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.