1. Industrial Enzymes BIT 230

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 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 Mesophiles35-60 o C pH 4-8 Extremophilesorganisms that colonize niches with one or more extreme environmental parameters pH temp salinity pressure

10. Hyperthermophiles 90 o C 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 0 o C Antifreeze proteins- prevent ice crystal formation

13. Others Thermoacidophiles Barophiles Halophiles Alkaliphiles Acidophiles

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