Transcript Biocatalysis - Chatham University

BIOCATALYSIS
DISCUSSION
This Week Agenda
Today: Biocatalysis
Wednesday: Biodiversity
Final Paper Discussions
Exam 4 – Monday, October 27
What are limitations of enzyme catalysis?
What could be done to overcome these limitations?
METHODS OF ENZYME IMMOBILIZATION
A. Adsorption:
Suitable
adsorbents: ion-exchange matrices,
porous carbon, clays, hydrous metal
oxides, glasses and polymeric
aromatic resins
B. Covalent binding: cyanogen
bromide on sepharose, ethyl
chloroformate on cellulose,
carbodiimode on various COOmatrices, glutaroaldehyde, 3aminopropyltrioxysilane
C. Entrapment: fibers and gels
D. Membrane confinement: calcium
alginate gel
http://www1.lsbu.ac.uk/water/enztech/immethod.html
GENERALIZED COMPARISON OF DIFFERENT
ENZYME IMMOBILIZATION TECHNIQUES
Characteristics
Preparation
Adsorption
Covalent
binding
Entrapment
Membrane
confinement
Simple
Difficult
Difficult
Simple
Low
High
Moderate
High
Strong
Weak
Strong
Yes
No
Yes
No
Applicability
Wide
Selective
Wide
Very wide
Running Problems
High
Low
High
High
Matrix effects
Yes
Yes
Yes
No
Large diffusional barriers
No
No
Yes
Yes
Microbial protection
No
No
Yes
Yes
Cost
Binding force
Enzyme leakage
Variable
COVALENT BINDING
IMMOBILIZATION OF ENZYMES: CROSS
INKING WITH GLUTARALDEHYDE
1) Improves various aspects of stability:
- Heat
- pH
- Exogenous proteases
- Organic solvents
2) Elongates enzyme activity
3) Reuse of an enzyme
4) Increases final productivity
ENCAPSULATION OF ENZYMES
USE OF ENZYMES IN ORGANIC SOLVENTS
Advantages:
-Water-sensitive/ water-insoluble compounds could be used
-Thermal stability is improved
-Water-dependent side reactions are suppressed (including hydrolysis)
-Problems:
-Proteins are usually insoluble in organic solvents (or denaturated)
-Hydrogen bonding and 3D structure of enzyme could be altered
-Solvent could be active site inhibitor
-Rates drop dramatically
-Solvents used:
-Tetrahydrofuran, DMSO, sc CO2, acetonitrile, hexane, toluene,
methanol, ionic liquids
-Monomolecular layer of water should be
present on enzyme surface!
EXTREMOPHILES AND EXTREMOZYMES
Extremophiles - microorganisms, typically Archarea, populating extreme
habitats
Extreme conditions → Extreme enzymes
High-temperature stable enzymes
Food preparation: separation step is not required for
sterilization
Cold-temperature stable enzyme
Food preparation: tenderization of meat
Detergents used in cold water
STRUCTURAL FEATURES OF
HYPERTHERMOPHILIC ENZYMES
-Lower surface area/volume ratio
-High number of salt bridges
-Tungstopterin cofactor
Tungstopterin
Aldehyde ferredoxin oxidoreductase
CATALYTIC ANTIBODIES (ABZYMES)
A hapten is a small molecule that can elicit an immune response only when
attached to a large carrier such as a protein; the carrier
http://nptel.ac.in/courses/104103018/module3/lec8/images/3.png
THE RANGE OF BIOCATALYSIS
•Oxidation and Reduction
•Preparation and Hydrolysis of Esters
•Preparation of Acids, Amides, and Nitriles
•Compounds with Two or More Hydroxyl Groups
•Preparation of Aromatic Compounds
Product/Application
Starting material
Enzyme/organizm
Are there any products traditionally synthesized from petroleum?
ESSENTIALS STEPS IN DNA CLONING
1. Cutting target DNA at precise locations. Sequence-specific
endonucleases (restriction endonucleases) provide the
necessary molecular scissors
2. Selecting a small carrier molecule of DNA capable of selfreplication. These DNAs are called cloning vectors (typically
plasmids or viral DNAs).
3. Joining two DNA fragments covalently. DNA ligase links the
cloning vector and the DNA to be cloned. Resulting DNA –
recombinant DNA.
4. Moving recombinant DNA from the test tube to a host cell
that will provide the enzymatic machinery for DNA
replication.
5. Selecting or identifying host cells that contain recombinant
DNA.
Recombinant DNA technology or genetic engineering
E.coli is a hero of genetic engineering
CLEAVAGE OF DNA BY RESTRICTION
ENDONUCLEASES
CLONING VECTORS ALLOW AMPLIFICATION OF
INSERTED DNA SEGMENTS
1. Plasmids (E.coli)
2. Bacterial artificial chromosomes (E.coli)
3. Vector from yeasts
Plasmids-circular DNA, 5 kbp to 400 kbp long, replicates separately from host
chromosome, usually symbionts
Classic E. coli plasmid pBR322 constructed in 1977
CLONED GENES CAN BE EXPRESSED TO AMPLIFY
PROTEIN PRODUCTION
-Alteration of the sequence around a cloned gene to trick protein
overexpression
-Transcription and translation sequences must be inserted in the vector DNA
(expression vectors)
ALTERATION OF CLONED GENES PRODUCES ALTERED
PROTEINS
Site-direct mutagenesis –
replacement of individual
amino acid
Oligonucleotide-directed
mutagenesis creates a change
in specific DNA sequence
Fusion protein – the product of a
fused gene
PROBABLY THE MOST CONTROVERSIAL
COMPANY
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