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 fromPlant source – Papain, ficinAnimal source – RennetMicrobial source – amylase, proteases,

cellulase, xylanase etc.,

Application Enzymes UsesFood 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 Cellulases, pectinases Clarify fruit juices

Dairy industry Rennin, lipase, lactase Production of cheese and other diary products

Meat tenderizers papain 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 enzymeOsmotic stabilityAge of the myceliaInoculation periodRegeneration mediumRegeneration frequencyPEG concentrationOsmotic stabilityFusant 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.

1. to get multiple copies of specific gene

2. to get high amounts of specific protein or

product3. to integrate gene of interest of one organism

into another

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 pBR322- 107 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 10l of protoplasts were suspended in 100 l of PEG(4000-6000-30% 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 3Regeneration 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 

  

30.4%10 %

  

20.6%8.2%

   

6.10%Regeneration on RMMBeforePEG treatment After PEG treatment

 00

 00

 0

2%

Complementation Frequency (Fc)

 Fc = 2.00 =

0.33%6.10

   

 

RCM-regeneration on complete mediumRMM-regeneration on minimal mediumThe 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

OrganismMolecular weight

(Kda)(3 isoenzymes)

Laccase

production

(IU/mL/min)

P. leucospongia 64-39 13.8

T. versicolor 68-36 9.0

Fusant 66-39 18.7

OrganismMolecular weight

(KDa)

Xylanase

production

(IU/mL/min)

Aspergillus wentii 25 62.25

A. indicus 36 70.62

Fusant 29 91.42

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