1. Project Topic:
Structural analysis of various starch
debranching enzymes and their production
in Bacillus sp.
PRESENTED BY
Tushar Singh Barwal [123811]
Shubham Vashishtha [123802]
PROJECT SUPERVISOR
Dr. Saurab Bansal
Assistant Professor
Jaypee university Of Imformation
Technology

2. Introduction
• Starch: A major raw material
• In food washing detergent industries via
chemical or enzymatic degradation.
• Mixture of two polymers with high
molecular weight
• A linear chain molecule
amylose(alpha 1,4 linkage),
• A branched polymer of glucose
amylopectin(alpha(1, 6 linkage).
• Debranching enzymes cleaves either
1,4 , 1,6 linkage or both(eg.
amylopullulanase).
Source: Chemical structure of amylose and amylopectin (Buleon et al., 1998)

3. Actions of various amylases on starch
C
α-, β-, γ-
C
cyclodextrins
A
Cyclodextrin
A
glycosyltransferase
C
B
Panose
Endoamylase Maltose
(C) Debranching
Isopanose
enzyme
Pullulan hydrolase(A) α-amylase
α-(1, 4) links Exoamylase types I, II, and III
Maltotriose
Branching Pullulanase II
(B) β-amylase enzymeGlucoamylase
α-(1, 4) links α-glucosidase Pullulanase I
Maltotriose
α-(1, 4) and α-(1, 6) links
α-(1, 6) links
Maltose
α-limit dextrin Isoamylase Glucose
Maltose and Glucose
β-limit
dextrin
Linear oligosaccharides
Linear oligosaccharides
Glucose Maltose
FIGURE 4: Schematic presentation of the action of amylases. Black circles indicate reducing sugars (modified from [17]).Source: Siew Ling Hii, Joo Shun Tan, Tau Chuan Ling, and Arbakariya Bin Ariff (2012) Pullulanase: Role in Starch Hydrolysis and Potential Industrial
Applications Enzyme Research Volume 2012, Article ID 921362

4. Rationale
• Starch requires a combination of enzyme for de-polymerisation into
smaller sugars.
• Need for enzyme that have
• Debranching ability
• Bifunctionality
• Co-factor independent function

5. Objective-
• Comparative studies of active sites of various starch debranching enzyme by
sequence and structural analysis
• Multiple sequence alignment
• 3D Structural alignment
• Analysis of activity of various starch debranching enzymes from Bacillus
sources
• Optimization and production of starch debranching enzymes from Bacillus
sp.

6. Project Work plan (Wet and Dry lab)
Review of
literature
Experiment
design
Revival and
culturing selected
organisms
Qualitative and
Quantitative analysis
of Amylase activity by
the organisms
Qualitative and
quantitative
analysis of
pullulanase activity
by these organisms
Comparison of
activity of both the
enzymes. DNA
isolation from these
organisms.
Wet lab schedule

7. Review of literature
Sequence and Structural
analysis of selected
enzymes like Amylase,
different pullulanase
Study of active sites and
Catalytic sites that are
responsible for the enzyme
activity
Comparison of active sites
from different enzymes and
deducing the structural
difference that is responsible
for enzyme activity
Dry lab schedule

8. Results
Amylase activity Qualitative analysis (Starch Iodine test)
NCDC 71 1790 121
Bacillus Subtilis subspecies
Before
After

9. Amylase activity Qualitative analysis (Starch Iodine test)
Table 1
Numbering Organims Plate 1 Plate 2
1 121 + +
2 1790 ++ ++
3 NCDC71 +++ +++
4 2941 Very low Very low

10. Amylase activity Quantitative analysis (DNS test)
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
4.000
4.500
5.000
11 12 21 22 31 32 41 42
0.654 0.659 0.593 0.593
0.712 0.716
4.045
4.865
Enzymeactivity(U/ml)
Sample ID
Graph 1: Enzyme activity of various samples
11
12
21
22
31
32
41
42

11. Amylase activity at varying temperature (DNS test)
-0.20
0.30
0.80
1.30
1.80
2.30
2.80
3.30
3.80
4.30
4.80
11 12 21 22 31 32 41 42
Enzymeactivity(U/ml)
Sample ID
Graph 2:Enzyme activity at varying Temperature
25 Degree
30 Degree
40 degree
50 Degree
60 Degree

12. Protein purification by Ammonium Sulphate precipitation (DNS test)
Setup for ammonium precipitation

13. Enzyme activity after ammonium Sulphate precipitation (DNS test)
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
311 321 411 421 312 322 412 422 413 423
1.441 1.462
0.318
0.367
1.073
1.134
0.970 0.946
1.064
1.015
Enzymeactivity(U/ml)
Sample ID
Graph 3: Enzyme activity after each cut of ammonium precipitation
311
321
411
421
312
322
412
422
413
423

14. Pullulanase activity Qualitative analysis (Pullulan degrading test test)

15. Table 7
Numbering Organims Plate 1 Plate 2
1 121 + +
2 1790 + +
3 NCDC71 + +
4 2941 No growth No growth
Pullulanase activity Qualitative analysis (Starch Iodine test)

16. Pullulanase activity Quantitative analysis (DNS test)
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
1.800
2.000
11 12 21 22 31 32 41 42
0.217 0.215 0.221 0.223 0.254 0.246
1.826 1.843
Enzymeactivity(U/ml)
Sample code
Graph 4: Pullulanase activity using DNS method
11
12
21
22
31
32
41
42

17. Comparative activity analysis of both the enzymes
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
11 12 21 22 31 32 41 42
0.217 0.215 0.221 0.223 0.254 0.246
1.826 1.843
0.481 0.465 0.496 0.484 0.484 0.492
3.616 3.686
Enzymeactivity(IUml)
Sample ID
Graph 5: Comparative activity analysis of enzymes
pullulanase activity Amylase act

18. DNA Isolation (Phenol chloroform method)
121 1790 NCDC71 2941
Gel image for isolated
DNA
Table 13
Sample ID DNA Concentration(ng/µl)
11 94.9
12 92
21 89.1
22 82.1
31 57.1
32 51.1
41 206
42 211
Concentration of DNA by Nanodrop
Spectrophotometer

19. Acquiring Protein sequence and structure.
Table 14
Name Organism Accession
number
Length Bonds
processed
PDB ID
Pullulanase
Type I
Bacillus subtilis 255767686 718 1,6 2E8Y
Pullulanase
Type II
Bacillus subtilis 460686 2032 1,6 & 1,4 -
Isoamylase Bacillus lentus 493116169 886 1,6 -
Alpha amylase Bacillus subtilis 142435 425 1,4 1BAG

20. Multiple sequence alignment using Clustalw

21. Multiple sequence alignment using Clustalw

22. Multiple sequence alignment using Clustalw
Identity Matrix

23. Structure alignment of the Proteins
Pic 13: Structural alignment Pullulanase type I vs
Alpha amylase
Pic 14: Structural alignment Pullulanase type I
vs Isoamylase

24. Domain analysis
Table 15
Enzyme Conserved Domain Role Interval
Pullulanase type I AmyAc_Pullulanase_LD-like Alpha amylase catalytic
domain found in pullulanase
213-613
Pullulanse type II AmyAc_CMD Alpha amylase catalytic
domain, cleaves 1, 4 and 1,
6 bond.
475-952
Isoamylase AmyAc_Pullulanase_LD-like Alpha amylase catalytic
domain found in pullulanase
386-787
Alpha amylase AmyAc_bac1_AmyA Alpha amylase catalytic
domain found in bacterial
Alpha-amylases
50-393

28. Name Composition Molecular
characteristi
c
Topology Molecular
weight
Optima
l pH.
Isoelectric
point
Extinction co-
efficient
Activators Inhibitors
Alpha-
amylase
Glycoprotein
with a single
chain of
475residue, 2
free thiol groups
with four
disulphide
bridge, tightly
bound Ca2+ ion.
Class- alpha,
beta, Gama.
Architecture-
alpha-beta
barrel,
sandwich
TIM barrel 51.0-54.0KDa
(cozzone et
al.1970)
55.4KDa(SDS
page)
(Alkazaz et al
1996)
7.0 PI1:7.5
(Ajandouz et
al.1995)
PI2:6.4
(Ajandouz et
al.1995)
133,870cm-
1M-1
(theoretical)
E1%,280=26
(Caldwell et
al.1952)
Chloride,
Calcium
ions
Phenolic
compounds
(Fuke and Melzig
2005)
Urea and
other amide
(toralballa and
eitingon 1967)

31. References
• Siew Ling Hii, Joo Shun Tan,Tau Chuan Ling (2012) “Pullulanase:Role in starch hydrolysis and
potential industrial applications”, Enzyme Research Volume 2012, Article ID 921362
• Chemical structure of amylose and amylopectin (Buleon et al., 1998)
• M Nisha , T Satyanaraynana 2013, Recombinant bacterial amylopullulanases Developments
and perspectives, Bioengineered 4:6, 388–400
• S. Zareian, K. Khajeh, B. Ranjbar, B. Dabirmanesh, M. Ghollasi, and N. Mollania,
“Purification and characterization of a novel amylopullulanase that converts pullulan to
glucose, maltose, and maltotriose and starch to glucose and maltose,” Enzyme and
Microbial Technology, vol. 46, no. 2, pp. 57–63, 2010.