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Industrial Enzymes. BIT 230. Introduction. Industrial vs Medicinal/Therapeutic Hydrolytic depolymerases Types $1.5 billion industry Detergents No 1. Classes. Lipases Proteases Carbohydrases. Non-recombinant Sources. GRAS Bacillus protein is secreted into fermentation medium

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Presentation Transcript
introduction
Introduction
  • Industrial vs Medicinal/Therapeutic
  • Hydrolytic depolymerases
  • Types
  • $1.5 billion industry
  • Detergents No 1
classes
Classes
  • Lipases
  • Proteases
  • Carbohydrases
non recombinant sources
Non-recombinant Sources

GRAS

Bacillus

protein is secreted into fermentation medium

easier purification

Aspergillus

Yeast

recombinant sources
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

protein engineering
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 vs Extremophiles

Mesophiles 35-60oC

pH 4-8

Extremophiles organisms that colonize niches with

one or more extreme

environmental parameters

pH

temp

salinity

pressure

hyperthermophiles
Hyperthermophiles

90oC plus

typically anaerobic

found at tectonically active sites

vents

springs

geysers

enzymes are beneficial

substrate more susceptible

discourages microbial growth

hyperthemophiles
Hyperthemophiles

Difficult to culture

Move to recombinant production of their enzymes

Purification easier -Raise temp

psycrophiles
Psycrophiles

Extreme Low temperatures 0oC

Antifreeze proteins- prevent ice crystal formation

others
Others

Thermoacidophiles

Barophiles

Halophiles

Alkaliphiles

Acidophiles

proteolytic enzymes
Proteolytic Enzymes

Largest group of Industrial Enzymes (50%)

Function - degrade proteins

What is hydrolysis?

Don’t want absolute specificity. Why??

classifications
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)

divalent ions
Divalent Ions

Calcium

Magnesion

Iron

These make water hard!

Cause precipitation of soaps

Need to sequester, chelate

detergents
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

homework assigment
Homework assigment

What is best protease to use in detergents?

Why?

protease for cheese manufacture
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)

meat tenderizers
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)

other applications of protease
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

application of carbohydrases
Application of Carbohydrases

Cellulase - hydrolyzes cellulose

Cellulose is a polysaccharide (glucose units)

Stonewashing genes

break down fabric (which is cellulose)

releases dye

enzymes use in molecular biology
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