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Cellulase I Group 1. Isolation of Cellulase Producing Organism

The document describes the process of isolating and characterizing cellulase-producing bacteria from samples. Key steps include: 1. Screening samples on congo red medium containing cellulose to isolate cellulase-producing organisms. 2. Identifying the organism through tests like gram staining, biochemical characterization and 16S rDNA sequencing. 3. Characterizing the organism and cellulase production through assays measuring growth in varying pH, temperature, and carbon sources as well as production of other enzymes. 4. Quantifying cellulase activity using the DNS method to measure reducing sugar release from cellulose.

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156 views4 pages

Cellulase I Group 1. Isolation of Cellulase Producing Organism

The document describes the process of isolating and characterizing cellulase-producing bacteria from samples. Key steps include: 1. Screening samples on congo red medium containing cellulose to isolate cellulase-producing organisms. 2. Identifying the organism through tests like gram staining, biochemical characterization and 16S rDNA sequencing. 3. Characterizing the organism and cellulase production through assays measuring growth in varying pH, temperature, and carbon sources as well as production of other enzymes. 4. Quantifying cellulase activity using the DNS method to measure reducing sugar release from cellulose.

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aj_6
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Cellulase

I group
1. Isolation of Cellulase producing organism
1. Screening on congo red medium.
(Peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract, 1.5;
sodium chloride, 5.0, agar, 15, CMCellulose, 10) (g/l)
2. Filter paper assay
3. Single cell isolation
4. Storage
2. Identification of organism
1. Gram staining
The cultural characteristics observed were: colour, optical density,
pigmentation, margin, elevation and forms of colonies.
2. Bio-chemical Characterization
Bio chemical tests: motility, indole production, citrate utilization, urease,
oxidase, methyl red, voges proskauer, H2S production, beta galatosidase,
phenylalanine deaminase and fermentation tests
3. 16srDNA sequencing
3. Characterization of organism producing cellulase
1. Colony growth in different pH 3, 4,5,6,7,8,9
2. Colony growth at different temperature- 25, 30,37, 42, 50
3. Colony growth with different Carbon sources- Xylan, Pectin, Glucose, chitin
etc
4. Antibiosis
5. Production of other enzymes amylase, pectinase, chitinase, protease etc
4. Enzyme assay- Quantitative assay
Enrichment of cellulolytic bacteria was achieved in mineral salt solution of (in
g/l):
NaCl,6.0;(NH4)2SO4,1.0;KH2PO4,0.5;K2HPO4,0.5;MgSO4,0.1;CaCl2,0.1and
supplemented with 0.1% CMC as cellulose source
Endo--1,4-glucanase. Endo--1,4-glucanase (EG) activity was determined by
incubation of 1.0 ml of 0.2% (w/v) CMC in 0.025 M phosphate bu"er (pH 7.0)
with 1.0 ml of appropriate concentration of enzyme at 37C. A%er 30 min of
reaction, 1 ml of dinitrosalicylic acid (DNS) was added and boiled in a water
bath for 5 min to stop the reaction. #e resulting samples were then cooled to
room temperature and the absorbance measured at 540 nm (A540). One unit
of EG activity was defined as the amount of enzyme that could hydrolyze
CMC and release 1 g
of glucose within 1 min at 37C (Nelson, 1944).
5. Characterization of enzyme
1. Effect of pH
2. Effect of temp
3. Heat stability
4. Storage and Shelf life period
5. Inhibitors
6. Stimulators

The activity of Cellulase was assayed using DNS method. The bacterial
crude was prepared by
following method. 10 ml of culture was centrifuged at 5000 rpm for 15
minutes. The cell free extract was subjected to enzyme assay. The DNS
assay was carried out as follows. 0.2 ml of culture filtrate was mixed with
1% CMC in a test tube and incubated at 40C for 30 minutes. The reaction
was terminated by adding 3 ml of DNS reagent. The tube was then
incubated at 100C for 15 minutes followed by the addition of 1 ml of
Rochelle salt solution. The OD was taken at 575 nm against blank. One
unit of the cellulase activity refers to the amount of enzyme that released
1 M of glucose [5,6].
DNSA reagent (DNSA -1g, crystalline phenol - 0.2g, sodium sulphite 0.05g mixed in 1% NaOH). This
method becomes convenient and sensitive for the reducing sugars
estimation. In this method, the
Cellulase culture sample was pipette out in test tubes at various
concentrations. Blank contain 1ml of
distilled water. Then it was equalized the volume to 3 ml with distilled
water. It was followed by adding 3ml of DNSA reagent. The content of the
tubes are heated in a boiling water bath for 5 minutes. After cooling, 1ml
of 40% rochelle salt solution. Finally it was cooled and reading taken at
510nm using VIS Spectrophotometer (Systronic, 119). The amount of
reducing sugars was calculated by using standard graph [7].

II group
1. Isolation and purification of Enzyme
Enrichment of cellulolytic bacteria was achieved in mineral salt solution of (in
g/l):
NaCl,6.0;(NH4)2SO4,1.0;KH2PO4,0.5;K2HPO4,0.5;MgSO4,0.1;CaCl2,0.1and
supplemented with 0.1% CMC as cellulose source.
a. Production of shake flask culture
b. Pellet the cells and save the supernatant
c. Try Ammonium sulphate precipitation method in purification
d. Dialysis
e. Zymogram to confirm the cellulose activity.
2. Production of enzyme with different carbon source- glucose, CMC, Sucrose,
Chitin, pectin etc- Qualitative assay
3. Optimization of enzyme production with different media chemicals.
4. Efficient production of enzyme in fruit waste, market waste, paper pulp,
Kitchen waste, Agri waste
5. Quantitative assay for the production of ethanol.
Production of ethanol from Cellulase producing Bacteria.
The alcohol content of Cellulase culture sample was estimated by potassium
di chromate method [9]. In this method, 1ml of Cellulase culture sample was
taken. Blank contain 1 ml of distilled water. It was make the volume to 5 ml
with distilled water. It was followed by the addition of 1ml potassium
dichromate (10g of potassium dichromate was dissolved in distilled water
make up to 100 ml in volumetric flask) solution. Then 4ml of concentration
sulfuric acid was added. Finally the intensity of colour was read at 660 nm in
VIS spectrophotometer (Systronic, 119). Ethanol production was assayed by
comparing alcohol standard (Ethanol was dissolved in distilled water to get
10mg/ml concentration) graph.
6. Quantitative assay for the conversion of glucose
DNSA reagent (DNSA -1g, crystalline phenol - 0.2g, sodium sulphite - 0.05g
mixed in 1% NaOH). This method becomes convenient and sensitive for the
reducing sugars estimation. In this method, the Cellulase culture sample was
pipette out in test tubes at various concentrations. Blank contain 1ml of
distilled water. Then it was equalized the volume to 3 ml with distilled water.
It was followed by adding 3ml of DNSA reagent. The content of the tubes are
heated in a boiling water bath for 5 minutes. After cooling, 1ml of 40%
rochelle salt solution. Finally it was cooled and reading taken at 510nm using
VIS Spectrophotometer (Systronic, 119). The amount of reducing sugars was
calculated by using standard graph [7].

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