Immobilisation of Biomolecules

Question 1

Define immobilisation

Answer 1

A molecule whose movement has been restricted either completely or partially by attachment to a solid structure (e.g. beads)

Question 2

Give 5-6 examples of biomolecules that have been immobilized.

Answer 2

• Enzymes
• Ag/Ab
• Cells
• Metals
• Drugs
• Carbohydrates

Question 3

State 3-5 advantages for immobilized enzymes

Answer 3

• economical
• can control to stop rxn rapidly
• product not contaminated w/ enzyme
• easy separation
• enzyme stability from pH, temp, solvents, impurities, contamination

Question 4

State 3 criteria for successful immobilization.

Answer 4

1. enzyme must retain biological activity/sensitivity after attachment
2. must have a long-term stability
3. Avoid overloading enzymes per bead = immobilized enzymes can block or inactivate the active site

Question 5

State 2 protective methods that you can use during the covalent immobilization process?

Answer 5

• presence of inhibitor or add substrate

- Modified soluble enzyme link to matrix

Question 6

advantages of immobilised enzymes vs free enzymes

Answer 6

• saves time & money

bc enzymes can be separated from product and reused. (not lost after first use = expensive)

Question 7

5 types of immobilisation techniques

Answer 7

• Adsorption: held by weak physical bond
• Covalent: held by strong chemical bond
• Encapsulation: contain enzymes in semi-permeable membrane
• Entrapment: enzymes trapped in gel matrix = pores allow entry of R & exit of P but not Enz
• Cross-linking: bind enzyme tog.

Question 8

What are the 2 main advantage of covalent immobilization over the other techniques?

Answer 8

• stable

- minimal leaching

Question 9

Precautions for covalent immobilisation

Answer 9

• may alter conformational strcuture & active site = dec activity
• functional groups not essential to catalytic activity must bind to support matrix

Question 10

State 3-6 shapes of carrier to be used for the immobilization process?

Answer 10

• *Disc
• Bead
• fibres
• hollow spheres
• thin films
• membranes

Question 11

State 3-4 properties for an ideal carrier to be used for the immobilization process?

Answer 11

• CHeap
• Innert – just bind to enzyme
• Physically strong
• stable

Question 12

the carrier for enzyme immobilisation should ideally… (3)

Answer 12

• increase enz specificity (kcat/Km)
• shift pH optimum to desired pH value
• Discourage microbial growth and non-specific adsorption

Question 13

Simple/rapid S/R tests for pregnancy detection and HIV antibodies is based on which of the following techniques?

Answer 13

indirect ELISA

Question 14

how are antibiotics produced

Answer 14

1. Penicillin G [Penicillin G Acylase] -> 6-APA (B-lactam) + PAA
2. 6-APA = precursor for B-lactam antibiotics

Question 15

describe 4 types of lactose intolerance

Answer 15

1. 1º lactose intolerance: getting older = # of lactase decrease
2. 2º “ “: injury to SI from infection or disease
3. Developmental “ “: occur in premature babies and usually improves over a short period of time.
4. Congenital “ “: little or no lactase is made from birth (extremely rare genetic disorder)

Question 16

describe the lactose tolerance BLOOD test

Answer 16

• fast overnight
• 5 blood samples taken = 1x initial & 4x after ingesting 50g of lactose
• lactose intoleranct if blood glucose lvls rise <20mg/dL (1.11 mmol/L)

Question 17

Blood lactose intolerant test should not be done on and why?

Answer 17

1) ppl w/ Diabetes bc already have hyperglycemia

2) babies bc causes them severe diahorrea

Question 18

describe the lactose tolerance HYDROGEN BREATH test

Answer 18

• fast overnight
• 25g lactose given
• lactose intolerant = enteric bacteria breaks it down = H (& methane) => detects H in breath
• diabetic ppl can do this test

Question 19

describe the lactose tolerance STOOL ACIDITY test

Answer 19

• baby given lactose to drink

- if lactose not digested = bacteria in colon breaks down = acidic stools = pH <5.5

Question 20

Discus how can you manage lactose intolerant. (4)

Answer 20

• avoid lactose
• substitute to maintain nutrient intake,
• regulate calcium intake,
• use of enzyme substitute (immobilized lactase)
• ingest yoghurt w/ lactobacilli

Question 21

3 tratments for lactose intolerance

Answer 21

• drugs w/ lactase
• lactobacillus acidophilus
• lactose-free products

Question 22

how is fructose produced

Answer 22

inuline [inulinase] -> fructose

Question 23

characteristics of fructose

Answer 23

• 4x sweeter than glucose
• easily absorbed
• suitable for diabetics

Question 24

what is a biosensor?

Answer 24

• analytical device
• has a biological component with a physicochemical detector.
  > detection of an analyte (target)

Question 25

In biosensors, the bio-recognition molecule for detection of 
Urea is ---------------.
Cholesterol is -------------.
Penicillin is ---------------------
Glucose is --------------.

Answer 25

urea = urease
cholesterol = cholesterol oxidase
penicillin = panicillinase
glucose = glucose oxidase

Question 26

Usinga chemical equation, discus the principle of glucose meter for detecting glucose

Answer 26

1. glucose + O2 [glucose oxidase] ->gluconic acid + H2O2
2. H2O2 -> 2H+ + O2 + 2e-
   * e- is detected

Question 27

Reference range for:

a) fasting blood sugar
b) 2hrs after a meal
c) random blood glucose

Answer 27

a) fasting blood sugar = 3.5 - 5.5 mmol/L
b) 2hrs after a meal = 4.4 - 6.1 mmol/L
c) random blood glucose = <7.8 mmol/L

Question 28

Discus the principle of Immulite 1000.

Answer 28

• chemiluminescent substrate: dioxetane

- reacts w/ ALP-labelled reagent (COMPETETIVE to sample)