industrial enzymes bit 230. introduction industrial vs medicinal/therapeutic hydrolytic...
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Industrial Enzymes
BIT 230
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Introduction
Industrial vs Medicinal/Therapeutic
Hydrolytic depolymerases
Types
$1.5 billion industry
Detergents No 1
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Classes
Lipases
Proteases
Carbohydrases
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Non-recombinant Sources
GRAS
Bacillusprotein is secreted into fermentation mediumeasier purification
Aspergillus
Yeast
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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
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Protein engineering
Make oxidation resistant
make enzymes tolerant of processes used in industry
less substrate specificity
more thermostable
more stable in presence of detergent
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Mesophiles vs Extremophiles
Mesophiles 35-60oCpH 4-8
Extremophiles organisms that colonize niches with one or more extremeenvironmental parameterspHtempsalinitypressure
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Hyperthermophiles
90oC plus
typically anaerobic
found at tectonically active sitesventsspringsgeysers
enzymes are beneficialsubstrate more susceptiblediscourages microbial
growth
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Hyperthemophiles
Difficult to culture
Move to recombinant production of their enzymes
Purification easier -Raise temp
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Psycrophiles
Extreme Low temperatures 0oC
Antifreeze proteins- prevent ice crystal formation
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Others
Thermoacidophiles
Barophiles
Halophiles
Alkaliphiles
Acidophiles
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Proteolytic Enzymes
Largest group of Industrial Enzymes (50%)
Function - degrade proteins
What is hydrolysis?
Don’t want absolute specificity. Why??
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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)
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Divalent Ions
CalciumMagnesionIron
These make water hard!Cause precipitation of soapsNeed to sequester, chelate
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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
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Homework assigment
What is best protease to use in detergents?
Why?
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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)
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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)
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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
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Application of Carbohydrases
Cellulase - hydrolyzes cellulose
Cellulose is a polysaccharide (glucose units)
Stonewashing genesbreak down fabric (which is cellulose)releases dye
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Enzymes use in Molecular biology
1. Restriction endonucleasesdefensespecific sequencemethylationblunt vs staggered4, 6, 8 base cutters
2. Ligase catalyze formation of bonds of nucleic acids (DNA)
3. DNA polymerasetaqDeep Vent DNA polymerase