Expansins are the small extracellular cell wall loosening proteins, which are universal in the plant kingdom and are also found in a set of phylogenetically diverse bacteria, fungi, and other organisms. It plays an important role in the physiological process requiring cell modification that loosens plant cell wall and cellulosic material without any lytic activity.

1. Student
N.Shobana
2016801104
Chairman
Dr. U. Sivakumar
Professor & Dean (SPGS)
Dept. of Agrl. Microbiology
Tamil Nadu Agricultural University,
Coimbatore - 03
MCB 891 – Credit seminar (0+1)

2. INTRODUCTION
Expansins are plant derived
proteins, identified in the early
1990s -primarily known for
‘loosening’ effect on the cellulosic
network within plant cell walls
during growth. (Li et al., 1993)
Expansins five families: EXPA ( α-
expansin), EXPB (β-expansin), EXLA
(expansin-like), EXLB (expansin-
related), and EXLX (miscellaneous).
Milestones in Expansins discovery
(Cosgrove, 2005).

3. Microbial Expansins
Reported - Xanthomonas campestris, Clavibacter michiganensis, Ralstonia
solanacearum and Aspergillus niger or plant-colonizing bacteria such as Bacillus
subtilis (BsEXLX1) and the marine bacterium Hahella chejuensis.
 Helps to penetrate plants as pathogens or colonizers or digest plant tissues or
residues in the soil.
Cooperation with cellulase in cellulose hydrolysis
(Georgelis et al., 2014)

4. Conserved Structural Features
• N-terminal domain D1 has a double-ψ β-barrel fold similar to glycosyl
hydrolase family-45 (GH45) enzymes - lacks catalytic residues usually
required for hydrolysis.
• C-terminal domain D2 has a unique β-sandwich fold with three co-linear
aromatic residues that bind β-1,4-glucans by hydrophobic interactions.

5. Distribution of expansins and related proteins
Expansin-related sequences - found green
algae such as Spirogyra, Nitella and Penium
(Cosgrove, 2015)

6. CAZy Database
Family Organism Protein Name
CBM2 Clavibacter michiganensis subsp. michiganensis (CelA;pCM1_0020)
CBM63 Bacillus subtilis subsp. subtilis str. 168 (Exlx1;BsEXLX1;YoaJ;ExpA;BSU18630)
CBM63 Botrytis cinerea T4 Bofut4_p160720.1)
CBM63 Aspergillus niger ATCC 1015 (Asper EXP;ASPNIDRAFT_213462)
CBM63 Bacillus pumilus ATCC 7061 (BpEX;YoaJ;BAT_1917) (partial)
CBM63 Bacillus sp. E11CB expansin
CBM63 Hahella chejuensis KCTC 2396 (Exlx2;HcEXLX2;HCH_03669)
CBM63 Micromonospora aurantiaca ATCC 27029 (MaEX;Micau_1184)
CBM63 Pectobacterium carotovorum subsp. carotovorum PC1 expansin (PcEX;PC1_2092)
CBM63 Ralstonia solanacearum UW551 expansin (RRSL_02398)
CBM63 Clostridium clariflavum DSM 19732 (CclEXL2;CcEXL1298;Clocl_1298)
CBM63 Xanthomonas campestris pv. campestris expansin (XCC3535)
GH5 Xanthomonas campestris pv. campestris expansin (XCC3535)
CBM63 Schizophyllum commune RVAN10 expansin protein, partial (Exlx1)

7. Expansin-like proteins
• Fungi -swollenin, cerato-platanin, EXPN and loosenin expansin-like
proteins-cellulose aggregates without evidence of lytic activity.
• Cerato-platanins (CPs) - small fungal proteins - found in
basidiomycetes and ascomycetes (Yu and Li 2014)- plant
pathogenic fungus Ceratocystis platani.
• Swollenin (SWO) is a fungal protein that is about twice the size of
expansin (493 versus 225 amino acids) and is likewise reported to
disrupt cellulose aggregates.
(Saloheimo et al. 2002)

8. Activities of Microbial Expansins
• Expansins - loosen cell wall matrix polysaccharides by disruption of
non-covalent bonds between cellulose microfibrils or cellulose and
other matrix polymers.
• Decrystallization of cellulose by expansin - binding of the protein
onto H-bonded cellulose coated with other polysaccharides.
• Protein drifts along the chain, disruption of intermolecular and
intramolecular H-bonding occurs within the interacting matrix
carbohydrates to facilitate slippage.

9. Binding characteristics of Expansins
•Binds to several types of substrates - cellulose,
xylan and lignin.
•Binding capacity of microbial expansins propotional
- degree of crystallinity of the cellulosic substrates
and prefer - amorphous hemicellulose matrix than -
highly ordered cellulose fibers.
(Bunterngsook et al., 2015).

10. Role of expansins in Virulence
• C. michiganensis, an actinomycete - vascular wilt, canker, and
other diseases of tomato, potato, and other crops.
• Many expansin-containing pathogens proliferate and obstruct
the water-conducting xylem, resulting in leaf wilt as one of the
early symptoms of disease.
• Cerato-platanin family protein, contributes Botrytis cinerea
virulence and elicits the hypersensitive response in the host.

11. Role of expansins in Virulence BcSpl1, a cerato-platanin
Expression levels of bcspl1 under different
growth conditions Effect of the bcspl1 mutation on virulence
Induction of necrosis in plant leaves by BcSpl1.
Induction of reactive oxygen species
Electrolyte leakage from plant leaves
Frıas et al., 2011

12. • Expansins participate - formation and
maintenance of the interface, whereas other
act as major cell wall-loosening agents -
required intracellular colonization.
• Presence of EXPA proteins in mycorrhizal cells
- fungus induced enlargement of cortical cells
is achieved by the activity of EXPAs.
Expansins in Plant Colonization
(Balestrini et al., 2005)

13. • EXPA gene, MaEXP1 characterized in
process of nodule formation - sweet clover–
Sinorhizobium interaction.
• Expression of MaEXP1 - increased
inoculation of Sinorhizobium in both roots
and nodules.
• LjEXP, an expansin gene from Lotus
japonicus, expressed in infected roots and
nodules.
Expansins in Plant Colonization
(Flemetakis et al., 2004)
A, C, E, G, I, and K –uninoculated
B, D, F, H, J, and L, inoculated with Sinorhizobium meliloti
Whole-mount in situ hybridization
analysis

14. Synergistic enhancement of cellulase and expansins
Expansins and expansin-like proteins can cooperate with cellulase to
improve cellulose hydrolysis – key factor in efficient hydrolysis.
The yield of reduced sugars in a reaction mixture containing expansins
or expansin like proteins is 1.4–12.3 times that of the control sample
containing cellulase and substrates.

15. Synergistic enhancement of cellulase and expansins

16. Expansins from Bacillus subtilis for Enhanced Enzymatic Hydrolysis of Cellulose
Kim et al., 2009
EXPANSINS
Urea
Buffer
alone

17. Biochemical properties of expansins

18. • Cellulosome – integration of expansins proteins promotes the cellulose
degradation.
• Expansins as additives in lignocellulolytic enzyme cocktails in industrial
bioprocesses - EXLX family proteins.
Expansins as additives to lignocellulolytic
enzyme cocktails
(Chen et al., 2015)

19. Expression and purification of expansins
• The expression level of expansin - native organisms - lower than -
major cellulases (Saloheimo et al. 2002).
• Expansin from bacteria expressed in E. coli (Wang et al. 2011) -
yield 10 mg protein l-1 culture broth.
• Filamentous fungi and yeasts (Saccharomyces cerevisiae and
Pichia pastoris) expression level ranged 0.025 to 300 mg l-1 ( Jager
et al., 2011 & Kang et al., 2013).
• Purified by cellulose-affinity chromatography and Immobilized
metal ion affinity chromatography is convenient and is extensively
used to purify various proteins.
• Recombinant proteins - histidine affinity chromatography

20. Expression and purification of expansins

21. Industrial applications

22. Corresponding Literature

23. Hydrolytic activities of EXLX1 and
cellulase combinations
Contn........

24. Structure of EXLX1
Cell wall loosening activity of EXLX1.
Root colonization effect of EXLX1

25. Conclusion
• Many expansins and expansin like proteins were found to enhance
enzymatic cellulose hydrolysis.
• Productive interaction occurs between the expansin and its target-
move along the polymers rupturing hydrogen bonds under the
pressure exerted by the expanding cells.
• Addition - expansins in enzymatic saccharification of cellulose –
lignocellulose promising strategy improving hydrolysis process -
resulting cost reduction with respect to cellulase usage - eases and
quickens the process of hydrolysis.

26. Future thrust
The sites of action and physiochemical properties of microbial
expansins need to be clarified for understanding their role and
it pave the way for expanding its application in agricultural
biotechnology, crop protection, and biofuel production.

27. THANK YOU