Formation of low mass protostars and their circumstellar disks
Debabrat
1. Thermostable cellulose degrading bacteria from Dirang hotspring and its
utility in Bioethanol production from agrowaste
Dr. Debabrat Baishya
Assistant Professor
Department of Bioengineering and Technology (GUIST)
Gauhati University
Guwahati, Assam, India
2. Introduction
Dirang has many naturally proclaimed diseases healing hot springs in Arunachal Pradesh,
India.
The warm sulfur rich water in this hot springs cures numerous skin sicknesses.
Necto-benthic water content is a pristine habitat for diverse microbial population.
The optimum temperature of the water environment is approximately 40oC
Bacteria from hotsprings are capable of producing thermostable and thermophilic enzymes.
Water sample was collected from the deep groove of the hotspring of Dirang.
Dirang
3. Colony no.
Size (in mm) Colour Shape Elevation Margin Opacity
D1. 2.0 Off white Irregular Raised Undulate Opaque
D2. 2.0 White Rod Raised Undulate Opaque
D3. 2.2 Off white Round Raised Irregular Opaque
D4. 2.8 Off white Round Raised Undulate Opaque
D5. 2.7 Pale white Round Flat Irregular Translucent
D6. 2.5 Pale white Irregular Raised Irregular Translucent
D7. 2.0 White Rod Raised Undulate Opaque
D8. 3.5 Off white Irregular Flat Serrated Opaque
D9. 1.8 White Irregular Flat Irregular Opaque
D10. 1.9 White Rod Raised Irregular Opaque
D11. 2.2 White Round Flat Undulate Opaque
Isolation of Bacterial Colonies
Table 1. Isolation of bacterial colonies from the water sample of Dirang Hot-Spring
The microbial load of water samples collected from Dirang was found about 3.0 x 104 CFU/ml
4. Isolates Gram’s staining reaction Motility test
DB1 Gram +ve Motile
DB2 Gram +ve Motile
DB3 Gram +ve Non Motile
DB4 Gram +ve Motile
DB5 Gram +ve Non Motile
Screening of Cellulase Producing Colonies
Out of 5 colonies showing only two showed high extracellular cellulase activity in plate
assay containing 1% (w/v) CMC, stained with 0.1% (w/v) congo red.
5. Molecular Identification of Bacterial Colonies
DB1
DB2
Molecular phylogeny revealed that both DB1 and DB2 belongs to Genus
Bacillus
7. Production and Purification of extracellular cellulase enzyme from DB1 and DB2
DB1 DB2KDa
36 KDa
Bacterial isolates Protein concentration(µg/ml)
LB TB TY
DB1 452.18 1178.5 696.01
DB2 298.52 301.08 282.73
Table 2. Purification of partially purified cellulases from different media
Production of the extracellular cellulases were performed in LB, TB and TY medium where in
TB showed highest protein concentration while purified partially in ammonium sulfate.
Comparatively, DB1 showed significant 36 KDa pure protein band in SDS PAGE
8. Temperature optimization of partially purified cellulases from DB1 and DB2
Temperatures were found to be optimum 50oC for cellulases obtained from DB1 and DB2
9. pH optimization of partially purified cellulases from DB1 and DB2
pH was found to be optimum 6.5 and 6.0, respectively, for cellulases obtained from DB1 and DB2
10. Bacterial samples Filter paper assay (U/ml) Cotton assay (U/ml)
DB1 1.124 ±0.2 1.245±0.4
DB2 0.96±0.2 0.134±0.1
Enzyme Activity On Commercial Substrates
Substrates CelDB1 CelDB2
Vmax (µmole/min) Km (mg/ml) Vmax (µmole/min) Km (mg/ml)
Carboxymethyl Cellulose
(CMC)
1.8±0.2 1.2±0.3 0.9±0.1 0.7±0.3
BMCC 0.5±0.1 0.4±0.2 0.3±0.1 0.2±0.1
Konjac Mannan -- -- -- --
Galactomannan -- -- -- --
Birchwood xylan 0.4±0.2 0.3±0.1 -- --
Beechwood xylan 0.41±0.3 0.2±0.1 -- --
Glucuronoxylan -- -- -- --
-- = No activity found, SD (±) = standard deviation of triplicate samples
Table 3. Enzyme activities on various commercial substrates
Table 4. Enzyme activities based on filter paper and cotton assay
Partially purified celulase showed highest enzyme activity against 1% (w/v) CMC of DB1.
Which is comparatively higher than that of DB1. No pNP Glucosidase activity of the
enzyme signifies the endo-glucanase activity.
11. Functional ontology describes the evolutionary pattern of functional activity primarily similar with cellulase activity
Functional Network Modeling
12. Insilico prediction of structural and functional analysis of cellulase from DB1
(CelDBL1)
Structurally and functionally cellulsase from DB1 has similarity with the activities of
(family 12 Glyoside hyrolase, GH12) xyloglucanase from Bacillus licheniformis (Gloster et
al., 2007).
13. Molecular interaction of CelDBL1 with Cellulose substrates
CelDBL1 showed highest binding energy ΔG = -4.5 Kcal/mol with cellotriose
14. Comparative analysis of dock binding energies and MM/PBSA
Substrate -ΔG (Kcal/mol) docking -ΔG (Kcal/mol) MM/PBSA
Glucose 3.5 12
Cellobiose 3.6 15
Cellotriose 4.5 21
Cellotetrose 4.1 20
Cellopentose 2.1 8.0
Xylobiose 1.2 5.0
Xylotriose 1.3 6.0
Xylotetrose 1.1 4.6
Xylopentose 0.7 3
Table. Comparative analysis of dock score and MM/PBSA
Semi Empirical electron density function (Quantum Mechanics)
Complex Energy (Hartree) Energy (Kcal/mol)
Cellobiose-CelDB1 -0.024 -15
Cellotriose-CelDB1 -0.028 -18
Equation for semi-empirical calculation:
Binding energy (-ΔG) = MM-Energy CelBD1+ MM-Energy Substrate- MM-Energy CelDB1-Susbtrate
Table. Molecular mechanics (MM) and electron density function Binding energies of protein-oligosaccharide complex
