teri and meth - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/8139/11/11...54 4. Solid state fermentation medium 4.1 Nutrient solution (pH 6.0) Yeast extract Sucrose KH 2PO 4

50

� Materials and methods

51

Chapter 3

Materials and methods

3.1 Materials

3.1.1 Chemicals

Chemical Company Acetic acid (CH3COOH) SD fine chemicals (Mumbai, India)

Acetone [(CH3)2CO] SD fine chemicals

Acrylamide SRL Chemicals

Agar Qualigens Fine chemicals (Mumbai, India)

Ammonium persulfate Qualigens Chemicals

Ammonium sulfate ([NH4]2SO4) SD fine chemicals

Bisacrylamide SRL Chemicals

Bovine serum albumin (BSA) Sigma Chemicals (St.Louis)

Bromophenol blue Qualigens Chemicals

Calcium chloride (CaCl2) Qualigens Chemicals

Carboxymethylcellulose (CMC) Hi-Media (Mumbai, India)

Cellobiose Lobachemie

Congored SD fine chemicals

Coomassie Brillant blue R250 Bio-Rad laboratories (Richmond,

California)

3,5-Dinitrosalicylic acid (DNS) SD fine chemicals

Ferrous sulphate (FeSO4) SD fine chemicals

Folin-Ciocalteu reagent (FCR) or

Folin's phenol reagent

Qualigens Chemicals

Glucose (C6H12O6) SD fine chemicals

Glycine Qualigens Chemicals

Isopropanol SD fine chemicals

Lactose SD fine chemicals

Lead acetate [Pb(CH3COO)2] SD fine chemicals

Magnesium choloride (MgCl2) Qualigens Chemicals

Maltose SD fine chemicals

Manganese sulphate (MgSO4) SD fine chemicals

4- methylumbelliferyl-β-D-glucoside Sigma Chemicals

Mercuric chloride (HgCl2) SD fine chemicals

Na-EDTA SD fine chemicals

Peptone Hi-Media

Phenol (C6H5OH) SD fine chemicals

p-Nitrophenol SRL Chemicals

52

p-Nitrophenol-β-D-glucopyranoside SRL Chemicals

Potassium chloride (KCl) SD fine chemicals

Potassium di hydrogen phosphate SD fine chemicals

Potassium sodium tartrate SD fine chemicals

Silver nitrate (AgNO3) SRL Chemicals (Mumbai, India)

Sodium alginate SD fine chemicals

Sodium choloride (NaCl) SD fine chemicals

Sodium hydroxide (NaOH) SD fine chemicals

Sodium nitrate (NaNO3) SD fine chemicals

Sodium meta bisulphate SD fine chemicals

Sodium sulphate (Na2SO4) SD fine chemicals

Sosium lauryl sulphate SD fine chemicals

Sucrose SD fine chemicals

TEMED Ferak (Berlin, West Germany)

Tris SRL Chemicals

Triton X-100 SD fine chemicals

Tween 20 and 80 SD fine chemicals

Urea SD fine chemicals

Yeast extract Hi-Media

β-mercaptoethanol Qualigens Chemicals

All chemicals were of analytical grade

3.1.2 Major equipment

• Cooling centrifuge, Remi Instruments

• Polyacrylamide gel electrophoresis (PAGE) apparatus, (Broviga, Mini slab gel

electrophoresis), Balaji Scientific Services, Chennai.

• Electrophoresis power supply (Genei mini powerpack - 250), Bangalore

• Spectrophotometer, (UV-1800, Shimadzu).

• Gel doc system (UVP, CA, USA)

• Dry Bath, Labnet International

53

3.1.3 Media

Table 3.1 Media used for isolation, screening, maintainance and enzyme production by

Aspergillus flavus

Medium Composition

1. Isolation medium, pH 6.0 (Cz-D medium, Czapek-Dox)

Sucrose

NaNO3

KCl

MgSO4

K2HPO4

KH2PO4

FeSO4

Distilled water to

30.0 g

2.0 g

0.5 g

0.5 g

1.0 g

0.5 g

0.1 g

1000 ml

Streptomycin/Penicillin (0.1 g/ml) solution of 0.1 ml was added after

sterilization of medium and before pouring into the petri plates

2. Maintenance medium, pH 6.0 (Sabouraud’s agar medium)

Peptone

Dextrose

Agar

Distilled water to

10 g

40 g

20 g

1000 ml

Streptomycin/Penicillin (0.1 g/ml) solution of 0.1 ml was added after

sterilization of medium and before pouring into the petri plates

3. Screening and production medium for submerged fermentation i.e.,

Modified CMC-CD medium, pH 6.0 (Carboxymethylcellulose-Czpeck Dox

medium)

CMC

Sucrose

Yeast extract

NaNO3

KCl

MgSO4

K2HPO4

KH2PO4

FeSO4

Tween 20*

Distilled water to

5.0 g

10.0 g

1.0 g

2.0 g

0.5 g

0.5 g

1.0 g

0.5 g

0.01 g

2.0 ml

1000 ml * :

Tween 20 was added to sterilized and cooled medium before inoculation

under sterile conditions.

54

4. Solid state fermentation medium

4.1 Nutrient solution (pH 6.0)

Yeast extract

Sucrose

KH2PO4

FeSO4

Tween 20*

Distilled water to

10.0 g

20.0 g

1.0 g

0.01 g

2.0 ml

1000 ml

* :

Tween 20 was added to sterilized and cooled medium before inoculation

under sterile conditions.

4.2 Basal salt solution

NaNO3

KCl

MgSO4

Distilled water to

10.0 g

2.5 g

2.5 g

50 ml

3.1.4 Reagents, buffers and solutions

1. Congo red solution Congo red 0.1 g

Distilled water 100 ml

2. NaCl solution (2 M) NaCl 116.8 g

Distilled water to 1000 ml

3. Acetate buffer (0.1M, pH 4.8) Solution A : Acetic acid solution

Acetic acid 5.8 ml


Solution B : Sodium acetate solution Sodium acetate 8.2 g


Mix 20 ml acetic acid solution with 30 ml of sodium acetate solution, pH adjusted to

4.8 and the final volume was made upto 100 ml using distilled water.

4. CMC-substrate solution (0.5 %) Carboxymethylcellulose 0.5 g

Acetate buffer (pH 5.0, 0.1M) 100 ml

5. DNS reagent Solution A

3,5-dinitrosalicylic acid 1.06 g

55

NaOH 1.98 g


Solution B Potassium sodium tartarate 3.06 g

Phenol crystals 0.76 g

Sodium meta bisulphate 0.83 g


The above two solutions (prepared freshly) were mixed in equal proportions just

before use.

6. Citrate buffer (0.1 M, pH 5.0) Solution A: Citric acid solution

Citric acid 19.21 g


Solution B: Sodium citrate solution

Sodium citrate 29.4 g


Twenty ml of citric acid solution mixed with 30 ml of sodium citrate solution, pH

adjusted to 5.0 and the final volume was made up to 100 ml.

7. Glycine-NaOH buffer (0.25 M, pH 9.5) Solution-A Glycine 7.5 g

Distilled water to 1000 ml

Solution-B

NaoH 4.0 g

Distilled water to 1000 ml Fifty ml of solution A mixed with 45.5 ml of solution B, pH adjusted to 10.6 and the

final volume was made up to 100 ml.

8. pNPG-substrate solution pNPG 75.3 mg


9. Reagents for protein determination

I. Alkaline copper sulphate solution

Solution A. 3 g of sodium carbonate dissolved in 100 ml of 0.1 N NaOH.

Solution B. i. 0.2 g of copper sulphate dissolved in 10 ml of distilled water.

ii 0.4 g of potassium sodium tartarate dissolved in 10 ml of distilled water. Both

solutions (i) and (ii) were mixed to obtain solution B.

Solution C. The reagent C was obtained by mixing 96 ml of solution A and 4 ml of

solution B.

56

II. Folin phenol reagent

Ten ml of Folin phenol reagent was diluted with 10 ml of distilled water just before

use.

III. Preparation of standard stock solution

Ten mg of bovine serum albumin was dissolved and made up to 100 ml in a

volumetric flask with 0.1N NaOH.

10. Polyacrylamide gel electrophoretic analysis of enzyme preparation

10.1 Acrylamide – Bisacrylamide solution

Acrylamide 30.0 g

Bisacrylamide 0.8 g

Dissolved the above chemicals in distilled water and made up to 100 ml. Filtered and

stored at 40C in a amber colored bottle.

10.2 Tris-HCl buffer (1.5 M, pH 8.8)

Tris base 18.5 g

The above chemical dissolved in 80 ml of distilled water, pH adjusted to 8.8 with 1N

HCl, the final volume made up to 100 ml with water and stored at 4oC.

10.3 Tris –HCL buffer (0.5 M, pH 6.8)

Tris base 6.0 g

The above chemical dissolved in 60 ml of distilled water, pH adjusted to 6.8 with 1N

HCl, the final volume made up to 100 ml with water and stored at 4oC

10.4 SDS (10%)

SDS 10 g

SDS dissolved in 80 ml distilled water with gentle stirring, the final volume made up to

100 ml with water and stored at room temperature only.

10.5 Sample buffer (pH 6.8)

Distilled water 4.0 ml

0.5 M Tris HCL 1.0 ml

Glycerol 0.8 ml

10%-SDS 1.6 ml

β-Mercaptoethanol 0.4 ml

0.05% Bromophenol blue 0.2 ml

57

10.6 Electrode buffer/ Tank buffer (pH 8.3)

Tris Base 1.80 g

Glycine 8.64 g

SDS 0.60 g

The above chemicals dissolved in water and made up to 600 ml. It was stored at room

temperature.

10.7 Staining solution

Commassie brilliant blue 100 mg

(CBB-R250)

Methanol 50 ml

CBB-R250 dye dissolved first in methanol. Then 10 ml of acetic acid and 40 ml of

distilled water were added to it. The prepared stain solution filtered through filter paper

and the filtrate stored at room temperature.

10.8 SDS-PAGE gel renaturation solution

Isopropanol 25 ml


10.9 Destaining solution

Methanol 40 ml

Acetic acid 14 ml


10.11 CBB stained gel storage solution

Acetic acid 10 ml


10.12 Mixing of stock solutions for resolving and staking gel preparation

Stock solution Resoving gel Stacking Gel

12% 10% 4%

Acrylamide/

bisacrylamide 4.0 ml 3.3 ml 0.65 ml

Tris-HCl, pH 8.8 2.5 ml 2.5 ml -

Tris-HCl, pH 6.8 - - 1.25 ml

Distilled water *

3.3 ml 4.03 ml 3.05 ml

10% SDS 0.1 ml 0.1 ml 50 µl

10% APS#

50 µl 50 µl 25 µl

TEMED 10 µl 10 µl 10 µl

# Prepared just before use

58

*: I. Endoglucanase activity assayed in SDS-PAGE gel by adding solubuized 1% CMC

solution (0.1 ml) to distilled water of resolving gel composition.

II. β-D-glucosidase activity assayed in SDS-PAGE gel by adding 0.5 M solution of 4-

methylumbelliferyl-β-D-glucoside (0.1 ml) to distilled waster of resolving gel

composition.

10.13 Sample preparation

The given enzyme preparation sample was diluted with sample buffer in 1:4 (v/v) ratio

and heated at 70oC for 3 min in water bath and cooled.

10.14 Protein molecular weight markers

I. Genei, Bangalore, Unstained

Standard protein Moleculor weight (kDa)

Phospharylase b 97.4

Serum albumin 66.0

Ovalbumin 43.0

Carbonic anhydrase 29.0

Trypsin inhibitor 20.1

Lysozyme 14.3

II. Fermentos life science, India, Unstained

Standard proteins Moleculor weight (kDa)

β-Galactosidase 116.0

Bovine serum albumin 66.2

Ovalbumin 45.0

Lactate dehydroginase 35.0

REase Bsp 981 25.0

β-Lactoglobulin 18.4

Lysozyme 14.4

11. Heavy metal salt stock solutions (10 mM)

CaCl2 2H2O Solution CaCl2 2H2O 1.47 g


MgSO4 Solution

MgSO4 2.03 g


HgSO4 Solution HgCl2 2.96 g


ZnCl2 Solution

59

ZnCl2 1.36 g


CoCl2 Solution

CoCl2 2.37 g


CuSO4 Solution CuSO4 2.496 g


Pb(CH3COO) Solution Pb(CH3COO 3.79 g


12. EDTA stock solution (10 mM) EDTA 3.72 g


13. Surfactant stock solutions (1%)

Tween -20 solution (v/v) Tween - 20 1 ml


Tween -80 solution (v/v) Tween - 80 1 ml


Triton X-100 solution (v/v) Triton X-100 1 ml


SDS solution (w/v) SDS 1 g


14. Lactophenol cotton blue solution stain Solution A

Cotton blue 5 ml

(Saturated solution)

Glycerol 10 ml


Solution B

Phenol 20 g

Lactic acid 20 ml

Glycerol 40 ml

60

Water 40 ml

The above two solutions were mixed in equal proportions.

15. Immobilization

Sodium alginate solution (2%)

NaCl 0.1 g

Alginate power 2.0 g


CaCl2 Solution (4%)

CaCl2 4.0 g


3.2 Methods

3.2.1 Isolation, screening and selection of promising cellulolytic enzymes

producing fungi

Cellulolytic enzymes are produced by a wide variety of bacteria and fungi, aerobes,

anaerobic, mesophilic and thermophilic bacteria and fungi. However, relatively few fungi

and bacteria produce high levels of extracellular cellulase that can meet industrial needs.

Current study was focused on isolation of promising cellulolytic Aspergillus sp. isolates from

wheat flour mill and saw mill solid waste samples.

3.2.1.1 Sample collection, processing and isolation of fungi

The following samples were collected from different sites in sterilized polyethylene bags and

preserved in the refrigerator at 4 oC till processing

• Wheat flour mill solid waste samples were collected around Tirupati, Andhra

Pradesh, India.

• Saw mill solid waste samples were collected around Tirupati, Andhra Pradesh,

India.

61

Solid waste samples collected from flour mill and saw mill were suspended by mixing

1 g of sample in 10 ml (10-1

) of sterile distilled water with vigorous mixing and further 10-

fold dilutions were made by transferring 1 ml from 10-1

dilution to 9 ml sterilized water in

test tubes for preparing 10-2

dilution. Like wise, dilutions up to 10-5

were made. From 10-3

,

10-4

and 10-5

dilutions, 0.1 ml diluted suspension was spread inoculated on Cz-D medium

(Isolation medium 1). The petriplates were incubated at 30 oC for 5 days.

3.2.1.2 Screening for cellulolytic fungi by plate assay method

The cultured plates showing well isolated fungal colonies were purified by a series of

subculures on the same medium and the pure colonies were tested for endoglucanse

production by plate assay method. The screening test of Farkas et al. (1985) was carried to

detect the cellulase production by pouring a suitable amount of CMC-CD agar medium

(Table 1, medium 3) into petri dish and the fungal spores were streaked onto the agar. The

cultured plate was flooded with a congo red solution (0.1% w/v) for 15 min, and then

destained with 1 M sodium chloride by washing the plate with destaining solution several

times. Unstained areas indicate hydrolysis of CMC. The fungal colonies on medium showing

significant hydrolytic zone were identified and the corresponding stock cultures maintained

on SA medium in purified state for further processing and the stock cultures that lack the

desired property for enzyme production were killed by autoclaving and discarded

immediately.

3.2.2 Screening for endoglucanase, exogluconase and β-D-glucosidase

production by submerged fermentation

The promising fungal colonies showing maximum hydrolytic zone by plate assay were

further studied by submerged fermentation for production of enzymes.

62

3.2.2.1 Inoculum development

The promising selected fungal colonies were spot inoculated on to sterilized SA agar medium

slants (Sabouraud’s agar medium 2) and allowed for growth by incubating at 30 oC for 5

days. The profuse growth on SA medium slants was mixed with sterile distilled water and

thoroughly mixed on cylclomixture and the spore suspension transferred to sterile capped

bottle. The spore density in suspension was counted using haemocytometer. Haemocytometer

was thoroughly rinsed with autoclaved distilled water and dried with tissue paper. The two

chambers of the haemocytometer were loaded with spore suspension and allowed the spores

to settle for about 1 min. Cover slip was placed over the haemocytometer chambers and

excess fluid was wiped out with tissue paper. The haemocytometer was placed on stage of

the microscope and observed under 10X followed by 40X objective lens and 15X eye piece.

The spores per ml in the original suspension were calculated using the formula.

Number of fungal spores/ml =

Average number of fungal spores (4 corners +1 central larger compartments / 5) X 104

3.2.2.2 Fermentation

The spore suspension (0.5 ml) was inoculated into 50 ml of sterilized production medium (1

%, v/v) in 250 ml Erlenmeyer flask maintaining sterile conditions. The uniform spore count

(~ 16X104/mm

3) was maintained to each flask for enzyme production. The flasks were

incubated at 30oC with 120 rev min

-1. At intervals of 2

nd, 4

th, 6

th and 8

th day fermented culture

broth (50 ml) was collected and filtered through the whatman filter paper, the fungal

mycelium retained was used for dry weight estimation and from the filtrate four ml

centrifuged at 8000 rpm/15 min at 4oC and the supernatant was used for enzyme assay.

63

3.2.2.3 Dry weight determination

The fungal growth by dry weight determination was done by filtration through previously

dried and weighed whatman No-1 filter paper (W1) and the mycelium with filter paper was

dried in hot air oven at 60 oC for 20 h and the final weigh (W2) of filter paper was recorded.

The dry weight of fungus determined using the formula

Dry weight of fungus = Final weight (W2) – Initial weight (W1)

3.2.2.4 Assay of endoglucanse

Endoglucanase activity was quantified using carboxymethylcellulose as substrate (Ghosh,

1987). The reaction mixture contained 1.0 ml of 0.5 % carboxymethyl cellulose in 0.1 M

acetate buffer (pH 5.0). This substrate was pre-incubated at room temperature (30±2 oC) for

10 min. An aliquot of suitable diluted enzyme (0.1 ml) was added to the reaction mixture and

incubated at 50°C in water bath for 30 min. Appropriate control was simultaneously run. The

reducing sugar produced in the reaction mixture was determined by dinitro salicylic acid

method (Miller, 1959). 3,5-Dinitro-salicylic acid reagent was added to reaction mixture and

boiled for 5 min, the colour developed was read at A540 nm in a spectrophotometer. One unit

of endoglucanase activity was defined as the amount of enzyme releasing 1 µmole of

reducing sugar per min under standard assay conditions.

3.2.2.5 Assay of exoglucanase activity

Exoglucanase activity was quantified using whatman No-1 filter paper discs of 50 mg weight

suspended in 1.0 ml of 0.1 M sodium citrate buffer (pH 5) at 50°C in a water bath. Suitable

aliquots of enzyme source was added to the above mixture and incubated for 30 min at 50°C.

The reducing sugar produced in the reaction mixture was determined as described above.

64

3.2.2.6 Assay of β-D-Glucosidase activity

Activity of β-D-glucosidase in the culture filtrate was quantified according to the method of

Herr (1979). Its activity was measured in assay mixture containing 0.2 ml of 5 mM p-

nitrophenyl β-D-gluco pyranoside (pNPG) solution and 0.7 ml of acetate buffer (0.1 M, pH

5.0) and 0.1 ml of diluted enzyme solution with appropriate controls. After incubation for 30

min at 50°C, the reaction was stopped by adding 4 ml of 0.25 M NaOH-glycine buffer (pH

10.6). The yellow coloured p-nitrophenol liberated was determined at 420 nm. One unit of β-

glucosidase activity was defined as the amount of enzyme liberating 1 µmole of p-

nitrophenol per min under standard assay conditions.

3.2.3 Cultural and microscopic features of promising fungus

The promising fungal culture spot inoculated on to sterilized SA agar medium (Sabouraud’s

medium 2) and allowed for growth by incubating at 30 oC for 5-7 days. Colony characterstics

like size, pigmentation at early and late stages of mycelium growth were recorded. A drop of

lactophenol cotton blue stain placed on a clean microscope slide. A small tuft of fungus

containing mycelium on agar medium was transferred using a flame sterilized inoculation

needle on to a slide containing the stain. The fungal mycelium was thoroughly spread using a

dissection needle and mixed the fungus with stain properly. A cover slip was placed over the

slide taking care to avoid trapping of air bubbles. Then the slide was observed in microscope,

first under low power (10X) followed by high power (40X), and morphological features of

substratum and aerial mycelia were recorded.

3.2.3.1 Micrometry of Aspergillus sp (Brown, 2004)

The Aspergillus sp. was mounted in lactophenol cotton blue stain and covered with a cover

slip. The prepared slide was placed on the stage of the microscope. With the help of the

65

ocular micrometer placed in the eye piece (15X) the number of ocular divisions that are

equivalent to the hyphae, stipe (conidiophore), vesicle, phialides (conidiogenous cell) and

conidiospores were recorded. The number of state micrometers that are equivalent to one

ocular division were calculated as follows.

No. of divisions on stage micrometer

1 ocular micrometer division = X10

No. of ocular micrometer divisions.

3.2.3.2 Identification of Aspergillus sp.

The promising fungal culture obtained from the wheat flour mill solid waste was sent to

Microbioal Type Culture Collection Centre (MTCC), Chandigarh for identification. This

culture was used for further studies.

3.2.4 Bioprocess development: Optimization of cellulolytic enzymes production

in submerged fermentation

Enzymes occur in every living cell, hence in all microorganisms. Each single strain of

organism produces a large number of enzymes, hydrolyzing, oxidizing or reducing, and

metabolic in nature. But the absolute and relative amounts of the various individual

enzymes produced vary markedly between species and even between strains of the same

species. Hence, it is customary to select strains for the commercial production of specific

enzymes which have the capacity for producing highest amounts of a particular enzyme

desired. Either surface or submerged culture methods currently employed for most microbial

enzymes production. In theory, the fermentative production of microbial enzymes is a

simple matter, requiring an appropriate organism grown on a medium of optimum

composition under optimum conditions.

66

The optimization of cultural conditions and medium composition was done by shake-

flask-scale culturing. The submerged fermentation medium (CMC-CD medium) was used to

investigate the effects of physical and nutritional factors on fungal growth and cellulolytic

enzymes production. The medium was modified according to the conditions and factors that

were studied. The 50 ml submerged fermentation broth medium was prepared in 250 ml

Erlenmeyer flask and autoclaved at 121oC for 15 min. The fungal spore suspension (~

16X104/mm

3) was inoculated to the sterilized culutre broth and the flasks were incubated in

an orbital shaking incubator (120 rpm). At regular 2-days intervals culture medium (one

flask, 50 ml) was used for fungal dry weight determination (section 1.3.3) and enzyme

extraction by centrifugation at 8000 rpm for 15 min at 4oC and the clear supernatant was used

as crude enzyme source.

3.2.4.1. Physical factors

3.2.4.1.1. Incubation time

To determine the effect of incubation time on fungal growth and enzyme production, shake-

flasks cultured in incubator-shaker (120 rpm) at 30oC were sampled at regular intervals (2-

days) for fungal dry weight determination (section 1.3.3) and the enzyme extraction, and

endoglucanase, exoglucanase, β-D-glucosidase were assayed as described earlier (Section

3.2.2.4 - 3.2.2.6).

In view of highest production of endoglucanase and exoglucanase at 2nd

day and β-D-

glucosidase at 6th day incubation, the shake-flasks at 2

nd and 6

th day of incubation were

collected for optimization of remaining physical and nutritional factors.

3.2.4.1.2 Incubation temperature

67

To determine the optimum temperature for fungal growth and enzyme production, shake-

flasks cultured in incubator-shaker (120 rpm) at different incubation temperatures ranging

from 30-50oC, and enzyme extraction and endoglucanase, exoglucanase, β-D-glucosidase

were assayed as described earlier (Section 3.2.2.4 - 3.2.2.6).

3.2.4.1.3 Initial medium pH

To determine the optimum initial medium pH for fungal growth and enzyme production, the

production medium pH was adjusted from 5-7 using buffering agents (0.1M/L), flasks

cultured in a incubator-shaker (120 rpm) at 30oC and enzyme extraction and endoglucanase,

exoglucanase, β-D-glucosidase were assayed as described earlier (Section 3.2.2.4 - 3.2.2.6).

3.2.4.2 Nutrional factors

The optimized cultural conditions of incubation temperature (30oC) and production

medium (pH 6) were maintained for optimization of nutritional factors.

3.2.4.2.1 Carbon sources

To determine the effect of CMC (carboxymethylcellulose), sucrose and lactose, the

production medium (pH 6) was designed with different concentrations of carbon sources

ranging from 0.5-2.0 % (w/v), flasks were cultured in incubator-shaker (120 rpm), and

enzyme extraction and endoglucanase, exoglucanase, β-D-glucosidase assays were carried as

described earlier (Section 3.2.2.4 - 3.2.2.6).

3.2.4.2.2 Nitrogen sources

To determine the effect of yeast extract and tryptone , the production medium was designed

with different concentrations of nitrogen sources ranging from 0.2-0.6 % (w/v), flasks were

68

cultured in incubator shaker (120 rpm), and enzymes extraction and assays carried as

described earlier (Section 3.2.2.4 - 3.2.2.6).

3.2.5 Partial purification and characterization of endoglucanase, exoglucanase

and β-D-glucosidase

3.2.5.1 Enzyme production

The optimized cultural conditions and medium composition were used for enzyme

production and partial purification. In view of highest production of endoglucanase and

exoglucanase at 2nd

-day and β-D-glucosidase at 6th -day incubation, the shake-flasks at 2

nd-

and 6th

-day of incubation were collected, filtered separately and the filtrates centrifuged at

8000 rpm (4oC) for 15 min, and the clear supernatants were used for enzymes partial

purification.

3.2.5.2 Partial purification of enzymes

All the unit processes for enzymes partial purification were done at a temperature of 4oC

unless other wise specified. The centrifugal culture supernatant was subjected to ammonium

sulphate precipitation by 70 % saturation over night. The precipitate was collected by

centrifugation at 15000 rpm for 25 min and the pellet was dissolved in small volume of 0.1

M acetate buffer (pH 5.0). Dialysis was carried out in dialysis tubing which was boiled in 1%

sodium bicarbonate solution containing 0.01M Na-EDTA followed by three sterile distilled

water washes. The above precipitate was taken into dialysis bag, tied tightly the ends with

thread and dialyzed against the acetate buffer (0.1 M, pH 5.0), 3-4 changes in 24 h. The

enzyme activity was measured as described above (Section, 1.3.4, 1.3.5 and 1.3.6) and the

protein estimation was done by Folin’s-Phenol method using BSA as a standard (Lowry et al,

1951). Appropriately diluted enzyme (0.05 ml) was taken in clean test tube and made up to 1

69

ml by adding 0.1N NaOH solution. To each tube 5 ml of alkaline copper sulphate solution

was added and incubated for 10 min at room temperature. Folin Phenol reagent (0.5 ml) was

added to each tube, mixed thoroughly and incubated for 30 min at room temperature and the

intensity of blue colour was read at 660 nm using spectrophotometer. The concentration of

enzyme protein was estimated by intersecting the standard curve prepared for BSA under

similar conditions.

3.2.5.3 SDS-PAGE and zymogram analysis of endoglucanase and β-D-

glucosidase

Although several methods are available for determining purity, the easiest to apply and best

method is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins

migrate in an electric field because they are charged at the pH of buffer used in PAGE. SDS-

PAGE was performed essentially as described by Laemmeli (1970).

Vertical slab gel unit on the casting mold was assembled using 1 mm thickness

spacers. The gel cassette was vertically kept in a trough and molten bacteriological agar was

poured into the trough to seal the bottom end of gel cassette. Into 100 ml conical flask, the

required stock solutions were mixed (materials, 10.12) to obtain 12% resolving gel. The gel

solution transferred into the gel cassette without trapping air bubbles and the gel mix over

layered with distilled water to get uniform gelling surface. After gel, the upper water layer

was poured out and the residual water droplets were wiped out using filter paper. Depending

upon the number of wells required, the comb was inserted into cassette before pouring the

stacking gel solution. Into a separate conical flask, the stock solutions were mixed as shown

in the table (materials, section 10) to get 4% stacking gel. The staking gel solution was

layered using a pipette over the resolving gel and allowed it for solidification. After gelling

the comb was carefully removed from the gel and the gel cassette was carefully clamped to

70

the vertical electrophoresis unit with the help of clips. The tank buffer was poured into the

upper and lower buffer tanks without trapping air bubbles.

The protein samples were mixed with SDS-PAGE sample buffer (1:6) and boiled at

70oC for 3 min. The sample proteins and marker proteins were loaded into wells separately

using microsyringe and recorded the order of the samples loaded. The unit was connected to

the power pack and the current passed initially at 50 volts and later increased to 100 volts.

When the tracking dye (bromophenol blue) reached the other end of the gel, the current was

brought to ‘0’ volts and disconnected from the unit. The gel cassette was detached from the

unit, dismantled, and the gel was processed for further steps. After electrophoresis, the gel

stained with a dye to reveal the positions of the proteins in the gel. The enzyme activity stain

reveals the position of the enzyme in gel, while the total protein stain reveals the position of

all the proteins. The molecular weight (MW) of marker proteins (Y-axis) is plotted against

the electrophoretic mobility (X-axis) of the marker proteins on semi-log graph paper. Using

this curve, the Mτ of the unknown protein was determined.

3.2.5.3.1 Endoglucanase

SDS - PAGE was performed to analyze partially purified enzyme as described by Laemmli

(1970) using 12% resolving slab gel. For endoglucanase detection, carboxymethylecellulose

was added to the resolving gel mixture to 0.01% final concentration. After electrophoresis

the enzyme protein band was visualized by staining with Coomassie brilliant blue R-250. The

gel resolved endoglucanase activity in polyacrylamide gel (activity staining) was performed

by renaturaion of SDS-PAGE gel in 25% isopropanol (Blank et al. 1982) solution for 2 h in

shaking condition followed by distilled water and acetate buffer (0.1M, pH 5) washes each of

two times for 10 min at room temperature. The protein after renaturation associated with

71

endoglucanase activity was seen by incubating gel at 50 oC for 30 min in 20 ml of acetate

buffer (0.1 M, pH 5.0), the gel was flooded with congo red solution (0.1%), destained with 2

M NaCl solution to detect the enzyme activity (clear zone formation) and the image was

captured under UVP gel doc system.

3.2.5.3.2 β-D-glucosidase

SDS - PAGE was performed to analyze partially purified enzyme as described by Laemmli

(1970), using 12% resolving slab gel. For β-D-glucosidase detection, 4-methylumbelliferyl-

β-D-glucoside (Hosel et al 1978) was added to a final concentration of 5 mM to the resolving

gel mix. After electrophoresis the enzyme protein band was visualized by staining with

Coomassie brilliant blue R-250. The gel resolved β-D-glucosidase activity in polyacrylamide

gel (activity staining) was performed by renaturaion of SDS-PAGE gel in 25% isopropanol

solution (Blank et al. 1982) for 2 h in shaking condition followed by distilled water and

acetate buffer washes each of two times for 10 min at room temperature. The protein after

renaturation associated with β-D-glucosidase activity was seen (clear zone formation) by

incubating the gel at 50oC for 30 min in 20 ml of acetate buffer (0.1 M, pH 5.0) and the

image captured under UVP gel doc system.

3.2.5.4 Optimization of enzyme assay conditions

To find the optimum assay conditions for endoglucanase, exoglucanase and β-D-glucosidase

enzymes, enzyme assays were carried as described earlier and the relative activity

determined.

Percent relative enzyme activity =

Optical density value of test X 100

Optical density value of control

72

3.2.5.4.1 Effect of pH on stability and activity of endoglucanase, exoglucanase

and β-D-glucosidase enzymes

To find the optimum pH for stability and activity of partially purified endoglucanase,

exoglucanase and β-D-glucosidase enzymes, pH ranging from 4-9 (0.1 M acetate buffer, pH

4, 5, 0.1 M potassium phosphate buffer, pH 6,7, 8, and 0.1 M glycine- NaOH buffer, pH 9,)

was used. The pH stability was measured by incubating the enzyme in selected buffers

without substrate for 12 h and the residual enzyme activity measured as described earlier.

The effect of pH on enzyme activity was determined by varing the assay buffer pH under

standard conditions as described earlier.

3.2.5.4.2 Effect of temperature on stability and activity of endoglucanase,

exoglucanase and β-D-glucosidase enzymes

To find the optimum temperature for stability and activity of partially purified

endoglucanase, exoglucanase and β-D-glucosidase enzymes, temperature ranging from 30–

60oC was tested. The temperature stability was tested by incubating the enzyme in acetate

buffer (pH-5) without substrate from 30-60 o

C for 5 h and the residual enzyme activity

measured. The effect of temperature on enzyme activity was determined by varing the assay

temperature from 30–60oC under standard conditions as described.

For subsequent experiments, optimum assay temperature of 50oC and pH 5.0 were

selected.

3.2.5.4.3 Effect of surfactants on endoglucanase, exoglucanase and β-D-

glucosidase activities

The effect of surfactants such as Tween-20, Tween-80, TritonX-100 and SDS, at 0.5, and

1.0% was determined by incorporating them into the enzyme assay mix, and the activity of

endoglucanase, exoglucanase, β-D-glucosidase was determined as described earlier.

73

3.2.5.4.4 Effect of heavy metal ions and EDTA on endoglucanase, exoglucanase

and β-D-glucosidase activities

To the optimized enzyme assay mix, the salt solution of heavy metal ions such as Ca2+

, Co2+

,

Mg2+

, Zn2+

, Cu2+

, Hg2+

and Pb2+

, and the chelating agent EDTA were made up to 5 and 10

mM, and the three enzymes were assayed as described earlier.

3.2.5.5 Kinetics (Km and Vmax) of endoglucanase, exoglucanase and β-D-

glucosidase

The study of the rate at which an enzyme works is called enzyme kinetics. Enzyme kinetics

determined as a function of the concentration of substrate available to the enzyme. Plotting

the reciprocals of the enzyme velocity [V] and substrate concentration [S] yields a "double-

reciprocal" or Lineweaver-Burk plot. This provides a more precise way to determine Km and

Vmax. The effect of substrate concentration on activity is usually expressed in Km and Vmax

values using a double reciprocal Lineweaver-Burke plot. The data was analyzed and plots

were drawn using GraphPad Prism 5.0 software package.

3.2.5.5.1 Kinetics of endoglucanase

In order to determine the kinetics of the partially purified endoglucanase (Km and Vmax),

CMC was used as a substrate at concentrations ranging from 1 - 7 mg/ml under optimal

conditions (pH 5, temperature 50oC) and the enzyme activity assayed as described earlier.

The apparent Km and Vmax values were calculated by using GraphPad Prism 5.0 software

package.

3.2.5.5.2 Kinetics of exoglucanase

In order to determine the kinetics of the partially purified exoglucanase (Km and Vmax),

whatman filter paper No-1 was used as a substrate at concentrations ranging from 10-70 mg

74

under optimal conditions (pH 5, temperature 50oC), and the enzyme activity assayed as

described earlier.The apparent Km and Vmax values were calculated by using GraphPad Prism

5.0 software package.

3.2.5.5.3 Kinetics of β-D-glucosidase activity

To determine the kinetics of the partially purified β-D-glucosidase (Km and Vmax), pNPG was

used as a substrate at concentrations ranging from 0.03 to 0.3 mg/ml under optimal

conditions (pH 5, temperature 50oC), and the enzyme activity assayed as described earlier.

The apparent Km and Vmax values were calculated by using GraphPad Prism 5.0 software

package.

3.2.5.6 Product inhibition (Feed back inhibition) of β-D-glucosidase activity

To determine the feed back inhibition of the partially purified β-D-glucosidase, increasing

concentrations of product i.e., glucose raninging from 0.05-0.3 M incorporated into the

enzyme assay mixture. Under optimal enzyme assay conditions β-D-glucosidase activity was

measured as described earlier.

3.2.6 Bioprocess development: Optimization of cellulolytic enzymes

production in solid state fermentation

3.2.6.1 Selection and enzyme production using different cellulosic substrates

Solid-state fermentation (SSF) is a process whereby an insoluble substrate is fermented with

sufficient moisture, but without free flowing water. In order to select a suitable substrate for

the production of cellulolytic enzymes, various substrates were collected from different

sources. Substrates used in the present study were wheat bran, oat bran, rice bran, sugarcane

bagasse and sawdust. The wheat bran and oat bran were purchased from local market

75

(Tirupati, Chittoor district). The rice bran and sawdust were collected from rice mill and saw

mill in Tirupati. Sugarcane bagasse was collected from sugarcane juice making shops at

Tirupati.

3.2.6.1.1 Pretreatment and moisture content determination of solid substrates

In order to select a suitable substrate, five different substrates i.e., wheat bran, oat bran, rice

bran, sugarcane bagasse and sawdust were screened for the production of cellulolytic

enzymes. The substrates were sieved individually through a ~1 mm screen mesh for uniform

particle size, autoclaved at 121oC for 10-15 min, dried in an hot air oven at 60

oC for 5 h and

the substrates used for 100 % moisture determination. 10 g of each solid substrate was taken

in separate glass funnels lined with moistened filter paper and to the substrate distilled water

added with continuous mixing of substrate with glass rod till water percolates down from the

nozzle. The volume of distilled water required for complete moistening was considered 100

% moisture for 10 g (w/v) of substrate.

3.2.6.1.2 Enzymes production using different cellulosic solid substrates and

assay procedure

The enzymes production was carried out in 250 ml Erlenmeyer flasks containing 10 g of each

solid substrate. To select the best solid substrate for cellulase production, the pretreated

substrates were moistened to 60 % initial moisture level with basal salt and nutrient solution

(table 3.1, section 4), cotton-plugged and autoclaved at 121oC for 30 min. Sterile solid

culture medium in the flasks was inoculated with 0.5 ml spore suspension (~ 16X104/mm

3)

of A. flavus and incubated at ambient temperature (30 ± 2oC). At regular intervals (2 days)

the cultured fermented substrate was used for enzyme extraction and assay. The cultured

fermented substrate (1:10; w/v) was mixed thoroughly with acetate buffer (0.1M, pH 5.0 and

0.1 % T-20) for 30 min, the extract filtered thorough Whatman filter paper No. 1 and the

76

filtrate centrifuged at 10000 rpm at 4oC for 10 min and the crude centrifugal supernatant used

for enzyme assay. The enzymatic activities were assayed as described earlier. One unit (U) of

enzyme activity was defined as the amount of enzyme required to release 1 µmol of product

from the appropriate substrate per minute under assay conditions. The enzyme activities are

expressed as units per gram of fermentated moldy bran (U/g FMB).

3.2.6.2 Optimization of cellulolytic enzymes production by solid state

fermentation

Production of cellulolytic enzymes by solid-state fermentation (SSF) using various cellulosic

soild substrates was found to have several advantages such as higher productivity as well as

lower operational and capital costs. Because the cost of the enzyme is the major factor for

broad application, approaches that either decrease the medium cost or increase production

efficiency should be investigated. Since SSF can be performed on a variety of lignocellulosic

materials, the cost of cellulolytic enzyme production can be reduced greatly.

77

Table 3.2 Preparation of solid substrate medium components for endoglucanse and β-D-

glucosidase production under solid state fermentation

Si.

no Solid substrate

100 %

moisture

content

(ml/10 g)

60 %

moisture

content

(ml/10 g)

SSF medium preparation

(Final moisture to 60%)

Nutrient

solution

(ml)

BSS

(ml)

Spore

suspen-

sion (ml)

dH2O

(ml)

1 Oat bran (OB) 10 6 4 1.5 0.5 -

2 Wheat bran (WB) 27 16.2 4 1.5 0.5 10.2

3 Rice bran (RB) 22 13.2 4 1.5 0.5 7.2

4 Sugarcane bagasse (SCB) 43 25.8 4 1.5 0.5 19.8

5 Saw dust (SD) 20 12 4 1.5 0.5 6

Nutrients and BSS - Basal salt solution ( Table 3.1, section 4)

78

3.2.6.2.1 Optimization of enzyme production by ‘one variable at a time’

In view of highest production of endoglucanse and β-D-glucosidase on wheat bran by A.

flavus in SSF, subsequent experiments were carried out only with wheat bran. The ‘one

variable at a time’ approach for optimizing microbial products production may be effective in

some situations, but is inadequate as it is a time consuming process and it further neglects the

interaction between variables and does not guarantee attaining optimal points. The statistical

design is efficient and effective because it provides a good coverage of the experimental

space with as few measurements as possible. Thus the use of response surface methodology

(RSM) in biotechnological processes is gaining immense importance for the optimization of

enzyme production and several other processes (Sanjeev Kumar and Satyanarayana, 2004).

In the present investigation for cellulolytic enzyme production by ‘one variable at a time’

approach, a statistical experimental design (response surface methodology, RSM) was

applied to further maximize enzyme production and to understand the interactive effects of

most effective cultural parameters (substrate concentration, incubation time and inoculum

level).

3.2.6.2.1.1 Incubation time Vs enzyme production

To find the suitable incubation time for endoglucanse and β-D-glucosidase production, using

wheat bran as substrate, the enzyme extraction and assays were carried at different incubation

times as described above.

3.2.6.2.1.2 Effect of inoculum size on enzyme production

To find the suitable inoculum size for cellulolytic enzymes production in wheat bran solid

state fermentation, fermentation carried at different inoculum size, and enzyme extraction

and assays were carried as described above.

3.2.5.2.1.3 Effect of substrate concentration

To find the suitable substrate concentration for cellulolytic enzymes production, fermentation

carried at different concentrations of wheat bran, and enzyme extraction and assays were

carried as described above.

79

3.2.6.2.2 Optimization of enzyme production by RSM

The statistical design RSM was generated for endoglucanase and β-D-glucosidase

production. Three factors selected by ‘one variable at a time’ approach, were used in

designing the experiment: wheat bran concentration (A), incubation time (B) and inoculum

level (C). . The medium was modified according to the conditions and factors studied. The

results of RSM were used to fit a second order plynominal equation (1) as it represents the

behaviour of response (Kumar and Satyanarayana, 2001)

Y= βo+ β1A+ β2B+ β3C +β1β1 A2 + β2 β2 B

2 + β3 β3C

2 + β1 β2AB + β1 β3 AC + β2 β3

BC (1)

Y = response variable, βo = intercept, β1, β2, β3, = linear coeeficients, β1β1, β2 β2, β3 β3 =

Suqared coeeficients, β1 β2, β1 β3, β2 β3 = interaction coeficients, and A, B, C, A2, B

2,

C2, AB, AC, BC = level of independent variables.

3.2.6.2.2.1 Optimization of endoglucanase production

Three factors selected by ‘one variable at a time’ approach, were used in designing the

experiment: wheat bran concentration (A), incubation time (B) and inoculum level (C). The

ranges of the variables investigated in this study are given in table 3.3. A central composite

quadratic design containing 20 experiments (20 runs) was generated using Design-Exeper®

software (7.1.6 version), endoglucanase production was taken as an independent variable or

response (table 3.4).

3.2.6.2.2.1 Optimization of β-D-glucosidase production

Three factors selected by ‘one variable at a time’ approach, were used in designing the

experiment: wheat bran concentration (A), incubation time (B) and inoculum level (C). The

medium was modified according to the conditions and factors studied. The ranges of the

variables investigated in this study are given in table 3.5. A central composite quadratic

design containing 20 experiments (20 runs) was generated using Design-Exeper®

software

(7.1.6 version), β-D-glucosidase production was taken as an independent variable or response

(table 3.6).

80

Table 3.3 The level of factors chosen for the central composite quadratic design

for endoglucanase production.

Factor Name/Units Low Actual (Coded) High Actual (Coded)

A Wheat bran (g) 5.0 (-1.0) 10 (1.0)

B Incubation time (days) 2.0 (-1.0) 4 (1.0)

C Inoculum volume (ml) 0.5 (-1.0) 1 (1.0)

Table 3.4 The central composite quadratic design for endoglucanase production for three

independent factors.

Run no. Factor 1

A: Wheat bran (g)

Factor 2

B: Incubation time (days)

Factor 3

C: Inoculum level (ml)

1 5.00 2.00 0.50

2 7.50 3.00 0.75

3 10.0 4.00 0.50

4 5.00 2.00 1.00

5 7.50 4.00 0.75

6 7.50 3.00 0.67

7 7.50 3.00 1.17

8 5.00 4.00 1.00

9 7.50 1.32 0.75

10 7.50 3.00 0.75

11 10.00 2.00 0.50

12 7.50 3.00 0.75

13 7.50 3.00 0.75

14 7.50 3.00 0.75

15 10.00 3.00 0.75

16 7.50 3.00 0.33

17 10.00 4.00 1.00

18 5.00 4.00 0.50

19 10.00 2.00 1.00

20 3.30 3.00 0.75

81

Table 3.5 The level of factors chosen for the central composite quadratic

design for β-D-glucosidase production.

Factor Name/Units Low Actual (Coded) High Actual (Coded)

A Wheat bran (g) 5.0 (-1.0) 10 (1.0)

B Incubation time (days) 4.0 (-1.0) 8 (1.0)

C Inoculum volume (ml) 0.5 (-1.0) 1 (1.0)

Table 3.6 The central composite quadratic design for β-D-glucosidase production for

three independent factors.

Trial

No.

Factor 1

A: Wheat bran (g)

Factor 2

B: Incubation time (days)

Factor 3

C: Inoculum level (ml)

1 7.50 6.00 0.75

2 7.50 6.00 0.75

3 5.00 4.00 0.50

4 7.50 6.00 0.30

5 10.00 8.00 0.50

6 10.00 6.00 0.75

7 10.00 4.00 1.00

8 10.00 4.00 0.50

9 7.50 6.00 1.00

10 7.50 6.00 0.75

11 7.50 8.00 0.75

12 10.00 8.00 1.00

13 7.50 6.00 0.75

14 7.50 4.00 0.75

15 7.50 6.00 0.75

16 3.30 6.00 0.75

17 5.00 4.00 1.00

18 5.00 8.00 1.00

19 5.00 8.00 0.50

20 7.50 6.00 0.75

82

3.2.7 Immobilization

The 2% sodium alginate mix (16 ml) was autoclaved for 10 min at 120 oC at 15 lbs and

cooled to room temperature. To the sodium alginate mix, the partially purifed enzyme (4 ml)

was added and mixed uniformly without trapping air bobbles. Thus prepared mix released

slowly into CaCl2 solution drop-wise using surgical syringe (5 ml). The alginate drops form

beads immediately by contact with CaCl2 solution. The prepared beads in CaCl2 solution

allowed for 5 h in refrigerator at 4-6 oC for curing i.e., till the beads harden. The alginate

beads after curing process washed with sterile distilled water for 3-4 times to remove bound

CaCl2 solution. The final weight of the beads was measured (12 g) after blotting with coarse

filter paper (to exclude water) and the beads were preserved in acetate buffer (pH-5) at 4oC.

3.2.7.1 Substrates

The substrates selected for hydrolysis by immobilized enzyme are CMC, cellobiose, wheat

bran, oat bran and rice bran. The wheat bran, oat bran and rice bran were prepared as

described earlier. The substrates were taken in clean conical flasks at 1 % in acetate buffer.

CMC (1%) in acetate buffer allowed for overnight for solubulization before use as substrate

for immobilized enzyme.

3.2.7.2 Hydrolytic activity of immobilized cellulolytic enzymes

Hydrolysis of different cellulosic substrates was carried in 250 ml Erlenmeyer conical flasks

containing 50 ml of selected substrate and alginate immobilized enzyme beads (12 g) in

shaking condition (120 rpm) at room temperature (30 ± 2oC) for 6 h per batch, and at the end

of each batch hydrolyzed substrate solution (2 ml) was centrifuged at 5000 rpm for 10 min in

cooling centrifuge, the clear supernatant was used for assay of reducing sugars. The

concentration of reducing sugars formed in the hydrolyzed substrate solution was quantified

by DNS method (Miller, 1959). The reaction mixture contained 0.4 ml of hydrolyzed

substrate and 0.6 ml of distilled water and 1 ml DNS (3,5-Dinitro-salicylic acid). Appropriate

83

control (unhydrolyzed substrate) was simultaneously run. DNS reagent was added to aliquots

of the reaction mixture and the colour developed was read at A540 nm in a spectrophotometer.

One unit of immobilized enzyme activity was defined as the amount of enzyme releasing 1

µmole of reducing sugar per gram (alginate beads) of immobilized enzyme per hour.

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teri and meth - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/8139/11/11...54 4. Solid state fermentation medium 4.1 Nutrient solution (pH 6.0) Yeast extract Sucrose KH 2PO 4