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Enzymes are proteins specialized to catalyze biological reactions. Most remarkable biomolecules due to their extraordinary specificity and catalytic power.

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Presentation on theme: "Enzymes are proteins specialized to catalyze biological reactions. Most remarkable biomolecules due to their extraordinary specificity and catalytic power."— Presentation transcript:

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2 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

3 Report ID : BIO 030 D, Published : December 2004, Analyst : Yatin Thakore

4 Enzymes are obtained from Plant source – Papain, ficin Animal source – Rennet Microbial source – amylase, proteases, cellulase, xylanase etc.,

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6 ApplicationEnzymesUses Food processing Amylase, proteaseTo produce sugars and to digest the proteins in flour Baby foods TrypsinTo 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, pectinasesClarify fruit juices Dairy industry Rennin, lipase, lactaseProduction of cheese and other diary products Meat tenderizers papainTo soften meat for cooking. Starch industry Amylase, glucoisomerasesConvert starch into glucose and simple sugars Paper industry Amylase, cellulase, xylanase, ligninasesDegrade starch, aid in sizing, decolorizing, soften the paper Biofuel industry Cellulase, xylanase, ligniniase, lipaseProduction of ethanol and biodiesel Detergent industry Amylase, protease, lipase, cellulaseRemove starch, protein and lipid stains and as fabric conditioner Photographic industry ficinDissolve gelatin off scrap film, allowing recovery of its silver content.

7 Stages involved in commercial production of enzymes. 1. Isolation of microbes. 2. Screening of microbes. 3. Fermentation. 4. Increase the yield of the enzymes.

8 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.

9 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

10 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

11 1.Spontaneous mutation: Occur spontaneously at the rate of and 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)

12 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.,

13 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).

14 Mix oligonucleotide with recombinant vector ssDNA. Carried out at low temperature (0-10 o C) 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!!!

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16 Karana and Medicherla (2006)- lipase from Aspergillus japonicus MTCC mutation using UV, HNO 2, NTG showed 127%, 177%, 276% higher lipase yield than parent strain respectively Sandana Mala et al., lipase from A. niger - Nitrous acid induced mutation – showed 2.53 times higher activity.

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18 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

19 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).

20 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.

21 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.

22 1. 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

23 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

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25 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 pBR times higher yield than parent strain. Calado et al., 2004 – cutinase enzyme – Arthrobacter simplex fold higher.

26 Genetic instability Genetical information of the industrially employed organism is unknown Costlier than other methods

27 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.

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29 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

30 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.

31  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

32  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

33 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)

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39  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)

40 Incubation period pH, Temperature Carbon Nitrogen sources Purification of enzymes FPLC (Fast protein liquid chromatograpy )

41 1. Delignification and bleaching of hardwood kraft pulp(HWKP) 2. Deinking of waste papers 3. Dechlorination of chloroaromatics 4. Paper mill effluent treatment

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43 Environmental requirements during pretreatments Sterility conditions Necessary exposure times.

44 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

45 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( % w/v), with 10mM CaCl 2 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

46 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 30.4% 10 % 20.6% 8.2% 6.10% Regeneration on RMM BeforePEG treatment After PEG treatment % Complementation Frequency (Fc) 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

47 Tramates versicolorPolyporus leucospongia

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49 Organism Molecular weight (Kda) (3 isoenzymes) Laccase production (IU/mL/min) P. leucospongia T. versicolor Fusant

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52 Organism Molecular weight (KDa) Xylanase production (IU/mL/min) Aspergillus wentii A. indicus Fusant

53 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.

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59 Enzyme production (IU/mL) G. putredinisT. harzianumFusant Amylase Cellulase Xylanase Protease

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