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" 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.

1. IMMOBILIZATION
TECHNIQUE & SECONDARY
METABOLITE PRODUCTION
BY- DURGASHREE M D
MEDICINAL PLANT BIOTECHNOLOGY
M PHARM, DEPT OF PHARMACOGNOSY
KLE COLLEGE OF PHARMACY, BENGALURU
1

2. CONTENT
• IMMOBILIZATION TECHNIQUES OF 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
IMMOBILIZED ENZYMES
• Continuous use
• less labor
• Minimum reaction time
• Less chance of contamination in products,
• Improved process control and
• High enzyme: substrate ratio.
4

5. 5

6. IMMOBILIZATION TECHNIQUES
FOR PLANT CELL
• 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 PLANT CELL
• 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

8. METHODS OF ENZYME
IMMOBILIZATION
8

9. ADSORPTION
• An enzyme may 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

10. 10

11. COVALENT BONDING
• Covalent bond 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 are used 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 OF COVALENT
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 can be 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

15. 15

16. CROSS-LINKING/CO-
POLYMERIZATION
• Cross-linking is characterized 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 is the 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

18. 18

19. 19

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 Cells Reaction
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 PRODUCTION OF
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 material which 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 PLANT CELL
• 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
Short sequences 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
PCR product 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
 Short sequences 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
Short sequences 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
DNA fragment 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 DISADVANTAGE OF
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 CLONING OF
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
TISSUE CULTURE
• 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

35. 35

36. 36

37. 37

38. 38

39. Sec. metabolite from Picrorhiza kurroa by Tissue
culture
39

40. Sec. metabolite- artemisinin production by Tissue
culture 40

41. EFFECT OF PRECURSOR AND
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

42. 42

43. Elicitor
• Abiotic elicitors comprise 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

44. 44

45. L-dopa production in suspension 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 cloning advantages 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)
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