Transcript Strain Improvement - Bharathiar University


Enzymes are proteins specialized to catalyze biological
reactions.

Most remarkable biomolecules due to their extraordinary
specificity and catalytic power

Far greater than those of man-made catalysts

Overall global value of industrial enzymes is about $2.0
billion – 2004 is expected to rise at an average annual
growth rate (AAGR) of 3.3% to $2.4 billion in 2009
Report ID:BIO030D, Published: December 2004, Analyst: Yatin Thakore
Enzymes are obtained from
 Plant source – Papain, ficin
 Animal source – Rennet
 Microbial source – amylase, proteases,
cellulase, xylanase etc.,



Over 2000 different enzymes are available and only few are used commercially.
Commercial enzymes include enzymes from plant, animal and microbial
sources.
From 1960s microbial source is preferred for several reasons,
Economical – produced on large scale
within limited space and time.
Can be easily extracted and purified.
Can grow in a wide range of environmental conditions
Capable of producing a wide variety of enzymes.
They can be genetically manipulated to increase the yield
of enzymes
Application
Enzymes
Uses
Food processing
Amylase, protease
To produce sugars and to digest the proteins
in flour
Baby foods
Trypsin
To predigest baby foods
Brewing industry
Amylase, glucanases, proteases,
Acetolactatedecarboxylase (ALDC)
To degrade proteins and polysaccharides,
improve the wort and fermentation process,
increase the flavour.
Fruit juices
Dairy industry
Meat tenderizers
Cellulases, pectinases
Rennin, lipase, lactase
papain
Clarify fruit juices
Production of cheese and other diary products
To soften meat for cooking.
Starch industry
Amylase, glucoisomerases
Convert starch into glucose and simple sugars
Paper industry
Amylase, cellulase, xylanase, ligninases
Degrade starch, aid in sizing, decolorizing,
soften the paper
Biofuel industry
Cellulase, xylanase, ligniniase, lipase
Production of ethanol and biodiesel
Detergent industry
Amylase, protease, lipase, cellulase
Remove starch, protein and lipid stains and as
fabric conditioner
Photographic industry
ficin
Dissolve gelatin off scrap film, allowing
recovery of its silver content.
Stages involved in commercial production
of enzymes.
1.
Isolation of microbes.
2.
Screening of microbes.
3.
Fermentation.
4.
Increase the yield of the enzymes.
The yield has to be increased in order to
minimize the production cost. This can be
done by,
(i) developing a suitable medium for
fermentation
(ii) refining the fermentation process and
(iii)improving the strain for higher production.
The potential productivity of the organisms is
controlled by its genes and hence their genome
must be altered for the maximum production of
enzymes.
 The techniques involved are

* Mutations
* Recombination – Protoplast fusion
* Recombinant DNA technology
One of the most successful approaches for strain
improvement.
 A mutation is any change in the base sequence
of DNA - deletion, insertion, inversion,
substitution.
 The types include
- Spontaneous mutation
- Induced mutation
- Site directed mutation

1.Spontaneous mutation:


Occur spontaneously at the rate of 10-10 and 10-15 per generation and per
gene.
Occur at low frequency and hence not used much in industrial strain
improvement.
2. Induced mutation:
 The rate of mutation can be increased by various factors and agents called
mutagens.
 ionizing radiations (e.g. X-rays, gamma rays)
 non-ionizing radiations (e.g. ultraviolet radiations)
 various chemicals (e.g. mustard gas, benzene, ethidium bromide,
Nitrosoguanidine-NTG)
3. Site directed mutations(SDM) (site-specific
mutagenesis ):

Change in the base sequence of DNA

changing the codon in the gene coding for that amino acid.

Can be done by protein engineering method

Desired improvements might be
*increased thermostability
*altered substrate range
*reduction in negative feedback inhibition
*altered pH range, etc.,
Isolate required enzyme gene, e.g. via mRNA and its conversion into cDNA.
Sequence the DNA of the gene (in order to decide on change required for primer in stage 5).
Splice gene into M13 vector dsDNA and transduce E. coli host cells.
Isolate ssDNA in phage particles released from host cells.
Synthesize an oligonucleotide primer with the same sequence as part of the gene but with altered codon
(mismatch/mispair) at desired point(s).
For example, one of the codons in DNA coding for the amino acid Alanine is CGG. If the middle base
is changed by SDM from G to C the codon sequence becomes CCG which codes for a different
amino acid (Glycine).
Mix oligonucleotide with recombinant vector ssDNA.
Carried out at low temperature (0-10oC) and in high salt concentration to allow
hybridization between oligonucleotide and part of gene.
Use DNA polymerase to synthesize remainder of strand. (Oligonucleotide acts as a
primer for the DNA synthesis). Then add ligase to join primer and new strand
dsDNA molecule.
Transform E. coli cells and allow them to replicate recombinant vector molecule.
DNA replication is semi-conservative, therefore two types of clone are produced each
of which excretes phage particles containing ssDNA:
 Type 1: contain the wild-type gene (i.e. unaltered)
 Type 2: contain the mutated gene!!!
Karana and Medicherla (2006)- lipase from
Aspergillus japonicus MTCC 1975- mutation
using UV, HNO2, NTG showed 127%, 177%,
276% higher lipase yield than parent strain
respectively
 Sandana Mala et al., 2001- lipase from
A.
niger - Nitrous acid induced mutation – showed
2.53 times higher activity.

Protoplast fusion depends on the following criteria,
 Lytic enzyme
 Osmotic stability
 Age of the mycelia
 Inoculation period
 Regeneration medium
 Regeneration frequency
 PEG concentration
 Osmotic stability
 Fusant formation

Intraspecific hybridization
Interspecific hybridization
Intergeneric hybridization
o
The organism selected for fusion should be genetically related.
o
As the distance increases in genetic relationship between the
two mating isolates, the less successful protoplast fusion will
be (Anne and Peberdy, 1976).


Kim et al., 1998 did a comparative study on
strain improvement of Aspergillus oryzae for
protease production by both mutation and
protoplast fusion.
 UV radiation – 14 times higher yield.
 Ethyl methanesulphonate – 39 times higher
yield.
 Protoplast fusion – using PEG and CaCl2 – 82
times higher yield.

The more advanced method
 to increase the yields and consistencies of enzymes.
 Genetic material derived from one species may be
incorporated into another where it is expressed
 Increases the production of heterologous proteins by
- increasing the gene expression using
strong promoters
- deletion of unwanted genes from the
genome
- manipulation of metabolic pathways.

to get multiple copies of specific gene
2. to get high amounts of specific protein or
product
3. to integrate gene of interest of one organism
into another
1.
Steps involved:

Preparation of desired DNA

Insertion of desired DNA into vector DNA

Introduction of recombinant DNAs into host
cells

Identification of recombinants

Expression of cloned genes
Sidhu et al., 1998 tried both mutagenesis and
cloning in E.coli for increased production by
amylase in Bacillus sp. MK716.
Mutation – ethyl methane sulphonate –
40 times higher.
Mutated gene – cloned in E.coli pBR322107 times higher yield than
parent strain.
 Calado et al., 2004 – cutinase enzyme –
Arthrobacter simplex - 205 fold higher.


Genetic instability

Genetical information of the industrially
employed organism is unknown

Costlier than other methods
The task of both discovering new microbial
compounds and improving the synthesis of
known ones have become more and more
challenging.
 Newer genetic methods have been developed to
obtain higher yields.
 The basic genetic information for all the
organisms used industrially is not available
 The steps have been taken by firms in order to
gap the bridge between basic knowledge and
industrial application.


Raw material: Eucalyptus grandis wood chips,
Bamboo, sugarcane bagasse etc.

Chemical component: Lignin, Hemicellulose,
Cellulose

Lignin - Heterocyclic phenolic polymer.

Hemicellulose - Polymer of pentoses.

Cellulose - Polymer of hexoses(glucose)

Needed component - Cellulose

Unwanted – Lignin, Hemicellulose

Pulping and bleaching

Pulping: Wood chips cooked in alkali(kraft
pulping) or Sodium sulfite (sulfite pulping)
removal of Lignin. Lignin content measured in
terms of Kappa number

Bleaching: Alkali or Chlorine bleaching.
Removal of hemicellulose measured in terms of
brightness.

Important Parameters: Brightness, Tensile
strength, Tearing ability etc.

Cooking requires high amount of heat energy.

Cost of the chemicals and recycling of waste
papers – not economical.

Environmental problem due to black liquor
(lignin related compounds) and
Organochlorines
Hence need for alternative methodology
 White rot
Basidiomycetous fungi  Brown rot
 Soft rot
White rot fungi posses the enzymes needed for
degradation of lignin compounds :
Enzymes- Lignin peroxidase(LiP),
Manganase peroxidase(MnP), Laccase
Aryl alcohol oxidase(AAO),Other polyphenol
oxidases
1.
Isolation
2.
Surface sterilised plant parts showing
fructification.
3.
Medium for growth
4.
2% Malt agar medium
5.
Enzyme production medium
6.
C - Limited medium of Janshekar and
Feichter (1988)
 Dye
degradation (Poly R dye, Ramazole
brilliant blue)
 Utilization
of Lignin amended in synthetic
medium
 Production
of ethylene from KTBA (2-keto-4
thiomethyle butyric acid)

Incubation period
•
pH, Temperature
•
Carbon
•
Nitrogen sources

Purification of enzymes

FPLC (Fast protein liquid chromatograpy)
1.
Delignification and bleaching of hardwood
kraft pulp(HWKP)
2.
Deinking of waste papers
3.
Dechlorination of chloroaromatics
4.
Paper mill effluent treatment
•
Environmental requirements during
pretreatments
•
Sterility conditions
•
Necessary exposure times.
Isolation of protoplasts
(fungal culture four days old )
500mg of mycelium were washed in sterile water and osmotic stabilizer
(0.6M KCl in sequence)
The mycelia were then incubated in 5ml of 0.6M KCl with 5mg/ml of
Novozyme for 3 h at room temp
Release of protoplasts was checked at every 30 min interval
Centrifuge the protoplasts(300rpm for 3 min.)
Discard supernatant to remove the mycelial debris
The protoplasts pellet was suspended in osmotic stabiliser (0.6M KCL) 2ml
The protoplasts were viable and was observed using Light microscope
Fusion treatment
0.6M KCl centrifuged for 10min. At 700rpm at 4º C
From the above 10l of protoplasts were suspended in 100 l of PEG(4000-600030% w/v), with 10mM CaCl2 and 50mM Glycine buffer (pH 5.8) ,and incubate 20ºC
for 10min. diluted with 5ml of osmotic stabiliser .
Centrifuge for 700rpm for 10 min.
To the pellet added 50 l of osmotic stabiliser ,plated in the regeneration medium minimal medium and complete medium
Table 3
Regeneration and Complementation frequencies in the cross
Tramates versicolor and Polyporus leucospongia
P. leucospongia
T. versicolor
Mixed protoplasts of two
strains
Regeneration on RCM
Before PEG treatment
After PEG treatment
Regeneration on RMM
BeforePEG treatment
After PEG treatment
Complementation
Frequency (Fc)
30.4%
10 %
20.6%
8.2%
6.10%
0
0
0
0
0
2%
Fc = 2.00 = 0.33%
6.10
RCM-regeneration on complete medium
RMM-regeneration on minimal medium
The frequency of fusion was determined as the ratio between the number of colonies formed on minimal
medium and colonies formed on the same medium
Tramates versicolor
Polyporus leucospongia
Organism
Molecular weight
(Kda)
(3 isoenzymes)
Laccase production
P. leucospongia
64-39
13.8
T. versicolor
68-36
9.0
Fusant
66-39
18.7
(IU/mL/min)
Molecular weight
Xylanase production
(KDa)
(IU/mL/min)
Aspergillus wentii
25
62.25
A. indicus
36
70.62
Fusant
29
91.42
Organism

Intergeneric hybridization between Graphium putredinis
and Trichoderma harzianum

It was done to enhance the production of industrially
important hydrolytic enzymes like cellulase, xylanase,
amylase and protease.

Morphological study, protein profiling, restriction
digestion pattern and RAPD analysis was carried out.
Enzyme production
(IU/mL)
G. putredinis
T. harzianum
Fusant
Amylase
10.52
8.17
12.39
Cellulase
2.89
5.35
7.46
Xylanase
153.22
148.35
161.50
Protease
0.35
0.32
0.38