Post on 10-Apr-2019
Introduction
n Industrial vs Medicinal/Therapeutic
n Hydrolytic depolymerases
n Types
n $1.5 billion industry
n Detergents No 1
Non-recombinant Sources
GRAS
Bacillusprotein is secreted into fermentation mediumeasier purification
Aspergillus
Yeast
Recombinant Sources
Most industrial enzymes are produced recombinantly
Why?A. Higher expressionB. Higher purity (%protein:other junk)C. cheapD. can engineer proteinE. can express enzymes which are found
in pathogenic organisms
Heterologous Protein Expression
Homologous Protein Expression
Protein engineering
Make oxidation resistant
make enzymes tolerant of processes used in industry
less substrate specificity
more thermostable
more stable in presence of detergent
Mesophiles vs Extremophiles
Mesophiles 35-60oCpH 4-8
Extremophiles organisms that colonize niches with one or more extremeenvironmental parameterspHtempsalinitypressure
Hyperthermophiles
90oC plus
typically anaerobic
found at tectonically active sitesventsspringsgeysers
enzymes are beneficialsubstrate more susceptiblediscourages microbial growth
Hyperthemophiles
Difficult to culture
Move to recombinant production of their enzymes
Purification easier -Raise temp
Proteolytic Enzymes
Largest group of Industrial Enzymes (50%)
Function - degrade proteins
What is hydrolysis?
Don’t want absolute specificity. Why??
Classifications
Site of HydrolysisA. Endopeptidase
cleaves peptide bonds internallyB. Exopeptidase
cleaves peptides at C or N term
Mechanisms - based on active site (on enzyme)Serine proteasesCysteine protease (cys and his)Asparatic proteasesMetalloprotease (need metal ions, Ca, Mg)
Divalent Ions
CalciumMagnesionIron
These make water hard!Cause precipitation of soapsNeed to sequester, chelate
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 bleachmet and cys are sensitiveprotein engineering
Protease for Cheese manufacture
Rennin (Chymosin) is a aspartic protease
coagulation of milkcleave casein to form curds (micelles)
PreprorenninProrennin
Rennin (active)
First food product produced by rDNA technology approved for human use (1990)
Meat Tenderizers
Collagen makes meat tough
Younger animal less cross linking of collagen
Papain -cysteine proteasedegrades muscle fiber and connective proteinsactive at high temperature (cooking)
Other applications of protease
Aspartame
Phenylalanine and aspartic acid
Metalloprotease actually forms peptide bonds rather than breaks them
Contact Lens Solutionsto remove proteins in tearslysozyme, antibodies
Application of Carbohydrases
Cellulase - hydrolyzes cellulose
Cellulose is a polysaccharide (glucose units)
Stonewashing genesbreak down fabric (which is cellulose)releases dye