media optimization and characterization of thermos table amylase

8/6/2019 Media Optimization and Characterization of Thermos Table Amylase

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Media Optimization andMedia Optimization and

Characterization ofCharacterization ofThermostable Amylase fromThermostable Amylase fromAspergillusAspergillusnigernigerusing Riceusing Rice

bran.bran.

SWATI KUMARIM.Sc Industrial Biotechnology

Roll.No- IB1610

Reg.No-1546/2009


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INTRODUCTION: Amylases are starch degrading enzymes widely distributed

, .in microbial plant and animal kingdoms

Initially the term amylase was used originally to designate

enzymes capable of hydrolyzing - , -1 4 glucosidic bonds

, ,of amylose amylopectin glycogen and their degradation

.products

, .Amylase occurs in two forms designated and

- amylase only attacks the nonreducing ends of a starch

, ( - )molecule successively hydrolysing alternate l 4 linkages

.and releasing maltose molecules - - ( - amylase differs from amylase in that it can attack l

) .4 bonds within the starch molecule It thus degrades- .amylopectin more completely than amylase


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The production of the amylase enzyme under solid state conditionsformed the basis of this project. The work focused on the followingobjectives.

v Isolation and screening of amylase producing fungi from rhizospheresoil.

vDesigning of optimum media conditions favoring the growth of theselected fungal culture.

vPartial purification of the amylase by Ammonium sulphateprecipitation.

vDetermination of the factors affecting the enzyme activity.vDetermination of Molecular weight by SDS-PAGE .

OBJECTIVES


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.ollection of sample solation of amylolytic fungi on Potato Dextrose Agar.edium dentification of the isolates by colony morphology.nd microscopy .creening of isolates for amylase production .election of the Solid substrate .nalytical methods mylase assay ( ) .initro Salicylic Acid DNS method .election of the carbon supplement % .election of the best of carbon supplement .election of the nitrogen supplement % .election of the best nitrogen supplement .election of the optimal pH

METHODOLOGY


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.election of the optimal temperature .election of the percentage of inoculum election of the incubation time roduction of Amylase under optimum conditions .roduction of Amylase under optimum conditions .rotein assay :artial purification of enzymes .mmonium sulphate precipitation :haracterization of enzymes .ptimum temperature :ptimum pH :ffect of varying enzyme concentrations ffect of varying enzyme concentration onmylase activity .ffect of varying substrate concentrations .DS Poly Acrylamide Gel Electrophoresis


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RESULTSS. No Isolates OD at 540nm Enzyme Activity(U/gds)

1 A.niger

1.05 0.700

2 A.oryzae 0.47 0.313

3 A.flavus 0.32 0.257

4 Penicillium 0.08 0.053

Table 1: Results of initial screening of amylase production by fungal isolates

Figure 1: Results of initial screening of amylase production by fungal isolates

Screening of isolates for amylase production:


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S.No Solid substrate OD at 540nm Enzyme Activity(U/gds)

1. Corn cob 1.06 0.706

2. Rice bran 1.40 0.933

3. Sugarcane bagasse 0.781 0.521

4. Rice Straw 0.493 0.329

Table 2: Effect of solid substrate on Enzyme Production

Figure 2: Graph showing the effect of solid substrate on Enzyme Production

edia Optimization :ptimization of Solid Substrate


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S.No Carbon Source OD at 540nm Enzyme Activity(U/gds)

1. Maltose 0.37 0.246

2. Glucose 0.41 0.273

3. Sucrose 0.50 0.333

4. Soluble starch 0.53 0.353

65. Fructose 0.41 0.273

Table 3: Effect of Carbon sources on Enzyme Production

Figure 3: Graph showing effect of Carbon sources on Enzyme Production

ptimization of:arbon Source


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S.No % of Carbon Source OD at 540nm Enzyme Activity(U/gds)

1. 0.1 0.39 0.26

2. 0.3 0.945 0.63

3. 0.5 1.44 0.96

4. 1.0 1.56 1.04

5. 1.5 1.38 0.92

6. 3.0 1.14 0.76

7. 5.0 1.11 0.74

Table 4: Effect of percentage of soluble starch on Enzyme Production

Figure 4: Graph showing effect of Carbon sources on Enzyme Production

% :ptimization of of Carbon Source


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S.No Nitrogen sources OD at 540nm EnzymeActivity(U/gds)

1 Peptone 1.929 1.2862 Tryptone 1.239 0.8263 Beef Extract 1.369 0.9134 Ammonium chloride 0.979 0.6535 Ammonium sulphate 0.924 0.6166 Sodium nitrate 1.219 0.8137 Urea 1.053 0.702

Figure 5:Effect of different Nitrogen sources on enzyme production

Table 5:Effect of different Nitrogen sources on enzyme production

:ptimization of Nitrogen Source


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S.No % of Nitrogen Source OD at 540nm Enzyme Activity(U/gds)1. 0.1 1.659 1.1062. 0.3 1.354 0.9033. 0.5 1.215 0.814. 1.0 1.14 0.765. 1.5 1.11 0.746. 3.0 0.799 0.5337. 5.0 0.799 0.533

Table 6: Effect of percentage of peptone on Enzyme Production

Figure 6: Effect of percentage of peptone on Enzyme Production

% :ptimization of of Nitrogen Source


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S.No pH OD at 540 nm Enzyme Activity(U/gds)

1. 4.0 1.44 0.960

2. 5.0 1.40 0.936

3. 6.0 1.52 1.013

4. 7.0 1.37 0.913

5. 8.0 1.31 0.873

6. 9.0 1.35 0.900

7. 10.0 1.35 0.900

Table 7: Effect of different pH on enzyme production

ptimization for differentpH

Figure 7: Effect of different pH on enzyme production


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S.No Temperature (o C) OD at 540nm Enzyme Activity(U/gds)

1 25 1.587 1.058

2 30 3.699 2.466

3 35 1.470 0.984 40 1.465 0.976

5 45 2.208 1.472

Table 8: Effect of different temperature on enzyme production

:ptimization of the fermentation temperature

Figure 8: Effect of different temperature on enzyme production


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S.No Incubation Time(hrs)

OD at 540nm Enzyme Activity(U/gds)

1 24 0.376 0.251

2 48 0.477 0.318

3 72 0.703 0.469

4 96 1.701 1.134

5 120 2.538 1.692

6 144 1.927 1.285

7 168 1.893 1.262

8 192 1.378 0.919

Figure 10: Effect of different incubation time on enzyme production

Table 10: Effect of different incubation time on enzyme production

:ptimization of the incubation period


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ptimum temperature for enzyme activityS.No Temperature (o C) OD at 540nm Enzyme Activity(U/gds)

1 30 2.677 1.785

2 37 2.637 1.758

3 45 2.769 1.846

4 55 2.745 1.83

5 65 2.244 1.496

6 75 3.397 2.265

7 85 2.619 1.746

8 100 2.745 1.83

Table 11: Effect of different temperature on enzyme activity

Figure 11: Effect of different temperature on enzyme activity


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ptimum pH for enzyme activityS.No pH OD at 540 nm Enzyme Activity(U/gds)

1. 4.0 1.675 1.117

2. 5.02.494 1.663

3. 6.0 2.13 1.42

4. 7.0 1.344 0.896

5. 8.0 1.935 1.29

6. 9.0 2.118 1.412

7. 10.0 1.444 0.963

Table 12: Effect of different pH on enzyme activity

Figure 12: Effect of different pH on enzyme activity


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Figure 13: Effect of different enzyme concentrations on enzyme activity

S.No Enzyme concentration (l) OD at 540 nm Enzyme Activity(U/gds)

1. 20 0.726 0.484

2. 40 1.062 0.708

3. 60 1.300 0.867

4. 80 1.32 0.885. 100 1.885 1.257

6. 120 1.842 1.228

7. 140 2.523 1.682

8. 160 2.895 1.9309. 180 2.550 1.700

10. 200 2.095 1.397

ptimum enzyme concentration for enzymeactivity :

Table 13: Effect of different enzyme concentrations on enzyme activity


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:ptimum substrate concentration for enzyme activityS.No Substrate

concentration (l)OD at 540 nm Enzyme Activity(U/gds)

1. 20 1.723 1.149

2. 40 1.756 1.171

3. 60 1.923 1.2824. 80 1.962 1.308

5. 100 2.097 1.398

6. 120 2.175 1.449

7. 140 2.301 1.534

8. 160 2.310 1.540

9. 180 2.107 1.405

10. 200 1.995 1.330

Table 14: Effect of different substrate concentrations on enzyme activity

Figure 14: Effect of different substrate concentrations on enzyme activity


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:-stimation of Protein by Lowry s MethodTest tubes BSAvolume

in (ml)Conc ofBSA(g)

Distilledwater(ml)

Alkalinecopperreagent

Incubatefor 10min at

roomtemperature

FCreagent

Incubatein darkconditio

n for 20minutes

OD at660 nm

Blank12345

Unknown(0.5ml)Crude sample(0.5 ml)Ammoniumsulphate pptsample (0.5 ml)Dialyzedsample(0.5ml)

00.20.40.60.81

----

02004006008001000

----

21.81.61.41.21.0

1.51.51.51.5

5ml0.5ml

00.2180.3660.5260.6480.835

0.4180.4150.2160.229

Table 15: O.D values for standard graph for protein and partially purified enzyme at different stages


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olecular weight determination of the enzyme:

Figure 15: Protein profile of the partially purified amylase by SDS-PAGE.

Lane M, standard protein marker;Lane 1, amylase fromA. niger.The molecular sizes of the marker proteins (in kDa) are shown on the left.


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The results indicate that rice bran proved to be the best solid substratum might

be because of the ability of the substrate to retain high moisture and the ability toprovide air pockets to the organism due to its fluffy nature even under moistcondition.Our study proved that soluble starch as a carbon supplement increased the yieldof the enzyme and better enzyme activity when compared to the other usedsugars.A suitable amount of nutrient supplementation is required for the fermentationprocess, so that the enzyme can react with the substrate in appropriate amountand bring about the accumulation of the reaction product.Soluble starch was utilized at a percentage of 1% (w/w) to give the highest titerof the amylase production.In the present study, peptone was found to be the best nitrogen source for -amylase production byA.niger.

Nitrogen in the media results in the formation of the peptides and supplies theamino acids and the amounts at which they are present have got a steep role toplay in the formation of the desired product.The presence of 0.1% peptone (w/w) in the media resulted in the optimum C: Nratio which facilitated the highest production of the enzyme.


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In our study it was found that the best pH for the production of amylase under solidstate condition is 6.0. pH is one of the important factors that determine the growth and

morphology of microorganisms as they are sensitive to the concentration of hydrogenions present in the medium. Earlier studies have revealed that fungi required slightlyacidic pH and bacteria required neutral pH for optimum growth. pH is known to affect thesynthesis and secretion of a-amylase just like its stability (Fogarty M.W, 1983).

The influence of temperature on amylase production is related to the growth of the

organism. Hence, the optimum temperature depends on whether the culture ismesophilic or thermophilic. SinceA nigeris a mesophilicfungi the temperaturedemonstrating the highest enzyme activity was found to be 30 C. At this temperaturethe membrane was highly permeable and the enzyme synthetic machinery of theorganism was highly functional with the the maximum conversion rate of the starchpolymer into reducing sugars.

It is necessary that a particular microbial load has to be introduced into thefermentation media since any inappropriate amount would affect the level of the productformation.


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Our study had demonstrated that 0.5% inoculum size resulted in the highest

production of the enzyme, which is a promising finding since a small amount of themicrobial load can utilize the nutrients in the media to give the highest enzyme titre.

The maximum productivity of amylase was achieved in 120 hrs. Similar to our resultalso obtained the same result with his study on solid state fermentation ofAspergillus oryzae for glucoamylase production on agro residues. Incubationbeyond this period resulted in the decrease of enzyme activity).

The marketability of amylase is determined by its characteristics (Aunstrup,

1983). It is important to select the sources as thermostable, since amylases fromdifferent sources could have different product profiles.Different buffers of pH were used to study the effect of pH on amylase activity,

of which pH5 and 9 resulted in maximum enzyme activity. This suggests that theenzyme would be useful in processes that require wide range of pH change fromslightly acidic to alkaline range and vice versa. This property of the enzyme can beexploited for its industrial application. i.e., in food industry and in the manufacture of

detergents.

The SDS-PAGE analysis of the enzyme showed that the band appearedapproximate to 55 kDa, thus confirming it to be of -amylase. Similar result wasalso observed by found that the molecular of partially purified -amylase producedbyA. oryzae under SSF condition was 56 kDa.


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CONCLUSION -Production of amylase under solid state fermentation by the

rhizosphere isolate of Aspergillus niger was investigated using,rice bran one of the major solid waste released from the

.agricultural sector

A detailed investigation was used to evaluate the effect of the main

, . ., ( / ),variables i e additional carbon source 1w w soluble starch( . / ), (additional nitrogen source 0 1w w peptone incubation period 120

), ( ) (h pH 6 and temperature 300 ) .C on enzyme production Varying the

( . - %)inoculum concentration 0 1 5 of Aspergillus nigershowed that. % - .0 5 inoculum was the optimum for amylase production

The result of the stability of the partially purified enzyme at

( ) ( )higher temperature 75 C and at acidic and neutral pH pH 5 and 9

indicated that Aspergillus niger could be a potential fungal-candidate for amylase production in the food and detergent

.industry


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media optimization and characterization of thermos table amylase

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