Industrial Enzymes

BIT 230

Introduction

n Industrial vs Medicinal/Therapeutic

n Hydrolytic depolymerases

n Types

n $1.5 billion industry

n Detergents No 1

Classes

n Lipases

n Proteases

n Carbohydrases

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

Psycrophiles

Extreme Low temperatures 0oC

Antifreeze proteins- prevent ice crystal formation

Others

Thermoacidophiles

Barophiles

Halophiles

Alkaliphiles

Acidophiles

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

Homework assigment

What is best protease to use in detergents?

Why?

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

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