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Immobilization Tech- Durgashree Diwakar
The document discusses immobilization techniques and their application in the production of secondary metabolites from plant cells, highlighting various methods such as adsorption, covalent bonding, entrapment, and encapsulation. It also explores cloning methods for plant cells, the advantages and disadvantages of cloning, and the role of elicitors in enhancing metabolite production. The document emphasizes the importance of these techniques in improving the efficiency and yield of valuable compounds in biotechnology.
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Immobilization Tech- Durgashree Diwakar
- 1. IMMOBILIZATION TECHNIQUE & SECONDARY METABOLITEPRODUCTION BY- DURGASHREE M D MEDICINAL PLANT BIOTECHNOLOGY M PHARM, DEPT OF PHARMACOGNOSY KLE COLLEGE OF PHARMACY, BENGALURU 1
- 2. CONTENT • IMMOBILIZATION TECHNIQUESOF PLANT CELL • APPLICATION OF IT IN SECONDARY METABOLITE PRODUCTION • CLONING OF PLANT CELLS: DIFFERENT METHODS AND APPLICATIONS • ADVANTAGES AND DISADVANTAGES OF PLANT CELL CLONING • SECONDARY METABOLISM IN TISSUE CULTURE • PRECURSOR AND ELICITOR OF PRODUCTION OF SEC. METABOLITES. 2
- 3. INTRODUCTION • Immobilization is"the imprisonment of an enzyme in a distinct phase that allows exchange with, but is separated from the bulk phase in which the substrate, effector or inhibitor molecules are dispersed and monitored" 3
- 4. ADVANTAGES OF USING IMMOBILIZEDENZYMES • Continuous use • less labor • Minimum reaction time • Less chance of contamination in products, • Improved process control and • High enzyme: substrate ratio. 4
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- 6. IMMOBILIZATION TECHNIQUES FOR PLANTCELL • Plants are natural sources of valuable secondary metabolites used in pharmaceuticals, agrochemicals, the food industry, etc. • There is an increasing demand to obtain these metabolites through more productive plant tissue applications and cell culture methods. 6
- 7. IMMOBILIZATION TECHNIQUES FOR PLANTCELL • Immobilization of plant cells is a method used in plant cell cultures to induce secondary metabolite production. • In this method, plant cells are fixed in or on a supporting material or matrix such as agar, agarose, calcium alginate, glass, or polyurethane foam4. 7
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- 9. ADSORPTION • An enzymemay be immobilized by binding to either the external or internal surface of a carrier or support. • Bonds of low energy are involved e.g. Ionic interactions, hydrogen bonds, van der Waals forces, etc. • Types of absorption methods a. Static process b. Dynamic batch process c. Reactor loading process 9
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- 11. COVALENT BONDING • Covalentbond is formed between the chemical groups of enzyme and chemical groups on the surface of the carrier. • Covalent bonding is thus utilized under a broad range of pH, ionic strength, and other variable conditions. • Immobilization steps are the attachment of the coupling agent followed by an activation process or attachment of a functional group, and finally attachment of the enzyme 11
- 12. • Carriers areused in immobilization such as carbohydrates proteins and amine-bearing carriers, inorganic carriers, etc. • Covalent attachment may be directed to a specific group (e.g. amine, hydroxyl, tyrosyl, etc.) on the surface of the enzyme. • Hydroxyl and amino groups are the main groups of the enzymes with which it forms bonds, whereas the sulphydryl group is the least involved. 12
- 13. DIFFERENT METHODS OFCOVALENT BONDING a. Diazoation (bonding between the amino group of the support e.g. Aminobenzyle cellulose ), b. Formation of the peptide bond (bond formation between the amino or carboxyl group of the support and amino or carboxy group of the enzyme), c. Group activation (use of cyanogen bromide to support containing glycol group i.e. Cellulose, syphadex, sepharose, etc), d. Polyfunctional reagents (use of a bifunctional or multifunctional reagent e.g. Glutaraldehyde which forms bonding between the amino group of the support and amino group of the enzyme). 13
- 14. ENTRAPMENT • Enzymes canbe physically entrapped inside a matrix (support) of a water-soluble polymer such as polyacrylamide type gels and naturally derived gels • e.g. Cellulose triacetate, agar, gelatin, carrageenan, alginate, etc. • The form and nature of the matrix vary. • Pore size of the matrix should be adjusted to prevent the loss of enzyme from the matrix due to excessive diffusion. 14
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- 16. CROSS-LINKING/CO- POLYMERIZATION • Cross-linking ischaracterized by covalent bonding between the various molecules of an enzyme via a polyfunctional reagent such as glutaraldehyde, diazonium salt, hexamethylene disocyanate, and N-N' ethylene bismaleimide. • The demerit of using polyfunctional reagents is that they can denature the enzyme. • This technique is cheap and simple but not often used with pure proteins because it produces very little of immobilized enzyme that has very high intrinsic activity. • It is widely used in commercial preparation 16
- 17. ENCAPSULATION • Encapsulation isthe enclosing of a droplet of solution-of enzyme in a semipermeable membrane capsule. • The capsule is made up of cellulose nitrate and nylon. • The method of encapsulation is cheap and simple but its effectiveness largely depends on the stability of enzyme although the catalyst is very effectively retained within the capsule. 17
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- 20. IMMOBILIZATION OF CELLS •In cell immobilization –the main important feature is that enzymes are active and stable for a long period. • All cells constitute will be in the cellular domain in the resting state. • The methods of whole-cell immobilization are the same as described for enzyme immobilization 20
- 21. Support material CellsReaction A. Adsorption Gelatin Lactobacilli Lactose/lactic acid Porous glass Saccharomyces carlsbergensis Glucose/ethanol Cotton fibers Zymomonas mobilis Glucose/ethanol Vermiculite Z. mobilis Glucose/ethanol DEAE-cellulose Nocardia erythropolis Steroid conversion B. Covalent Bonding Cellulose + cyanuric chloride S.cerevisiae Glucose/ethanol Ti (IV) oxide, etc. Acetobacter sp. Wort/vinegar Carboxymethylcellulose + carbodiimide Bacillus subtilis L-histidine/uronic acid C. Crosslinking of cell-to-cell Diazotized diamines Streptomyces Glucose/fructose Glutaraldehyde E.coli Fumaric acid/L-aspartic acid Flocculation by chitosan Lactobacillus brevis Glucose/fructose D. Entrapment Al alginate Candida tropicalis Phenol degradation Ca alginate S. cerevisiae Glucose/ethanol Mg pectinate Fungi Glucose/fructose K-carrageenan E.coli Fumaric acid/L-spartic acid Chitosan alginate S. cerevisiae Glucose/ethanol E. Encapsulation Cellulose acetate Comamonas sp. 7-ACA production Ethylcelhilose Streptomyces sp. Glucose/fructose Polyester Streptomyces sp. Glucose/fructose Alginate-polylysine Pancreas cells - Alginate-polylysine Hybridoma cells Monoclonal antibodies 21
- 22. APPLICATION IN PRODUCTIONOF SECONDARY METABOLITES 22
- 23. PRODUCTION OF SEC.METABOLITE- CAPSAICIN • Immobilized cells of capsicum frutescens were found to produce more capsaicin as compared to the cells in suspension under similar conditions. • Using gel entrapment method and even by using elicitor. 23
- 24. • Any materialwhich when introduced in a cell in culture, increases the production of a particular compound, is called elicitor and the process is known as elicitation. • Biotic elicitor: cellulose, pectin, etc • Abiotic elicitor: pH, Cu2+, Cd2+, etc. 24
- 25. CLONING OF PLANTCELL • Plant cloning is the production of a cell, cell component, or plant that is genetically identical to the unit or individual from which it was derived. • Cells from meristems can be cloned • These cells can be removed from a plant and grown in tissue culture. 25
- 26. DIFFERENT METHODS OF CLONING •Restriction enzyme-based cloning. • PCR cloning. • Ligation independent cloning (lic) • Seamless cloning (sc). • Recombinational cloning. 26
- 27. 1. Restriction enzyme-based cloning Shortsequences containing restriction sites are added into the 5’ ends of primers for DNA amplification by PCR. Both the vector and DNA fragment are digested with restriction enzymes to create cohesive ends. The vector and DNA fragment are ligated. The recombinant DNA enters the 27
- 28. 2. PCR Cloning PCRproduct with a-tailed ends is combined with t-tailed vector. During ligation, pcr product is inserted into the vector. 28
- 29. 3. Ligation-independent cloning Shortsequences which match with sequences on the plasmid are added into the 5’ ends of primers for DNA amplification by PCR. Plasmid is linearized by using a restriction enzyme. Treated with 3’ to 5’ exonuclease to create cohesive overhangs. Both DNA and vector are annealed. After transformation, the host cell repairs the nicks on the recombinant DNA. 29
- 30. 4. Seamless cloning Shortsequences are added into the 5’ ends of primers for DNA amplification by PCR. Vector is digested by a restriction enzyme. Both DNA fragments and vector are treated with an enzyme with 5’ to 3’ exonuclease activity to create cohesive overhangs. During ligation, the DNA fragment is inserted into the vector. • 30
- 31. 5. Recombinational cloning DNAfragment is inserted into an entry vector to create an entry clone. Entry clone and destination vector are combined by a recombinase enzyme to create a destination clone6. 31
- 32. ADVANTAGES AND DISADVANTAGEOF PLANT CELL CLONING Advantages •It is quick •The stock created is disease-free •All the plants have the same, known phenotype •You can reproduce infertile plants •You can reproduce plants that are hard to grow from seed •You can create whole plants from genetically modified cells •They can be grown at any time, in any season, anywhere in the world •The plantlets are small so they can be transported easily and grown in small spaces •You can save rare/endangered species from extinction Disadvantages •It is an expensive and labour intensive process •The process can fail due to microbial contamination •There is no genetic variation •All of the offspring are susceptible to the same diseases or other environmental factors 7 . 32
- 33. APPLICATION OF CLONINGOF PLANT CELL • To produce identical plants quickly and economically. • Genetic engineering • Gene studies • Other applications in plant research • Both in discovery and applied settings. 33
- 34. SECONDARY METABOLISM IN TISSUECULTURE • Secondary metabolites (SMS) are generally defined as small organic molecules produced by an organism that is not essential for its growth, development, and reproduction. • Ex: toxins, gibberellins, alkaloids, antibiotics, and biopolymers, etc. 34
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- 39. Sec. metabolite fromPicrorhiza kurroa by Tissue culture 39
- 40. Sec. metabolite- artemisininproduction by Tissue culture 40
- 41. EFFECT OF PRECURSORAND ELICITOR IN PRODUCTION OF SECONDARY METABOLITE Precursor: a substance, cell, or cellular component from which another substance- cell/ cellular component/ Metabolite/ product is formed. Elicitor: An elicitor can be defined as a chemical or biochemical compound that is introduced in small concentrations to a living system to promote the biosynthesis 41
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- 43. Elicitor • Abiotic elicitorscomprise substances that are of nonbiological origin and are grouped in physical, chemical, and hormonal factors. • Biotic elicitors are the substances of biological origin that include polysaccharides originating from plant cell walls and micro–organisms. 43
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- 45. L-dopa production insuspension cultures of Mucuna pruriens L. • Methyl jasmonate, pectin, and yeast extract were dissolved in sterile distilled water • Chitin was solubilized in 1 or 2 drops of 50% H2SO4 and • L-tyrosine was prepared by dissolving initially with 1 or 2 drops of 1 N NaOH. • Later both chitin and tyrosine solutions were diluted with sterile double distilled water to get the final concentrations • Ph was adjusted to 5.8 45
- 46. REFERENCES 1. Pharmaceutical biotechnology-Vyas and Dixit 2. Elements of biotechnology- P K Gupta 3. A textbook of biotechnology- R C Dubey 4. Immobilization of Rubia tinctorum L. Suspension cultures and biomass production- pinar nartop. (https://pubmed.ncbi.nlm.nih.gov/27108315/#:~:text=Immobilization %20of%20plant%20cells%20is,%2C%20glass%2C%20or%20poly urethane%20foam). 5. In vitro production of capsaicin through plant tissue culture. (https://www.researchgate.net/publication/324020808_In_vitro_pro duction_of_capsaicin_through_plant_tissue_culture) 6. A quick overview in molecular cloning. (https://www.goldbio.com/articles/article/cloning-overview) 46
- 47. REFERENCES 7. Plant cloningadvantages and disadvantages. (https://getrevising.co.uk/grids/plant-cloning-advantages-and- disadvantages) 8. Elicitors and precursor induced effect on l-dopa production in suspension cultures of mucuna pruriens l. (https://www.tandfonline.com/doi/full/10.1080/21553769.2011.6491 88) 47
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