Immobilisation of Biomolecules Flashcards
Define immobilisation
A molecule whose movement has been restricted either completely or partially by attachment to a solid structure (e.g. beads)
Give 5-6 examples of biomolecules that have been immobilized.
- Enzymes
- Ag/Ab
- Cells
- Metals
- Drugs
- Carbohydrates
State 3-5 advantages for immobilized enzymes
- economical
- can control to stop rxn rapidly
- product not contaminated w/ enzyme
- easy separation
- enzyme stability from pH, temp, solvents, impurities, contamination
State 3 criteria for successful immobilization.
- enzyme must retain biological activity/sensitivity after attachment
- must have a long-term stability
- Avoid overloading enzymes per bead = immobilized enzymes can block or inactivate the active site
State 2 protective methods that you can use during the covalent immobilization process?
- presence of inhibitor or add substrate
- Modified soluble enzyme link to matrix
advantages of immobilised enzymes vs free enzymes
- saves time & money
bc enzymes can be separated from product and reused. (not lost after first use = expensive)
5 types of immobilisation techniques
- 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.
What are the 2 main advantage of covalent immobilization over the other techniques?
- stable
- minimal leaching
Precautions for covalent immobilisation
- may alter conformational strcuture & active site = dec activity
- functional groups not essential to catalytic activity must bind to support matrix
State 3-6 shapes of carrier to be used for the immobilization process?
- *Disc
- Bead
- fibres
- hollow spheres
- thin films
- membranes
State 3-4 properties for an ideal carrier to be used for the immobilization process?
- CHeap
- Innert – just bind to enzyme
- Physically strong
- stable
the carrier for enzyme immobilisation should ideally… (3)
- increase enz specificity (kcat/Km)
- shift pH optimum to desired pH value
- Discourage microbial growth and non-specific adsorption
Simple/rapid S/R tests for pregnancy detection and HIV antibodies is based on which of the following techniques?
indirect ELISA
how are antibiotics produced
- Penicillin G [Penicillin G Acylase] -> 6-APA (B-lactam) + PAA
- 6-APA = precursor for B-lactam antibiotics
describe 4 types of lactose intolerance
- 1º lactose intolerance: getting older = # of lactase decrease
- 2º “ “: injury to SI from infection or disease
- Developmental “ “: occur in premature babies and usually improves over a short period of time.
- Congenital “ “: little or no lactase is made from birth (extremely rare genetic disorder)
describe the lactose tolerance BLOOD test
- 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)
Blood lactose intolerant test should not be done on and why?
1) ppl w/ Diabetes bc already have hyperglycemia
2) babies bc causes them severe diahorrea
describe the lactose tolerance HYDROGEN BREATH test
- fast overnight
- 25g lactose given
- lactose intolerant = enteric bacteria breaks it down = H (& methane) => detects H in breath
- diabetic ppl can do this test
describe the lactose tolerance STOOL ACIDITY test
- baby given lactose to drink
- if lactose not digested = bacteria in colon breaks down = acidic stools = pH <5.5
Discus how can you manage lactose intolerant. (4)
- avoid lactose
- substitute to maintain nutrient intake,
- regulate calcium intake,
- use of enzyme substitute (immobilized lactase)
- ingest yoghurt w/ lactobacilli
3 tratments for lactose intolerance
- drugs w/ lactase
- lactobacillus acidophilus
- lactose-free products
how is fructose produced
inuline [inulinase] -> fructose
characteristics of fructose
- 4x sweeter than glucose
- easily absorbed
- suitable for diabetics
what is a biosensor?
- analytical device
- has a biological component with a physicochemical detector.
> detection of an analyte (target)
In biosensors, the bio-recognition molecule for detection of Urea is ---------------. Cholesterol is -------------. Penicillin is --------------------- Glucose is --------------.
urea = urease cholesterol = cholesterol oxidase penicillin = panicillinase glucose = glucose oxidase
Usinga chemical equation, discus the principle of glucose meter for detecting glucose
- glucose + O2 [glucose oxidase] ->gluconic acid + H2O2
- H2O2 -> 2H+ + O2 + 2e-
* e- is detected
Reference range for:
a) fasting blood sugar
b) 2hrs after a meal
c) random blood glucose
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
Discus the principle of Immulite 1000.
- chemiluminescent substrate: dioxetane
- reacts w/ ALP-labelled reagent (COMPETETIVE to sample)
