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 BansalAssistant Professor
Jaypee university Of Imformation Technology
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)
Actions of various amylases on starch
C
α-, β-, γ-
C c y c lo de x tr ins
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
enzyme
G lu c o a m y la se
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 G lu c ose
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
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
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.
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
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
ResultsAmylase activity Qualitative analysis (Starch Iodine test)
NCDC 71 1790 121
Bacillus Subtilis subspecies
Before
After
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
Amylase activity Quantitative analysis (DNS test)
11 12 21 22 31 32 41 420.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
4.000
4.500
5.000
0.654 0.659 0.593 0.593 0.712 0.716
4.045
4.865
Graph 1: Enzyme activity of various samples
Series1
Sample ID
Enz
yme
activ
ity (U
/ml)
Amylase activity at varying temperature (DNS test)
11 12 21 22 31 32 41 42-0.20
0.30
0.80
1.30
1.80
2.30
2.80
3.30
3.80
4.30
4.80Graph 2:Enzyme activity at varying Temperature
25 Degree30 Degree40 degree50 Degree60 Degree
Sample ID
Enz
yme
activ
ity (U
/ml)
Protein purification by Ammonium Sulphate precipitation (DNS test)
Setup for ammonium precipitation
Enzyme activity after ammonium Sulphate precipitation (DNS test)
311 321 411 421 312 322 412 422 413 4230.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.6001.441 1.462
0.3180.367
1.0731.134
0.970 0.946
1.0641.015
Graph 3: Enzyme activity after each cut of ammonium precipitation
Series1
Sample ID
Enz
yme
activ
ity (U
/ml)
Pullulanase activity Qualitative analysis (Pullulan degrading test test)
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)
Pullulanase activity Quantitative analysis (DNS test)
11 12 21 22 31 32 41 420.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
1.800
2.000
0.217 0.215 0.221 0.223 0.254 0.246
1.826 1.843
Graph 4: Pullulanase activity using DNS method
Series1
Sample code
Enz
yme
activ
ity (U
/ml)
Comparative activity analysis of both the enzymes
11 12 21 22 31 32 41 420.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
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
Graph 5: Comparative activity analysis of enzymes pullulanase activity Amylase act
Sample ID
Enz
yme
activ
ity (I
U\m
l)
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
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
Multiple sequence alignment using Clustalw
Multiple sequence alignment using Clustalw
Multiple sequence alignment using Clustalw
Identity Matrix
Structure alignment of the Proteins
Pic 13: Structural alignment Pullulanase type I vs Alpha amylase
Pic 14: Structural alignment Pullulanase type I vs Isoamylase
Domain analysisTable 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
Name Composition Molecular characteristic
Topology Molecular weight
Optimal 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)
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.
Top Related