1. Industrial Enzymes
BIOL 431

2. Introduction Industrial vs Medicinal/Therapeutic
Hydrolytic depolymerases
Types
$1.5 billion industry
Detergents No 1

3. Classes
Lipases
Proteases
Carbohydrases

4. Non-recombinant Sources
GRAS---Generally recognized as safe
Bacillus
protein is secreted into fermentation medium
easier purification
Aspergillus
Yeast

5. Recombinant Sources Most industrial enzymes are produced recombinantly
Why?
A. Higher expression
B. Higher purity (%protein:other junk)
C. cheap
D. can engineer protein
E. can express enzymes which are found
in pathogenic organisms
Heterologous Protein Expression
Homologous Protein Expression

6. Protein engineering Make oxidation resistant
make enzymes tolerant of processes used in industry
less substrate specificity
more thermostable
more stable in presence of detergent

9. Mesophiles vs Extremophiles
Mesophiles 35-60oC
pH 4-8
Extremophiles organisms that colonize niches with
one or more extreme
environmental parameters pH
temp
salinity
pressure

10. Hyperthermophiles 90oC plus typically anaerobic
found at tectonically active sites
vents
springs
geysers
enzymes are beneficial
substrate more susceptible
discourages microbial growth

11. Hyperthemophiles Difficult to culture
Move to recombinant production of their enzymes
Purification easier -Raise temp

12. Psycrophiles Extreme Low temperatures 0oC
Antifreeze proteins- prevent ice crystal formation

13. Others Thermoacidophiles Barophiles Halophiles Alkaliphiles

14. Proteolytic Enzymes Largest group of Industrial Enzymes (50%)
Function - degrade proteins
What is hydrolysis?
Don’t want absolute specificity. Why??

15. Classifications Site of Hydrolysis A. Endopeptidase
cleaves peptide bonds internally
B. Exopeptidase
cleaves peptides at C or N term
Mechanisms - based on active site (on enzyme)
Serine proteases
Cysteine protease (cys and his)
Asparatic proteases
Metalloprotease (need metal ions, Ca, Mg)

16. Divalent Ions Calcium Magnesion Iron These make water hard!
Cause precipitation of soaps
Need to sequester, chelate

17. Detergents Removes biological and non-biological stains
Removes all classes of organic molecules
What is found in detergents?
Soap - structure
Problems
Safety allergic reactions from microbial enzymes?
Oxidation caused by bleach
met and cys are sensitive
protein engineering

18. Homework assigment
What is best protease to use in detergents?
Why?

19. Protease for Cheese manufacture
Rennin (Chymosin) is a aspartic protease
coagulation of milk
cleave casein to form curds (micelles)
Preprorennin
Prorennin
Rennin (active)
First food product produced by rDNA technology approved for human use (1990)

20. Meat Tenderizers Collagen makes meat tough
Younger animal less cross linking of collagen
Papain -cysteine protease
degrades muscle fiber and connective proteins
active at high temperature (cooking)

21. Other applications of protease
Aspartame
Phenylalanine and aspartic acid
Metalloprotease actually forms peptide bonds rather than breaks them
Contact Lens Solutions
to remove proteins in tears
lysozyme, antibodies

22. Application of Carbohydrases
Cellulase - hydrolyzes cellulose
Cellulose is a polysaccharide (glucose units)
Stonewashing genes
break down fabric (which is cellulose)
releases dye

23. Enzymes use in Molecular biology
1. Restriction endonucleases
defense
specific sequence
methylation
blunt vs staggered
4, 6, 8 base cutters
2. Ligase catalyze formation of bonds of nucleic acids (DNA)
3. DNA polymerase
taq
Deep Vent DNA polymerase