Microsoft Word - Production of Protease Enzyme from Wheat Straw


 

 

Al-khwarizmi  
Engineering   

Journal 

Al-Khwarizmi Engineering Journal, Vol. 4, No. 2, PP 10 - 18 (2008) 

  

 

Production of Protease Enzyme from Wheat Straw 
 

Mohammed A. Atiya 
Biochem. Eng. Dept., Al-Khwarizmi Coll.Eng . 

Baghdad University 
 

(Received 9 September 2007; accepted 22 January 2008)                            
 

 

Abstract 

      Protease enzyme production was studied and optimized as  a first step to collect information about solid state 

fermenter) to produce protease enzyme. A local isolated Aspergillus niger was used for this study with constant spores 

feeding in every experiment at (105/g). Experiments carried out in conical flasks with (250 ml) containing (10 g) of 

wheat straw as a substrate with different conditions included temperature, pH, hydration ratio, and fermentation time, 

the results comprised by measuring protease activity (u). The results showed that the best activity can be obtained at (T 

= 32°C, t= 100 hrs, pH= 2.5 and hydration ratio is 1:3). On the other hand the results is courage to proceed to design a 

solid state protease fermenter from wheat straw. 

 

Keywords: Fermentation, Protease, Straw, pH, Hydration, Solid State Fermenter.  
 

 

Introduction 
      Protease enzyme is one of the most important 

industrial products also it used in many food and 

pharmaceutical applications. It is used in  

1.lether industries in dehairing and bating stages. 

2.detergents manufacturing 

3.cheese manufacturing 

4.protein hydrolysates 

      Most organisms (i.e. bacteria fungus) produce 

protease. The important thing to industry is to 

obtain extracellular enzymes. There are different 

type (acidic and basic)  of this enzyme depending 

on the organism and the cultivation conditions. 

The microps produce protease enzyme include 

type and six of bacteria and fungus differ in their 

microorganism, composition according to the 

products and the growing circumstances. Such 

microps can be isolated from natural sources like 

soil, crops material food by planting them in a 

amid contain protein source and preparing the 

proper circumstances to grow the microps, usually 

the milk agar is used for this purpose. The 

primarily isolation may be done by making a 

farms of raw materials by adding cheep protein 

materials to soil the amending the pH to produce 

the demanded enzyme and keep them into convent 

temperature. There are more than one definition to 

the substrates of solid states fermentation, one of 

them it: growing of microps on solid state without 

presence of water, or its substrate in which 

growing and producing microps on solid state 

were found (Michael, 1988, Agosin, 1985, 

Hameed, 1999, Battaglino, 1991, Cannel, 1980, 

Chen, 1977). 

      The aim of this research is to study the 

operating conditions of producing acidic protease 

enzyme from wheat straw by solid state 

fermentation to Aspergillus Niger and obtain the 

optimum conditions which can be used for future 

study of bioreactor design to produce this enzyme. 

 

Materials and Methods 
       Wheat straw used as a substrate for solid state 

fermentation hydrated by sodium acetate buffer, 

and a local isolated   Aspergillus niger was used 

as enzyme production source , sodium acetate, 

casein, TCA(trichloroacetic acid) were used as a 

chemicals ,also the following equipments were 

used for measures and analysis; sensitive balance, 

pH meter, autoclave (sterilizer), incubator, 

centrifuge, oven (dryer), spectrophotometer (UV), 

pipette, micro-pipette, test tubes, conical flasks. 

 



Mohammed A. Atiya                          Al-Khwarizmi Engineering Journal, Vol. 4, No. 2, PP 10-18 (2008) 

 

 ١١

Fermentation Procedure 
      The experiments were carried out in identical 

250 ml conical flasks each containing 10 g of 

wheat straw substrate which was hydrated by 

adding 0.02 molar of sodium acetate buffer 

solution with a certain value of hydration ratio as 

mentioned in attached (tables, 2, 3 and 4). The 

flasks were closed with cotton wool plugs. the 

medium was sterilized by an autoclave operating 

at 121°C of temperature, and pressure of 1.5 bar 

for 15 minutes. Proliferation was carried out by 

adding 1 ml inoculums to the medium and  

incubated at a certain temperature with respect to 

variables study as shown in tables (2, 3 and 4) and 

figures (1 to 8) later. Incubation periods ranged 

between (28-156 hr.), pH (1-4.5), temperature 

(24-38°C), hydration ratio           (1:1-5.5:1 

wt/vol), size of inoclum (spore number) was 

constant 105/g wet weight.  

     At the end of each of run, the samples from 

each flasks were prepare to the analytical step. 

 

Analytical Procedure 

Enzyme Activity  
      Protease activity was measured by degradation 

of casein. 2 mL of culture filtrate was added to 2 

mL of 1% (w/v) casein (pH 7.5) and incubated for 

10 min at 40°C. The reaction was stopped by 

adding a protein-precipitating agent, 4 mL of 0.4 

molar trichloroacetic acid (TCA). Solutions were 

centrifuged in speed of 2500 rpm for 20 minutes. 

The precipitated protein was removed by filtration 

and the absorption of filtrate was measured at 275 

nm. One unit  of protease activity (u) was defined 

as the amount of enzyme that released 1 micro 

gram  of tyrosine per min under assay condition 

(pH 7.5, 40°C). Blank  was prepared by the same 

way except adding TCA  before adding enzymes 

the values will compare with  prepared standard 

absorption tyrosine curve at 275 nm (Liy, 2002, 

Murachi, 1976). 

 

 Protein Concentration  
      Dissolving the precipitated protein which was 

removed as mentioned above by adding 0.05 

molar NaOH and the optical density of the 

solution was measured at 280 nanometer and 235 

nanometer. Blank solution of 0.05 molar NaOH 

was used. 

      The equation used to estimate protein 

concentration is (Whiteaker,and Granum 1980): 

 

Protein concen.(mg/ ml) = (light abs. at 235 nm–

light abs. at 280nm)/2.5                                 … (1)        

 

Results and Discussion 

Effect of Hydration Ratio  
      Aspergillus niger was cultured or inoculate in   

hydrated wheat straw by adding 0.02 molar of 

sodium acetate buffer in the range of (1:1–5.5:1 

wt/vol). The effect of  hydration ratios were 

studied. These studies were conducted at constant 

temperature (32oC), pH = 2.5  and cultivation time 

of  100 hours as shown in table (1) and figs. (1, 2).  

 
Table 1 

Hydration ratio effect on enzyme activity and 

enzyme yield at constant temperature of 32°C, 

incubation period of 100 hours and pH = 2. 

Hydration 

ratio 

Enzyme activity 

(×100) 

Enzyme yield 

(×1000) 

(weight/ 

volume) 
(u /g protein) (u/g substrate) 

1.00 6.30 0.48 

1.50 10.00 0.60 

2.50 11.90 0.70 

3.00 12.40 0.79 

3.50 11.50 0.75 

4.50 7.70 0.50 

5.50 2.80 0.30 

       

      From these results, the enzyme activity 

increased slightly with hydration ratio up to 1:3 

and then decreases. The results indicated that at 

1:3 hydrations ratio enzymes production with 

highest yield and activity of 790 units per g 

substrate and 1240 unit per mg protein 

respectively. The effect of hydration ratio can be 

divided in two partitions. In the first partition the 

increasing of hydration will increase mass transfer 

media needed for oxygen transformation and 

cetric acid produced dilution. But when hydration 

ratios more than (1:3) the concentration of 

nutrients will be decreased rapidly and that inhibit 

the yeast growth and also enzyme quality and 

quantity will be that decrease the activity of 

protease enzyme. 

 

Effect of pH  
      Different values of  pH medium were studied 

at constant temperature (32°C), proliferation  time 

of  100 hours and hydration ratio of 1:3 ,as shown 

in table (2) and figures (3 and 4). 

      The studied of  pH  values of growth medium 

were  initially adjusted to pH values of 1, 2, 2.5, 3, 

3.5, 4, 4.5 before sterilization. 

 

 



Mohammed A. Atiya                          Al-Khwarizmi Engineering Journal, Vol. 4, No. 2, PP 10-18 (2008) 

 

 ١٢

Table 2  

pH values effect on enzyme activity  and enzyme 

yield at constant other variables (temp. =32°C, 

incubation period of 100 hours  and hydration ratio 

1:3. 

pH 

Enzyme activity 

(×100), 

Enzyme yield 

(×1000) 

u /g protein u/g substrate 

1 8.8 0.41 

2 9.2 0.46 

2.5 9.8 0.58 

3 9 0.48 

3.5 7.3 0.39 

4 5.9 0.32 

4.5 4.4 0.242 

 

       The results showed that the activity of 

enzyme increases from (880 u per g protein to 980 

u per g protein) when the pH increases from 1-2.5 

respectively and then decreases to reach 440 u per 

g protein when the pH is 4.5. Also the results 

showed that the enzyme yield has the same 

behavior and reach the maximum value of 580 u 

per g substrate when pH value is 2.5 .The results 

indicated the best proliferation pH value of 2.5. 

 

Effect of Temperature 

      The effect of different temperature range 

between (24 to 38°C) were studied at constant pH 

2.5, proliferation time of 100 hours and hydration 

of 1:3 as shown in table (3) and figs. (5 and 6). 

      The enzyme activity increased from (1020 to 

1200 u per g protein) when temperature increased 

from 24 to 32oC and then decreased from this 

value to (620 u per g protein) when temperature 

reached 38oC.The reason of this behavior can be 

consider as the relation between organism growth 

and temperature in one hand and the relation 

between organism growth and enzyme production 

in the another hand. Aspergillus Niger can be 

grown well in middle temperature range around 

32oC. 

 
Table 3 

Effect incubation temperature on enzyme activity and 

enzyme yield at constant pH =2.5, incubation period of 

100 hours and hydration ratio 1:3. 

Temp.( oC) 

Enzyme activity 

(×100), 

Enzyme yield 

(×1000) 

u /g protein u/g substrate 

24 10.2 0.6 

28 11.5 0.68 

32 12 0.7 

34 10.4 0.64 

36 8.5 0.49 

38 6.2 0.28 

 

Effect of Cultivation Time  

      Proliferation of Aspergillus niger for several 

incubation time were studied at constant 

temperature (32°C), pH 2.5 and hydration ratio of 

1:3 in order to produce highest enzyme activity 

with highest yield  .the results indicated that when 

proliferation time increases that will cause an 

increase in enzyme activity and yield from 180 

and 130 to  reached its maximum values of 1200 

and 780 respectively at time equal to 100 hours  

during  increasing in cultivation time from 28 to 

100 hours  and then decreases to 700 and 600 at 

time equal to 156 hours .The results indicated the 

highest yield enzyme activity and highest enzyme 

activity obtained at incubation period of 156 hours 

as shown in table (4) and figures (7 and 8). 

 
Table 4. Effect of incubation time on enzyme activity 

and enzyme yield at constant other variables 

(temperature of 32°C, pH of 2.5 and  hydration ratio of 

1:3.   

Time 

(hours) 

Enzyme activity 

(×100), 

Enzyme yield 

(×1000) 

u /g protein u/g substrate 

28 1.8 0.13 

52 6.2 0.3 

76 11.3 0.6 

100 12 0.78 

124 11.1 0.74 

148 9 0.68 

156 7 0.6 

 

Conclusion  
      From this study it can be recommended that it 

is good to produce protease enzyme with high 

activity using wheat straw substrate plants as a 

solid state fermentation of Aspergillus Niger. So it 

is important to go ahead in studying other 

parameters such as hydrodynamic of bioreactor to 

produce this enzyme according to the parameters 

mentioned in this study. The results showed that 

the best activity can be obtained at (T=32°C, 

t=100 hrs, pH= 2.5 and hydration ratio is 1:3). 

 

 

 

 

 

 

 

 

 

 

 



Mohammed A. Atiya                          Al-Khwarizmi Engineering Journal, Vol. 4, No. 2, PP 10-18 (2008) 

 

 ١٣

 

Fig.1 Hydration ratio effect on enyzme activity  at constant temperature of 32 ,incubation  time of  100 

hours and pH of 2.5.

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6

hydration ratio(weight /volume)

E
n

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y

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c
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y
(*

1
0

0
),

  
  

  
u

 /
g

m
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r
o

te
in

 
 

 

 

 

Fig.2 Hydration ratio effect on  enzyme yield at constant temperature of 32 ,incubation  time of  100 hours 

and pH of     2.5.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1 2 3 4 5 6

 hydration ratio(weight /volume)

E
n

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y
m

e
 y

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 (
*
1

0
0
0

)u
/g

m
 s

u
b

st
r
a
te

 
 

 

 

 

 

 

 

 

 



Mohammed A. Atiya                          Al-Khwarizmi Engineering Journal, Vol. 4, No. 2, PP 10-18 (2008) 

 

 ١٤

 

Fig .3  pH values effect on enzyme activity  at constant other variables (temperature of 32 ,incubation  time  of  

100 hours and  hydration ratio of 1:3.

0

2

4

6

8

10

12

14

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

PH values

E
n

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y
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c
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y
(*

1
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0

),
u

 /
g

m
 p

r
o
te

in

 
 

 

 

 

Fig 4.pH values effect on enzyme yield  at constant other variables (temperature of 32 ,incubation  time of  100 

hours and  hydration ratio of 1:3.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

PH values

E
n

z
y
m

e
 y

ie
ld

 (
*
1
0

0
0

)u
/g

m
 s

u
b

st
r
a
te

 
 

 

 

 

 

 

 

 

 



Mohammed A. Atiya                          Al-Khwarizmi Engineering Journal, Vol. 4, No. 2, PP 10-18 (2008) 

 

 ١٥

Fig. 5 Effect of incubation temperature on enzyme activity at constant pH 2.5 ,  incubation  time of  100 

hours and hydration ratio of 1:3 .

0

2

4

6

8

10

12

14

22 24 26 28 30 32 34 36 38 40

Temperature.(c)

E
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(*

1
0

0
),

u
 /

g
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in

 
 

 

 

Fig. 6 Effect of incubation temperature on enzyme yield at constant pH 2.5 ,  incubation  time of  100 hours 

and hydration ratio of 1:3

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

20 22 24 26 28 30 32 34 36 38 40

Temperature.(c)

E
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y
m

e
 y

ie
ld

 (
*

1
0
0

0
)u

/g
m

 s
u

b
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a
te

 
 

 

 

 

 

 

 

 

 

 

 



Mohammed A. Atiya                          Al-Khwarizmi Engineering Journal, Vol. 4, No. 2, PP 10-18 (2008) 

 

 ١٦

 

 

Fig.7 Effect of incubation time on enzyme activity  at constant other variables (temperature of 32 ,pH of 2.5  

and hydration ratio of 1:3)

0

2

4

6

8

10

12

14

0 20 40 60 80 100 120 140 160 180

Time(hours)

E
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(*

1
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0

),
u

 /
g
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in

 
 

 

 

Fig.8 Effect of incubation time on  enzyme yield at constant other variables (temperature of 32 ,pH of 2.5  

and hydration ratio of 1:3.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 20 40 60 80 100 120 140 160 180

Time(hours)

E
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 (
*
1

0
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0
)u

/g
m

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Mohammed A. Atiya                          Al-Khwarizmi Engineering Journal, Vol. 4, No. 2, PP 10-18 (2008) 

 

 ١٧

 

 
 

Reference 
[1] Agosin, E., Solid state fermentation, 1985. 

[2] Battaglino, R. A., Culture requirements of 

production of protease by aspergillus orzyea 

in solid state fermentation. Appl. Microbial. 

Biotechnol. 35:292 , 1991. 

[3] Cannel, E., and Moo-young, M., solid state 

fermentation systems, 1980. 

[4] Cohen , B. L., ,The proteases of aspergillus. In 

genetic and pHysiology of aspergillus, p 281, 

1977. 

[5] Hameed, A.; Keshavarz, T.; Evans, C. S. 

Effect of dissolved oxygen tension and pH on 

the production of extracellular protease from 

a new isolate of Bacillus subtilis K2, for use 

in leather processing. J. Chem. Technol. 

Biotechnol., 74, 1999. 

[6] LiY,H, Hu, H. Kinetic model of neutral 

proteins fermentation process. J. Chem. Eng. 

Chin. Univ. 9, 404-408, 1995. 

[7] Murachi, T, Methods in Enzymology, Vol.19,   

P.273, 1976. 

[8] Whiteaker, J.R. and Granum, P.E., An 

absolute method of protein determination, 

Annl. Biochem, 1980. 

[9] Michael J. pelczar , Microbology ,1988. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



Mohammed A. Atiya                          Al-Khwarizmi Engineering Journal, Vol. 4, No. 2, PP 10-18 (2008) 

 

 ١٨

 

  انزيم البروتيز من نخالة الحنطة انتاج
 

  محمد عبد عطية
  جامعة بغداد / كلية هندسة الخوارزمي / قسم هندسة الكيمياء االحيائية

  

  
  الخالصة

المخم"ر  غ"رض تص"ميملتم دراسة عملية إنتاج انزيم البروتيز كخطوة اولى من أجل وضع المعايير االساسية وإيجاد الظروف التش"غيلية           

fermenter من خالل تنمية كائنات حية مجهرية وهي نوع من االعفان تسمى  في الحالة الصلبةAspergills niger)،( عي هو على وسط زر

تعادل"ة بنس"ب منخالة الحنطة . تتكون مراحل البحث من تغيير الحامضية للوسط الزرعي حيث نقوم بترطيبه بمحاليل قاعدية وحامضية واخ"رى 

لمع"د س"لفا" امل  حيث يوضع بها الوسط الزرعي وهو نخالة الحنطة وي"تم  اللق"اح بالك"ائن الح"ي  ٢٥٠مختلفة حيث توضع باوعية حجم ترطيب 

رارة عل"ى الفعالي"ة / غرام  ثم يتم اخذ النماذج ودراسة تاثير الحامضية ونسبة الترطي"ب،زمن التخمي"ر، ودرج"ة الح" ٥١٠بعدد من الخاليا عددها 

  لمتغيرات.الالنزيم المنتج وكميته  حيث يتم تثبيت احدى المتغيرات السابقة وتثبيت المتغيرات االخرى وايجاد الظروف المثلى لهذه 

، اعةس"" ١٠٠م، وال""زمن يس""اوي °٣٢المخم""ر ه""ي درج""ة الح""رارة تس""اوي تش""غيلية المثل""ى لغ""رض تص""ميم أظه""رت النت""ائج إن الظ""روف ال     

  .١:٣لترطيب تساوي ونسبة ا ٢٬٥والحامضية تساوي