipitation or ultrafiltration), purification (chromatography) and formulation (addition of stabilizers, etc.)
7
Q
- Briefly describe the function, operation and key features of an industrial disk stack centrifuge.
A
Function is to separate cells from a culture broth. Continuous flow centrifuge with 30 – 200 discs at an angle of 35 - 50º and kept 0.4 – 2 mm apart. Culture is fed in at bottom, with solid deposition on lower disc surface and cell-free liquid discharged at the top of the bowl.
8
Q
- Briefly explain the operation of a rotary drum filter. What cell type is this method especially suited for?
A
The rotary drum (0.5 – 3.0 m in diameter) is partially submerged in the culture fluid and as it revolves in the trough it ‘sucks up’ liquid (0.1-2 rpm), leaving the cells as a cake on the surface of a porous fabric. It is continous and suited to yeast and filamentous fungi
9
Q
- Briefly explain the operation of a filter press. What cell type is this method especially suited for?
A
comprises a variable number of cloth filters through which a broth may be forced under pressure. System comprises multiple plate ‘packs’ housed on a skeleton; packs are simultaneously filled with broth. Batch system and must be dismantled to collect accumulated solids. It is suited to yeast and filamentous fungi
10
Q
- Describe the process of tangential flow microfiltration as used in the large scale separation of cells from culture broths. List one advantage of this method
A
Cross-flow microfiltration (particle size 0.2 μm – 10 μm) uses pressures of 0.5 – 2 bar and features a fluid stream which is pumped at an angle to the filter medium. Advantages (any one): efficient separation (>99.9% cell retention); it is a closed system
(good containment); does not require the addition of a filter aid; simple process; low power consumption relative to centrifugation; independent of media or cell density.
11
Q
Within the context of the commercial production of a microbial protein product, discriminate between upstream and downstream processing. Name one upstream factor that should be considered with a view to simplifying downstream processing.
A
USP: all steps involved in growing the microbe. DSP: all steps subsequent to halting the fermentation, and centrally involving the purification of the protein
Influencing factors - any one of the following: select organism that does not produce proteases/glycosidases that will cleave product/carbohydrate chains; select organisms that have extracellular secretion mode; selection of growth media that will not
contain proteinaceous components that may complicate biomolecule purification (problem with mammalian cells and FCS); low viscosity growth medium (centrifugation); avoid inclusion body formation; avoid organisms with slime layer formation.
12
Q
- Providing one example, define broth conditioning as used in downstream processing.
A
any pre-treatment of a culture broth to improve the efficacy of the solids-liquid separation (filtration or centrifugation).
13
Q
- Provide one purpose and one example of broth conditioning.
A
ease solid-liquid separation by changing particle size, alter degree of interaction between particles or alter liquor viscosity. Filter aid or flocculation.
14
Q
- Providing one example, describe the use and purpose of a filter aid in the separation of cells from a culture broth.
A
this is an example of broth conditioning in which a slurry of inert incompressible particles (2-20 um) is coated onto a filter and also mixed with the broth so that the resulting filter cake will have a solid structure which will delay premature ‘blinding’ of the filter. Example: Kieselguhr, Perlite, Celite
15
Q
- Explain the operation of a filter aid.
A
Filter aid (fossilized diatoms or volcanic rock) is mixed with broth to be filtered and a layer also placed on top of filter membrane. As filtration proceeds, the filter aid gives structure to the compressible cell cake and so allows higher filtration pressures for longer
16
Q
- Briefly describe the principle of one method used for breaking open cells at the industrial scale. Why are Gram positive organisms among the cell types which are most resistant to disruption?
A
any one of the following: homogenization, wet milling, detergent solubilization, osmotic shock, enzyme treatment. Gram positive cells comprise about 80% peptidoglycan in their cell walls (tough ‘basket’).
17
Q
- Briefly, outline the principle of flocculation as a way of performing a solids-liquid separation. Name one salt commonly used for this purpose.
A
: addition of a polycation to a cell suspension will neutralize surface charge (cells normally negatively charged and so repel each other), disrupt the layer of surface water on the protein (which acts as a thermodynamic barrier to aggregation) and form
bridges between molecules. Salts used are ammonium sulphate, sodium sulphate or aluminium potassium sulphate
18
Q
- What is membrane polarization in ultrafiltration and how may it be reduced?
A
the unwanted accumulation of a protein gel layer on the surface of the membrane which begins to act as a secondary membrane. It may be reduced by cross-flow filtration mode or the use of a stirrer (small scale).
19
Q
- Explain the principle of spray drying.
A
involves generating an aerosol of tiny droplets which is shot into a stream of hot gas (150 - 250ºC). Atomisation is achieved by either a high pressure nozzle (700 – 1,000 kPa) or a centrifugal atomizer which pumps the sample through a spinning disk. The droplet water evaporates and this leaves behind solid product particles which are collected. The residence time of the droplet in the drying chamber is quick, and moisture content decreases from 60% to 5-10%.
20
Q
- Briefly, explain the process of spray drying.
A
involves generating an aerosol of tiny droplets (solution, suspension, emulsion or paste) which is shot into a stream of hot gas (150 - 250ºC). Atomisation is achieved by either a high pressure nozzle (700 – 1,000 kPa) or a centrifugal atomizer which pumps the sample through a spinning disk.
21
Q
- Explain the difference between dead-end and cross-flow microfiltration.
A
Both used for solids liquid separation (μm sized particles – bacteria, yeasts). Dead-end (conventional filtration) versus cross-flow (liquid feed-flow parallel to membrane).
22
Q
- Explain the key difference between dead-end and tangential flow filtration
processes.
A
feedstock flow is perpendicular to filter and cake will eventually blind the filter. Tangential flow features feedstock running parallel with filter (under pressure). Resultant scouring action delays filter blinding.
23
Q
- What is a ‘host cell protein’ (HCP) and how would one test for their presence?
A
Protein contaminants in a pharmaceutical preparation arising from the producing organism. Tested via polyclonal sandwich immunoassay (polyclonal anti-HCP antibodies which will recognize the broadest possible population of HCPs for a given cell type)
24
Q
- What are the ideal attributes of a chromatography resin for use in industrial scale processes?
A
(any 2) resin should be inert; it should withstand compression at high flow rates; it should be chemically stable vis-à-vis cleaning regimes; resin should be bead-shaped and porous to give good flow properties and a high surface area.
25
25. During an enzyme purification the specific activity should increase. Give two reasons why this might not happen.
loss of enzyme in the purification step (for example, unwanted adsorption to column); denaturation; loss of cofactor or metal ion; proteolysis; inhibition by buffer.
26
26. What information is conveyed to the protein purification scientist by measurement of the specific activity at the various steps of an enzyme purification? Should the value for specific activity decrease or increase as a purification proceeds?
provides a measurement of the purification fold for the target biomolecule, against a diminishing background of contaminant proteins as the latter are removed at each purification step. It should increase at each step.
27
27. What is the principle of precipitation methods used in the purification of proteins? Provide one advantage of ammonium sulphate as a precipitant.
Precipitants such as salts or organic solvents induce a decrease in molecule solubility by disrupting electrostatic charges that keep a protein in solution and increasing the hydrophobic forces that cause precipitation
Advantages of ammonium sulphate: no change in pH, inexpensive, many proteins stabilized by NH 4+ ion.
28
28. Sephacryl S-200 gel filtration chromatography has a biomolecule separation range of 5,000 Da to 250,000 Da. Providing a brief explanation of the principle of gel filtration, which of the following proteins would be most suitable to carry out a void volume determination using this chromatography medium? Glutamate dehydrogenase (Mr = 290,000 Da); Lactate dehydrogenase (Mr = 140,000 Da); Bovine serum albumin (66,000 Da); Ovalbumin (Mr = 12,400 Da); Cytochrome C (Mr = 12,400 Da).
Large proteins are excluded from the pore structure of the gel and elute before smaller proteins. Glutamate dehydrogenase will be totally excluded from the gel pores and elute in the void volume.
29
29. What is the operational basis for the use of Cibacron blue agarose in affinity chromatography purification methods?
The dye is structurally similar to nucleotides and will bind any protein that possesses a nucleotide-binding domain (kinases, dehydrogenases, some blood coagulation factors)
30
30. What is the use and operational basis for the use of agarose in affinity chromatography purification methods?
Agarose is used as a support matrix in affinity chromatography because it is inert, porous, and hydrophilic, minimizing nonspecific binding. It can be chemically activated to immobilize ligands like Protein A, which binds the Fc region of IgG for monoclonal antibody purification. Bound proteins are then eluted under mild conditions.
31
31. What is the principle of hydrophobic interaction chromatography (HIC)? Would you need to desalt a protein solution before applying a sample to a HIC column? Explain.
hydrophobic amino acid residues on the surface of a protein can bind to hydrophobic alkyl and aryl groups on the gel matrix. As salts will increase these hydrophobic forces, there is no need to desalt the protein solution to be separated.
32
32. In adsorption chromatography, what is the difference between isocratic and gradient elution? Provide one desirable attribute of a chromatography resin.
: for example in IEX: single buffer used to elute bound proteins versus buffer which features an increasing concentration of competing salt to effect sequential elution of different components. Attributes: inert; non-compressible; chemical stability; bead shaped-porous; high surface area.
33
33. Briefly outline the principle of any two assay methods commonly used by the biopharmaceutical sector for determining/measuring the protein concentration of a solution
UV Absorbance at 280 nm:
Proteins absorb UV light at 280 nm due to the presence of aromatic amino acids (mainly tryptophan and tyrosine). The absorbance is proportional to protein concentration.
Bradford Assay:
Based on the binding of Coomassie Brilliant Blue dye to proteins, which causes a shift in the dye’s absorbance maximum. The color intensity (measured at 595 nm) correlates with protein concentration.
34
34. Protein X is a monomeric glycoprotein with a molecular mass of 60 KDa. A single
N-linked glycan chain on the protein possesses 8 negatively charged sialic acid
residues in the terminal position. Protein X is treated with a sialidase that removes the sialic acid residues. What effect will this modification have on the analysis of protein X by SDS-PAGE and isoelectric focusing?
SDS-PAGE:
Removal of the 8 sialic acid residues reduces the glycosylation and overall size of the glycoprotein. As a result, Protein X will migrate faster on SDS-PAGE, appearing as a band of slightly lower apparent molecular weight.
Isoelectric Focusing (IEF):
Sialic acids are negatively charged, so their removal increases the protein’s net positive charge. This causes an increase in the isoelectric point (pI), and the protein will focus at a more basic pH on an IEF gel.
35
35. Protein Y is a monomeric glycoprotein with a molecular mass of 60 KDa. A single N-linked glycan chain on the protein possesses 8 negatively charged sialic acid residues in the terminal position. Protein Y is treated with a sialidase that removes the sialic acid residues. What effect will this modification have on the analysis of protein Y by SDS-PAGE and isoelectric focusing?
The protein will appear smaller (faster moving) on SDS-PAGE due to removal of the four sugar residues. It will also display an altered pI value on IEF
36
36. You wish to separate two proteins, X and Y, in a purification process. The pI values of Protein X and Y are 4.5 and 6.0 respectively. What buffer pH would you choose to ensure that protein Y binds to a DEAE column? What pH would be most suitable to maximize the effectiveness of the ammonium sulphate precipitation of protein X?
To bind Protein Y to a DEAE column (anion exchanger):
Choose a buffer pH of 7.0–8.0. Since Protein Y has a pI of 6.0, at pH values above its pI it will be negatively charged and can bind to the positively charged DEAE resin.
For ammonium sulphate precipitation of Protein X:
Use a buffer pH of 4.5, which is the pI of Protein X. Proteins are least soluble at their isoelectric point due to having no net charge, making precipitation most effective at this pH.
37
37. Protein Y is negatively charged in a chromatography mobile phase and is applied onto a hydroxyapatite column. What is the principal mechanism by which Protein Y will bind to this resin?
Protein Y binds to a hydroxyapatite column primarily through metal coordination (chelation) interactions. Negatively charged groups on the protein — such as carboxylate groups (from aspartate or glutamate) and side chains like histidine or cysteine — act as electron donors that interact with the calcium ions present in the resin.
There may also be minor ion exchange interactions, as hydroxyapatite has both cationic (Ca²⁺) and anionic (PO₄³⁻) sites.
38
38. Providing one example, explain what is meant by ‘strong’ and ‘weak’ ion exchange resins?
Most biomolecule purifications are usually performed in the region of physiological pH (stability). A strong IEX resin means that the charge on the bead will remain unchanged over the range of pH values at which the separation is performed. This is related to the pKa value of the charged species (strong cation exchangers such as sulfopropyl or sulfoethyl have pKa values in the region of 1.1-2.0. Alternatively, weak cation exchangers such as those based on carboxy groups, may lose charge within the normal operating pH range of protein separations (pKa values of 3.7 – 4.3).
39
39. What is the principle of ion exchange chromatography? Provide one example of a commonly used ion exchange matrix.
Ion exchange chromatography separates proteins based on their net charge. The resin contains chemically bound charged groups that attract and bind oppositely charged molecules through electrostatic interactions. Proteins bind to the resin depending on their charge at the working pH and are eluted by increasing salt concentration or changing pH.
DEAE-Sephadex (an anion exchanger) binds negatively charged proteins.
40
40. You wish to separate two proteins, X and Y, in a purification process. The pI values
of Protein X and Y are 4.0 and 6.0 respectively. What buffer pH would you choose to
ensure that protein Y binds to a DEAE column? What pH would be most suitable to
maximize the effectiveness of the ammonium sulphate precipitation of protein X?
A pH of around 7.5 to ensure that Y is negatively charged. Ammonium sulphate precititation of X might be expected to be most effective at pH 4.0 (the pI)
41
41. Biopharmaceutical legislation demands that unwanted changes in the charge or conformation of a biomolecule be monitored during purification. Outline two examples of possible modifications which might be encountered. What modifications may be revealed by electrophoresis?
Deamidation (IEF); protein aggregation (native PAGE); oxidation (IEF); proteolysis (SDS-PAGE); glycan modification (SDS-PAGE; IEF)
Two Common Modifications Encountered:
Deamidation
A chemical modification where asparagine or glutamine side chains are converted to aspartic acid or glutamic acid, respectively.
This adds a negative charge, altering the protein’s isoelectric point (pI).
Can occur during storage or under mildly basic conditions in purification buffers.
Oxidation
Typically affects methionine or cysteine residues, forming sulfoxides or disulfide scrambling.
May lead to loss of activity or altered tertiary structure.
Can increase susceptibility to aggregation or immunogenicity.
Modifications Revealed by Electrophoresis:
Isoelectric Focusing (IEF):
Detects changes in net charge (e.g., deamidation, glycosylation changes) by shifts in pI.
Deamidated forms will focus at more acidic pH (lower pI).
SDS-PAGE:
Reveals changes in apparent molecular weight due to conformational changes, truncations, or altered glycosylation.
Example: Loss of glycosylation makes proteins migrate faster, appearing “smaller” on the gel.
Aggregation or proteolysis may also be seen as high or low molecular weight bands.
42
42. In clean room manufacturing what is a HEPA filter and what are the recommendations for ceiling coverage in a European grade A GMP manufacturing environment?
High-Efficiency Particulate Air Filters: depth filters several inches thick and made from high-density glass fibre. Grade A requires 100% of ceiling coverage
43
43. What is a pharmaceutical excipient? List two functions of excipients. Provide one example of a commonly used excipient.
Inert substances added to a medicine (additional to the active principle). Any two of the following functions: stabilizers, binders, dyes, sweeteners, disintegrants, bulking agents, anti-caking agents, coatings on pills. Examples: lactose; HSA, amino acids, sucrose, trehalose.
44
44. In pharmaceutical manufacturing according to GMP guidelines, explain the job of a Qualified person.
Medicinal products are not sold or supplied before a Qualified Person has certified that each production batch has been produced and controlled in accordance with the requirements of the MA
45
45. In terms of quality control procedures, provide the meaning of each letter in the acronym ‘HACCP’.
Hazard Analysis and Critical Control Points.
46
46. Filters used for terminal sterilization of biopharmaceuticals must be validated. Briefly explain any one aspect of this validation process.
Compatibility of filter matrix with product: no product degradation, adsorption or production of particles in the product (‘extractables’) OR Microbial retention testing: the filter matrix is challenged with ‘worst case scenario’ bacterial contaminants (in a
scale -down model). The challenge usually involves 1 x 107 Brevundimonas diminuta (ATCC 19146) (a small rod of 0.3 μm diameter). The latter can pass through a 0.45 μm pore size filter, but should be retained by an intact 0.2 μm filter. This validation is performed every 12 months.
47
47. Outline either EU or US clean room classifications. What is HEPA filter? Outline the required HEPA filter usage for the different clean room classifications.
(in order of cleanliness ‘stringency’): EU: grades A, B, C or D. US: class 100 (equivalent to EU grade A and B) and class 10,000 and 100,000. High Efficiency particulate Air filter mounted in the ceiling to create downward (laminar) air flow. HEPA filters for grade B, C and D occupy about 20 - 25% of ceiling space. Grade A requires 100% of ceiling (or grade A cabinet in grade B room).
48
48. Explain the meaning of ‘media fill’ within in the context of the validation of sterilization in the manufacturing of biopharmaceutical products.
process simulation, filling a microbiological medium into product containers (instead of product; termed ‘media fill’). Tryptone Soy Broth usually the growth medium employed. Such procedures are typically performed every 6 months
49
49. Name the three major types of N-linked glycosylation found in many of the proteins of
mammalian cells. Give one function of protein glycosylation. What is meant by microheterogeneity of glycan chains?
high mannose; hybrid; complex. For functions, any one of the following: mediating biological activity; recognition role; increase molecule solubility; affect circulatory half-life; protein stabilization. Microheterogeneity refers to variations in sugar composition of glycoprotein antennary chains among a single glycoprotein type.
50
50. Provide the operating principle of one method commonly used for the drying of biomolecule products.
either principle of freeze drying (freezing followed by sublimation from ice to a vapour under low pressure) or spray drying (atomization of liquid stream into hot air).
51. Explain the principle of freeze drying.
51
51. Explain the principle of freeze drying.
freezing of protein solution followed by sublimation by vacuum under low pressure so that ice is converted directly to vapour without passing through a liquid phase.
52
52. What are pyrogens? Briefly, provide the principle of one method used in the pharmaceutical industry to test biopharmaceuticals for the presence of pyrogens
Fever inducing agents that if injected into the body cause the loss of hypothalamic regulation of body temperature. Limulus amoebocyte lysate assay or rabbit test
53
53. What is protein deamidation and how it may be controlled during biomolecule purification?
unwanted conversion of asparagine to aspartic acid or glutamine to glutamic acid by removal of amide groups via hydrolysis. Lessened by reducing final pH to pH 4.0 – 5.0 and using low temperatures (one method)
54
54. Which of the common twenty amino acids are most susceptible to oxidation and give an example of one antioxidant that may be incorporated into biopharmaceuticals to minimize this.
methionine or cysteine (or histidine, tyrosine and tryptophan. Antioxidants such as methionine thiosulfate, ascorbic acid or headspace filling of vial with inert gas.
55
55. Name two amino acids which are susceptible to oxidation during downstream processing and formulation of biopharmaceuticals. How may oxidation be minimized?
Methionine, cysteine, tyrosine, histidine, tyrptophan. Oxidation may be minimized by replacing air in a vial headspace with an inert gas (such as N2) and/or the incorporation of antioxidants into the product
56
56. Downstream processing operations are conducted at 4°C. Provide two reasons for this requirement.
retard metabolic activity of any remaining cells or microbial contaminants. Reduce unwanted trace protease activity.
57
57. Name two amino acids which are susceptible to deamidation during downstream processing and formulation of biopharmaceuticals. How may deamidation be minimized?
asparagine and glutamine. Lessened by reducing final pH to pH 4.0 – 5.0 and using low temperatures (one method)
58
58. Proteolysis may complicate the purification of a biomolecule. How are proteases classified and how may their unwanted activity be minimized?
Serine proteases I (mammalian) and II (bacterial); Cysteine proteases; Aspartic proteases; Metalloproteases I (mammalian) and II (bacterial). Controlled via inhibitors, low temperature, and fast processing.
59
59. What disease is Cerezyme® used to treat?
Gaucher’s disease – glucocerebrosidase defiency
60
60. Briefly explain the use of collagenase in medicine.
wound debridement.
61
61. What is an orphan disease? Give one example of an orphan drug product and the disease target of this treatment. What is the main provision of orphan disease legislation that acts as an incentive to companies to develop drugs for such diseases?
within a pharmaceutical context, a disease that affects not more then 5 in 10,000 persons (EU). Example: glucocerebrosidase deficiency (Gaucher’s disease). Main clause/incentive: market exclusivity for 10 years.
62
62. What is an orphan disease in pharmaceutical regulation?
within an EU pharmaceutical context, a disease that affects not more then 5-in-10,000 persons; US: 7.5 in 10,000 persons
63
63. What is meant by pegylation of a biopharmaceutical and what is its purpose? Give one example of a medicine which has undergone this modification.
Covalent linkage of polyethylene glycol to a protein medicine via linkage to terminal amino group of a protein or the epsilon amino group of lysine. It increases serum half-life of a parenteral medicine. Examples are asparaginase (cancer) or interferon alpha (hepatitis C).
64
64. What is the purpose of pegylation of biopharmaceuticals?
Increase the circulatory half-life of drugs.
65
65. What is meant by hesylation of a biopharmaceutical and what is its purpose? Give one example of an alternative strategy to achieve this purpose.
conjugation of a biopharmaceutical to hydroxyethyl starch (prolongation of the circulatory half-life, reduced renal clearance and reduced immunogenicity. Pegylation, PASylation or FcRn-mediated recycling.
66
66. What is the purpose of hesylation of biopharmaceuticals?
Increase the circulatory half-life of drugs.
67
67. What is the clinical use of asparaginase and how does the treatment work?
used in certain forms of childhood leukaemia. Asparaginase hydrolyzes L-asparagine to yield aspartic acid and ammonia. Unlike normal cells, leukaemic cells are unable to make their own asparagine, and so are starved of this essential amino acid by the use of asparaginase. Normal cells can make their own asparagine.
68
68. The symptoms of which disease are treated with asparaginase? What is the basis of
this therapy?
Used in the treatment of acute lymphoblastic leukemia (cancer). Cancer cells lose the ability to make their own asparagine, and must source this amino acid from the blood. Asparaginase hydrolyzes asparagine in serum to aspartic acid and ammonia
69
69. The symptoms of which disease are treated with DNase? Why is a deoxyribonuclease needed in this specific case, and what symptoms of the disease are alleviated by its use?
Used in the treatment of cystic fibrosis. Hydrolyzes DNA produced on degradation of bacteria and macrophages and which adds to the problems of unusually high mucous production by CF patients. It helps to loosen mucous and aids in chest percussion.
70
70. What is chemical wound debridement? Give one example of an enzyme used for this
purpose.
Removal of dead tissue using a topical application of proteolytic enzyme, usually in association with surgical debridement. Collagenase (Clostridium histolyticum), trypsin, papain
71
71. What is wound debridement? Give one example of an enzyme used for this purpose
Removal of dead tissue using a topical application of proteolytic enzyme. Collagenase (Clostridium histolyticum), trypsin, papain
72
72. What are the reactions catalysed by penicillin acylase? Name one microbial source of this enzyme. List two benefits of the enzymatic hydrolysis over chemical methods.
Removal of penicillin side chain under alkaline conditions and addition of new side chain under acidic conditions.
Source: E. coli, Pseudomonas, Proteus, Brevibacterium, Bacillus, Streptomyces.
Benefits: environmental (elimination of solvents and associated issues; no hazardous waste; gentle reaction conditions; good reaction control & monitoring; re-use of catalyst; better product quality; less impurities; improved yields; decreased manufacturing costs).
73
73. Briefly explain the use of papain in the food industry.
As an aid in meat tenderization for older animals. It disrupts the myofibrils and connective tissues on cooking. Also used in beer clarification (chill proofing).
74
74. Outline the catalytic specificity and the industrial uses of either lactase or invertase. In your answer, provide one example of a producing organism
Lactase: breaks down lactose to glucose and galactose. Produced by K. lactis, K. fragilis and A. niger. Used in whey degradation and generation milk for lactose intolerance.
Invertase: breaks down sucrose into glucose and fructose. Produced by S. cerevisiase and Aspergillus spp. Used in confectionary industry and marzipan manufacture.
75
75. What is the role and enzymatic specificity of chymosin in cheese manufacture? Name one microorganism which has been used for the recombinant expression of calf chymosin.
Chymosin is necessary for the coagulation of the milk protein, casein. Chymosin cleaves the Phe(105)-Met(106) linkage of kappa-casein resulting in the formation of the soluble glycomacropeptide which diffuses away from the casein micelle and para-kappa-casein, a distinctly hydrophobic peptide that remains on the micelle. The patch or reactive site that is left on the micelles after enzymatic cleavage is necessary before aggregation of the paracasein micelles can begin, and coagulation of casein occurs. Any of the following: E. coli K12, Aspergillus niger, Saccharomyces cerevisiae, Trichoderma reesei, Kluyveromyces lactis, Bacillus licheniformis, Lactococcus lactis
76
76. Describe one commercial use of a specific cysteine protease.
Cysteine proteases - papain, bromelain or ficin. Meat tenderization, leather production, clarification in brewing. Wound debridement (papain).
77
77. Outline the basis for the classification for the major groups of proteases.
classified on the basis of amino acid groups within active site into Serine (I and II), cysteine, aspartic and metallo-proteases (I and II).
78
78. Outline the basis for the classification for the major groups of proteases. List one way whereby unwanted protease activity may be controlled or reduced during protein purification.
classified on the basis of amino acid groups within active site into Serine (I and II), cysteine, aspartic and metallo-proteases (I and II). Control via: inhibitors, fast processing, low temperature.
79
79. Briefly outline the process for optical resolution of amino acids by immobilized L-amino acylase.
Acetylate a racemic mixture of D- and L-amino acids and pass over a bed of L-amino acylase. The enzyme deacetylates only the L-form and yields a free L amino acid and the intact N-acetyl-D-amino acid. Separate products by ion exchange.
80
80. α-amylases and proteases are commonly used in detergents. Outline three requirements for an enzyme to be used in such an environment.
Thermostability; protease resistance, bleach tolerance (perborates will oxidize susceptible amino acid residues), no metal ion requirements (water softening agents will chelate metals). Must show maximum activity at wash pH values (alkaline). Broad substrate specificity needed to handle range of stains encountered.
81
81. Name the three stages of starch hydrolysis to produce glucose syrups and identify the key enzymes at each step.
Gelatinisation (α-amylase); liquefaction (α-amylase) and saccharification (glucoamylase and pullulanase).
82
82. What is the dextrose equivalent and how is it used in the starch hydrolysis process?
Dextrose equivalent is a method of determining the level of reducing sugar as starch hydrolysis proceeds (formula in papers)
83
83. Explain one function of the use of fungal α-amylase in the baking industry.
Increasing the gassing power of the dough (and bread porosity) or retardation of staling.
84
84. What is the enzymatic specificity of pullulanase?
specific for the α1,6 link in amylopectin in starch.
85
85. Outline the classification of cellulases and also the reactions catalysed by each class. Name one major microbial source of commercial cellulases.
Endocellulases (cleave random internal links); cellobiohydrolases (remove cellobiose units from non-reducing end); β-glucosidases (yield glucose from short chain oligosaccharides and cellobiose).
Most important commercial producers: Trichderma (spp: viride, reesei, koningii), Penicillium funiculosum. Range of others.
86
86. Name one commercially important cellulase used in detergents. Explain one function
performed by cellulases in detergents. Outline one other commercial use of cellulases
from a different market sector.
Either Celluzyme or Carezyme. Textiles: Stain removal, colour revival, depilling, fabric softening, stonewashing. Other markets: extraction of fruit juice; clarification of fruits juice, enzymatic deinking, coffee processing.
87
87. Provide three differences between speciality and commodity enzymes. Give one example of a commodity enzyme and one example of a speciality enzyme.
Commodity [cheap, low purity (<10%) contaminants present; spray dried, etc.].
Therapeutic [expensive, pure (99.9%); often freeze dried; difficult purification].
Examples: wide range discriminated on end use (bulk or therapeutic/analytical).
88
88. Provide one example of a commercially important enzyme which is inducible. What is
a gratuitous inducer?
Amylase, invertase, lactase. A non-metabolizable inducer such as IPTG that can be used in place of lactose for the lac operon
89
89. Provide one example of a commercially important recombinant DNA lipase preparation used in detergents. What is the multi-cycle effect?
Either Lipolase or Lipomax. Multi-cycle effect: Water content of clothes must be reduced to 10 – 40% to get lipase action (ie, on drying)
90
90. Name any enzyme commonly used as a label in enzyme-linked immunoassay.
Horse radish peroxidase, alkaline phosphatase, β-galactosidase.
91
91. Name two genera of microbes that are used commercially for lipase production. Explain interfacial activation seen in lipases.
Candida, Pseudomonas, Rhizopus, Bacillus, Aspergillus, Munor, Humicola, Penicillium. Interfacial activation: the enzyme is activated at the oil-water interfaces and activity depends on the level of substrate aggregation, the use of emulsifiers and
agitation.
92
92. With respect to lipases, explain the meaning of the ‘multi-cycle effect’.
Answer relates to inclusion of lipases in biological washing powders. Major lipase action only occurs when water content of clothes
is reduced (during drying).
93
93. Providing one example, explain the basis of a bioassay as used to test the potency of a biomolecule.
a bioassay features the use of a whole animal or isolated cell to test the therapeutic efficacy of a biomolecule. Examples include the antibiotic diffusion test using susceptible test organisms or the cytopathic effect inhibition assay used with interferons.
94
94. Explain one method by which enzyme entrapment may be achieved.
Covalent binding (derivatize surface lysine residues by reaction with acryloyl chloride to give the acryloyl amides and then co-polymerise and cross-link with acrylamide and bisacrylamide to form a gel.
Physical caging: entrap in cellulose acetate fibres by making up an emulsion of the enzyme plus cellulose acetate in methylene chloride, followed by extrusion through a spinneret into a solution of an aqueous precipitant, or use calcium alginate technique
95
95. What enzyme is used in the production of high fructose corn syrups? Name one organism that is used for the industrial scale production of this enzyme
glucose isomerase. B. coagulans, Aerobacter, Streptomyces rubiginosus, S. albus, Lactobacillus brevis and Flavobacterium arborescens.
96
96. In immobilized enzyme technology, what is the difference between a packed bed reactor and a fluidized bed reactor?
In the PBR, the enzyme is immobilized onto a solid particle housed in a column-type reactor. The substrate feed is delivered to the column in a manner akin to column chromatography. In a fluidized bed reactor, the flow of substrate keeps the immobilized particles in a fluidized (suspended) state.
97
97. With the aid of a sketch, outline the principle of any one heterogeneous ELISA with which you are familiar, and name enzymes commonly used as reporter molecules in ELISA systems
any format featuring immobilization of antibody or antigen. Alkaline phosphatase; horse radish peroxidase or β-galactosidase.
98
98. Briefly explain the difference between a direct and indirect ELISA.
Direct ELISA, the molecule (sample) to be detected is immobilized on a 96-well plate. The target molecule is then detected directly by adding an antibody conjugated to an enzyme label. In indirect ELISA, the primary antibody (that recognizes the analyte) is not labelled with an enzyme, but rather an anti-species labelled secondary antibody is used in the amplification step (this makes it ‘indirect’).
99
99. What is the principle of a coupled enzyme assay? Provide one example of this type of assay.
the reaction products of one enzymic reaction are the substrates of a second enzyme. In this way, general or unspecific indicator reactions can be linked without individual optimization to various specific auxillary reactions. Example: glucose
determination using glucose oxidase and peroxidase.
100
100. Briefly, what is the industrial use of immobilized invertase?
production of glucose-fructose syrups from sucrose for addition to fizzy drinks, juices, marzipan, sweets and liquors
