Optimization, immobilization of extracellular alkaline ... · PDF fileThe enzyme was partially purified with 50% acetone and showed 4.1-fold purification. ... Streptomyces, application

Research Journal of Agriculture and Biological Sciences, 4(5): 434-446, 2008© 2008, INSInet Publication

Corresponding Author: N efisa M. A . El-Shayeb, Department of Chemistry of Natural and Microbial Products National

Research Center, Cairo, Dokki, Egypt.

E-mail: [email protected]

434

Optimization, Immobilization of Extracellular Alkaline Protease and Characterization of its Enzymatic Properties

Samia A. Ahmed, Ramadan A. Al-domany, Nefisa M.A. El-Shayeb, 1 2 3

Hesham H. Radwan and Shireen A. Saleh.4 5

Department of Chemistry of Natural and Microbial Products 1,3,5

National Research Center, Cairo, Egypt. Department of Microbiology and Immunology, Faculty of Pharmacy, 2,4

Helwan University, Egypt.

Abstract: Streptomyces avermectinus NRRL B-8165 was the most potent for production of extracellular

alkaline protease among 6 strains of Streptomycetes using shaken culture technique (180 rpm) after 72hat 37°C. Highest yield of extracellular enzyme (3.62 U/ml) was achieved using 7% sucrose and 0.5435%

alkaline extracted soybean as carbon and nitrogen sources, respectively. Addition of wheat bran extract

in concentration of 20 g/L increased alkaline protease production by 24.29% than control. Adding 0.25%

2corn oil and MnCl (0.1mM) as trace element to the medium caused a slight increase in enzyme activity.

The enzyme was partially purified with 50% acetone and showed 4.1-fold purification. Alkaline protease

was immobilized on different carriers by different methods and its specific activity for casein hydrolysiswas studied. Immobilization of alkaline protease on sponge (covalent binding) had the highest

immobilization yield (86.19%) using glutaraldehyde (0.25% concentration). It was further observed thatthermal stability of the immobilized enzyme was higher compared to free enzyme. The immobilized

m maxenzyme had higher K (Michaelis constant) and lower V (Maximum rate of reaction) than the free

Aenzyme. Activation energy (E ) of the free enzyme was 6.206 Kcal/mol which was higher than the

1/2immobilized enzyme. Half-life (t ) of immobilized enzyme was 808.6 and 108.6 at 65 and 70°C,

respectively, which was higher than that of free enzyme. Immobilized enzyme showed the highest

operational stability for up to 15 reuses with 49.74% residual activity. On the other hand, someapplications of enzyme were carried out (dehairing, decomposition of gelatin layers on X-ray films and

detergent stability).

Key words: Alkaline protease, immobilization, Streptomyces, application

INTRODUCTION

Proteases are the most important kind of industrial

enzymes and account for about 65% of the[22]

worldwide sale of industrial enzymes in the world

market . Microbial alkaline proteases dominate the[21]

worldwide enzyme market, about two-third share of thedetergent . Applications of alkaline protease are[18]

concentrated in laundry detergents, leather

processing, b re wing, food and pharmaceuticalindustries . The enzyme should be stable and active[26]

in the presence of typical detergent ingredients for usein detergent . Microorganisms served as an important[37]

source of proteases mainly due to their shorter

generation time, the ease of bulk production and theease of genetic and environmental manipulation .[39]

Streptomyces species are the most industrially

important actinomyces, due to their capacity to produce

numerous secondary metabolites and particularlyantibiotics. The ability of these bacteria to produce

large amounts of enzymes, as proteases with varied

substrate specificities, offers another potentiallyinteresting use . For industrial applications, the[40]

immobilization of proteases on a solid support can

offer several advantages, included repeated usage of theenzyme, ease of product separation and improvements

in enzyme stability. Enzyme stabilization will thus

continue to be a key issue in biotechnology.In the present study, certain environmental and

nutritional parameters controlling the biosynthesis ofextracellular alkaline protease from Streptomyces

avermectinus NRRL B-8165, partial purification and

the immobilization of partially purified enzyme ondifferent carriers using diffe re n t me thods of

immobilization including physical adsorption, ionic

binding, covalent binding and entrapment were fully

Res. J. Agric. & Biol. Sci., 4(5): 434-446, 2008

435

investigated. The immobilized enzyme by covalentbinding on sponge had the highest immobilization yield

(86.19%). The refore, this immobilized enzyme

preparation was used as a typical example forStreptomyces avermectinus NRRL B-8165 immobilized

alkaline protease and its properties was investigatedand compared with the free one. Also, in this paper,

we present potential applications of extracellular

alkaline protease for different industrial purposes.

MATERIALS AND METHODS

Organism: Streptomyces avermectinus NRRL B-8165

was obtained from Northern Regional Research

Laboratory (NRRL), Peoria, Illinois, USA.

Carriers for Enzyme Immobilization: Polystyrene,

Dowex 50 Wx4 (50-100) mesh, Dowex 1x8 (20-50)mesh, cellulose triacetate, DEAE-Sephadex A-50,

chitin, agarose, chitosan were from Fluka Company,

Switzerland. Sephadex G-100 was obtained fromPharmacia fine chemicals, Uppsala, Sweden. DEAE-

cellulose 53 was obtained from Whatman Biosystems

Ltd.

Medium Composition and Cultivation: The basalmedium for liquid cultures consisted of (g/L):

4 2 4 2 4 3(NH ) SO , 1; KH PO , 0.5; CaCO , 1; NaCl, 1;

4MgSO , 1; peptone, 1; dextrin (white pract), 30; .[2]

The pH was adjusted to 8.0. The same medium was

also used for inoculum preparation for all the

fermentation experiments. Cultivation was in 250Erlenmeyer flasks containing 50 ml of sterile medium.

The flasks were inoculated with 2 ml of 24h old

culture. The flasks were incubated at 37°C withshaking at 180 rpm. The cells were harvested by

centrifugation at 4000 rev/min. The clear culture

filtrates were assayed for enzyme activity.

Assay of Protease Activity: It was determined using

casein as a substrate according to the method reportedby Gessesse and Gashe with some modifications and[17 ]

protease activity was determined as released tyrosine.

One unit of enzyme activity was defined as the amountof enzyme that librates 1µmole of tyrosine per min

under the above mentioned conditions.

Protein Determination: This was estimated according

to the method of Lowry et al. .[32]

Partial Purification of the Enzyme: This was done by

fractional precipitation using ethanol or acetone or bysalting-out with ammonium sulphate using cell free

culture filtrate of the optimized fermentation medium.

Different acetone concentrations (30-60 %v/v) were

tested. The precipitation was done in a sequentialmanner. The precipitated fractions were removed by

centrifugation using a refrigerated centrifuge at 4000

rev/min for 15 min. The active fractions werelyophilized and assayed for caseinolytic activity.

Immobilization Techniques:

Physical Adsorption: It was carried out according to

Woodward (1985), one gram of each carrier wasincubated with the 50% acetone enzyme fraction (66.5

U/g carrier) dissolved in 5 ml glycine-NaOH buffer

(0.1 M, pH 10) overnight at 4°C.

Ionic Binding: It was carried out according to

Woodward in which 0.1 g of the cation or anion[50]

exchanger was equilibrated with glycine-NaOH buffer

(0.1 M, pH 10) and incubated with 1 ml of enzyme

solution (6.65 U) overnight at 4°C.

Covalent Binding: This method was carried out as

follows: One gram of were treated with 5ml of 0.25%(v/v) glutaraldehyde for 2 h at 30°C. Then washed with

distilled water to remove the excess glutaraldehyde.

The carriers were mixed with 5 ml of enzyme solution(66.5 U) and incubated overnight at 4°C .[3]

Entrapment:

In Agar and Agarose: 10ml of different concentrations

of agar (2, 3 and 4%) or agarose (1, 2 and 3 %)solutions were mixed with enzyme solution (2.66U).

The mixture was quickly cooled to 4°C and cut into

small cubes .[50]

In Ca-alginate Beads: 10ml of different concentrations

of sodium alginate solution (1, 2 and 3%) were mixedwith enzyme solution (2.66U). The entrapment was

carried out by dropping the alginate solution into 0.15

2M CaCl . The resulting beads (0.5-1.0mm diameter)were collected .[8]

Thermal Stability: The thermal stability of free andimmobilized protease were tested by incubating the

enzymes in 0.1 M glycine-NaOH buffer (pH 10) at

different temperatures (30-75°C) for different timeintervals (15-120 min) before activity assay.

Effect of the Reaction Time: The enzyme assay was

conducted at 55°C and pH 10 for different time

intervals (5-60 min).

Effect of the Addition of Some Salts of Metal Ions:

Free and immobilized enzymes were incubated, inabsence of substrate, with metal ions in different

concentrations (0.1, 1.0 and 10.0 mM) at 30°C

for 1 h.


436

Operational Stability of the Immobilized Enzyme:

2.5 grams of immobilized enzyme (wet) containing

about 66.5 U was incubated with substrate in glycine-

NaOH buffer (0.1M at pH 10) at 55°C for 20 min. At

the end of the reaction period, the carrier was squeezed

washed with buffer and the immobilized enzyme was

resuspended in freshly prepared substrate to start a new

run. The wash was assayed for alkaline protease

activity.

Some Applications:

Detergent Stability of Crude Extracellular Alkaline

Protease: This was carried out according to Hadj-Ali

et al. . [20]

Decomposition of Gelatin Layers on X-ray Films:

This was done according to Masui et al. method[34]

with some modifications.

Dehairing Agent: This was done according to Zambare

et al. method. [51]

RESULTS AND DISCUSSION

The ability of 6 Streptomycetes strains to produce

active extracellular alkaline protease was investigated.

They were cultured on the basal medium for different

incubation periods under shaking conditions and in

each case the culture filtrate was assayed for caseinase

activity. The results indicated that Streptomyces

avermectinus NRRL B-8165 was the most potent

producer of extracellular alkaline protease (2.04 U/ml)

after 72 h incubation at 37°C. These results are similar

to those reported by Mohamedin from Streptomyces[36]

thermonitrificans and Gupta et al. from Virgibacillus[19]

pantothenticus for alkaline protease production.

Substitution of the dextrin in the culture medium

by other carbon sources (sucrose, glucose, soluble

starch, lactose, maltose, molasses and casein) indicated

that lactose and casein significantly inhibited the

alkaline protease production. Sucrose proved to be the

most favorable carbon source for the production of

active alkaline protease by Streptomyces avermectinus

NRRL B-8165 (2.61 U/ml). On the other hand,

maltose, molasses, dextrin and glucose gave lower

levels of enzyme productivity compared to sucrose.

These results are similar to that reported by Chi and

Zhao who found that different carbon sources have[ 1 4 ]

different influence on extracellular enzyme production

by different strains. The results indicated that gradual

increase in sucrose concentration from 0.0 to 7.0%

resulted in 12.5-fold increase in alkaline protease

production. Higher levels however showed an adverse

effect on enzyme production. The result is highly

compared to t ha t found by Kathiresan and

Marivannan who reported the production of alkaline[24]

protease at 5.0% sucrose by Streptomyces sp.

Also, different nitrogen sources in the culture

medium were investigated. This included organic

(yeast extract, backer's yeast, peptone, casein, alkaline

extracted soybean and urea) and inorganic (ammonium

sulphate and ammonium chloride), nitrogen sources

used in the production medium on equal nitrogen basis.

It seemed that the alkaline extracted soybean was more

suitable for the enzyme production than the other

nitrogen sources. Ammonium chloride gave the least

enzyme productivity, while ammonium sulphate

inhibited the enzyme production completely. This result

is similar to that reported by Kaur et al. and[25]

Chauhan and Gupta . They found that organic sources[11]

were better suited to Bacillus sp. for enzyme

production. It has been reported that the effects of a

specific nitrogen supplement on protease production

differ from organism to organism although complex

nitrogen sources are usually used for alkaline protease

production . However, addition of inorganic sources[29]

resulted in decreased production of enzyme. Similar

results also have been obtained with other Bacillus

sp. . In contrast to our results, Sinha and[ 4 1 ]

Satayanarayana and Nascimento and Martins[ 4 4 ] [38]

reported that higher protease production with inorganic

nitrogen sources such as ammonium sulphate and

ammonium nitrate.

Therefore, alkaline extracted soybean was used as

the most suitable nitrogen source for maximal enzyme

production. This result is in agreement with that

obtained by Mabrouk et al. . The study also included[33]

the effect of different concentrations of alkaline

extracted soybean as nitrogen source in the culture

medium. The succeeding experiments were performed

using alkaline extracted soybean at 5.435 g/L

concentration in the culture medium. This concentration

was shown to be the most effective for the enzyme

production (3.62 U/ml).

It is worthy to note that calcium carbonate in the

basal medium was used as a buffering agent.

3 Therefore, the effect of different levels of CaCO was

also investigated. The maximal extracellular alkaline

protease production was obtained when its level was

30.5% g/L. Increasing CaCO level from 0.0 to 0.5 g/L

resulted in 10.44-fold increase in enzyme production.

However, the higher levels showed an adverse effect

on the enzyme production Fig3. In this respect, Laan

and Konings reported that higher concentration of[30]

Ca ions negatively affected the release of proteinase2+

3from Lactococcus lactis. This indicated that CaCO at

0.1% was below the critical level at which the

inhibition of protease occurred.


437

Some experiments were done in order to explore

the effect of external additives on the enzyme

production. The addition of wheat bran extract and

corn oil was studied. The results showed that wheat

bran extract in concentration of 20 g/L gave alkaline

protease of (5.03 U/ml) with increase in the enzyme

activity by 24.20% than the control.

Also, adding 0.25% corn oil to the medium as a

surfactant led to a little increase in productivity

(1.38%). This result is in agreement with that

obtained by Mabrouk et al. by Bacillus licheniformis[33]

ATCC 21415.

With regard to the effect of addition of some salts

of metal ions indicated that Mn (1mM) enhanced the2+

production of extracellular alkaline protease. On the

other hand, Zn , Fe and Cu had adverse effects on2 + 3+ 2+

the enzyme production. This result is similar to that

reported by Shafee et al. for a Bacillus sp. alkaline[ 42]

protease.

Production of extracellular alkaline protease by

Streptomyces avermectinus NRRL B-8165 was

considerably affected by the initial pH values. It can be

noticed that pH 7.5 was the most favorable for the

alkaline protease production. Above and below initial

pH values showed a gradual decrease in enzyme

production. This result is similar to those reported by

Banik and Prakash who produced alkaline protease[7]

maximally at pH 7.5 after 72h incubation from

Bacillus cereus.

After optimization of the chemical composition of

the production medium for the biosynthesis of

extracellular alkaline protease by Streptomyces

avermectinus NRRL B-8165, the results indicated that

the culture filtrate possessed extracellular alkaline

protease of 5.22 U/ml. This culture medium was

therefore used in all succeeding work.

Alkaline protease from Streptomyces avermectinus

NRRL B-8165 was partially purified by ethanol,

acetone and ammonium sulphate. The most of

proteolytic activity was recovered at 50% acetone

(21.02) and showed 4.10-fold purification. The results

presented in Table1 points to the most active fractions

produced by ethanol, acetone and ammonium sulphate.

Immobilization of the partially purified extracellular

alkaline protease was studied using the 50% acetone

fraction. This was done using different supports and

different methods of immobilization such as physical

adsorption, ionic binding, covalent binding and

entrapment. The efficiency of enzyme immobilization

was evaluated different parameters including the

retained enzyme activity, the specific activity of the

immobilized enzyme and the loading efficiency

(immobilized activity/g carrier). The immobilization

yield is the key parameter since it represents the

general output of the efficiency of the immobilization

process .[9]

The data for immobilization of the extracellular

partially purified alkaline protease by covalent binding

(Fig1) indicated a good immobilization yield with

sponge through a spacer groups (glutaraldehyde) which

showed good load ing e f f ic ienc y and good

immobilization yield. The good loading efficiency for

the immobilization by covalent binding may be due to

the formation of stable crosslinking between carrier and

enzyme through a spacer group (glutaraldehyde). In

addition, covalent binding through a spacer group

probably increased the surface area of the carrier and

consequently reduced the steric hindrance in the

immediate vicinity of the enzyme molecule. These

results are similar to those reported by Siso et al. .[45]

The results also showed that 0.25% glutaraldehyde was

the best concentration to activate support (sponge) for

giving the highest enzyme immobilization yield (77.47

%). Carrara and Rubiolo reported that the best[10]

concentration of glutaraldehyde was 1% for support

(chitosan) activation. While Tanksale et al. and[48]

Ferreira et al. found the maximum immobilization[16]

yield was obtained with 1.76 and 0.5% glutaraldehyde,

respectively. The decrease in immobilization yield by

increasing glutaraldehyde concentration may be due to

the reticulation among enzyme molecules favored by

the use of a bifunctional molecule and therefore,

affecting the overall activity .[16]

The data for the immobilization of alkaline

protease by ionic binding (Fig2) showed low

immobilization yield (11.03%) on DEAE- Sephadex.

On the other hand, the immobilized enzyme on

Amberlite IR-120 showed the highest immobilization

yield (34.25%). Similar results were previously reported

for Bacillus mycoides protease immobilized by ionic

binding on Amberlite IR-120 . [1]

Immobilization of Streptomyces avermectinus

NRRL B-8165 alkaline protease by physical adsorption

(Fig 3) was employed on different carriers including

wool, stone, sponge, loofa, corn pulps, chitin and

cellulose. The immobilization of the enzyme by

physical adsorption on sponge had the highest

immobilization yield (42.36%) and the highest loading

efficiency (37.77 U/g carrier).

The immobilization of the alkaline protease by

entrapment in calcium alginate, agar or agarose was

done using different concentrations (Fig 4). The

results indicated that agarose (1%) gave the highest

immobilization yield (49.79%). At higher carrier

concentration the immobilization yield decreased.

This may be due to decreased gel porosity with the

increase in gel concentration and consequently the

diffusion limitation was developed. Similar observations


438

were previously reported for other entrapped

enzymes .[31]

From all immobilization methods and carriers, the

enzyme immobilized by covalent binding on sponge

was used as a typical example for immobilized

Streptomyces avermectinus NRRL B-8165 alkaline

protease. The properties were investigated and

compared with those of the free one (Table 2).

The specific activity of immobilized was about

71.1% of the free enzyme which was higher than that

obtained by Mittal et al. with immobilized enzyme[35]

(56%). while Suh et al. reported that the specific[47]

activity of immobilized enterokinase was 25% .

Investigation of the effect of the reaction time of

immobilized enzyme and free enzyme were 20min and

10min (data not shown). In this respect, Mittal et al.[35]

re por t e d a c a lc iu m a lg in a t e im m o b i l i z e d

dipeptidylpeptidase which had maximum activity of

90min compared to 60min for the free. They explained

that this was possibly due to limited diffusion of

substrate initially, but once a sufficient amount of

substrate had entered the beads then the enzymatic

activity increased because of the close proximity of

substrate and enzyme.

The optimum temperature of the reaction was not

affected by immobilization process (55°C). This result

is similar to that reported by Abdel-Naby et al. who[1]

reported that both free and immobilized alkaline

protease covalently linked to chitosan had the same

optimum temperature (50°C).

The calculated values of the activation energies for

the free and immobilized enzymes were 6.206 and

5.688 Kcal/mol, respectively. The lower value of

activation energy of the immobilized enzyme compared

to the free enzyme may be attributed to the mass

transfer limitations. Kitano et al. and Allenza et al.[27] [5]

reported that the activation energies of the

immobilized enzymes were lower because the internal

diffusion is the rate limiting step. The decrease of the

activation energy of immobilized protease was also

reported by .[16,49]

The rate of heat inactivation of the free and

immobilized enzyme was investigated at 65 and 70°C.

In general, the immobilization process protected the

enzyme against heat inactivation. For example, the

calculated half-live values at 65 and 70°C for the

immobilized enzyme on sponge were 808.6 and 108.6

min, respectively, which were higher than that of the

free enzyme.

The result is higher than that reported by Ahmed

1 /2 et al. on wool immobilized alkaline protease (t at[4]

60 and 70°C were 100 and 25, respectively). Also,

Silva et al. reported lower half live of immobilized[43]

protease at 60°C (438 min) than our result.

The calculated deactivation rate constant showed

that the thermal stability of the immobilized enzyme

was superior to that of free one due to protection

that provided by immobilization to enzyme .[28]

The calculated deactivation rate constants at 65 and

70°C for the free enzyme were 4.20 x 10 and 9.33 x-3

10 and for the immobilized enzyme 0.857 x 10 and-3 -3

6.38 x 10 , respectively.-3

The investigation of the free and immobilized

enzymes at different pH values showed that the

optimum pH values of the free and immobilized

enzymes were the same (pH 10.0) Table (2). This

result is similar to that reported by Chellapandian and

Sastry who reported that alkaline protease covalently[12]

linked on nylon had optimum pH 8.5 which was

similar to that of free 8.5-9.0.

Thermal stability of the immobilized enzyme was

also studied (Fig 5 & 6). In general, the thermal

stability of the immobilized enzyme was significantly

improved in comparison to the free enzyme. Thus after

heating the enzyme, in the absence of the substrate, the

adverse effect of temperature began with the free

enzyme when incubated at 45°C for 60min and more.

On the other hand, the immobilized enzyme was

slightly affected when heated at 65°C for 90min

leading to loss of only 10.37% of its activity. On

heating free enzyme at 65°C and immobilized at 70°C

and higher, the adverse effect of temperature were

more pronounced in case of free than the immobilized

enzyme. These results agreed with those reported

for immobilization of Bacillus subtilis alkaline

protease by Davidenko . The immobilized enzyme[15]

showed 2-fold increase in thermal stability, compared

to free one, after 2h at 50°C. This result is similar to

Ferreira et al. . [16]

Chellapandian and Sastry stated that the increase[12]

in thermal stability of the immobilized protease is due

to reduction in autolysis and this is possibly due to (i)

enzyme-enzyme interaction and (ii) re st ricting

conformational flexibility. The restriction of molecular

flexibility might be due to multipoint covalent

interaction with the matrix.

The effect of the metal salts on the activity of

immobilized and free alkaline protease was investigated

in Tables (3). The activation by metal ions (Ca , Mg ,2+ 2+

Mn and Na ) became more pronounced with the2+ +

immobilize d e nzyme . On the o t h e r h a n d ,

immobilization protected the enzyme from the

inhibition by some metal ions (Co , Cu and Fe ).2+ 2+ 3+

These results similar to Wehidy . On the other hand,[49]

Amaral et al. reported the inhibition of trypsin by[6]

Al , Cu , Zn and Mg , but immobilization protected3+ 2+ 2+ 2+

the enzyme to some extent from Ca and Mg2+ 2+

inhibition.


439

Table 1: Partial purification of Streptomyces avermectinus NRRL B-8165 alkaline protease.

Purification Total protein Total activity Recovered Specific activity Purification

(mg/fraction) (U) activity (%) (U/mg protein) fold

Culture filtrate 247.00 807.69 100 3.27 1

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Ethanol 50% 19.82 77.79 9.63 3.92 1.20

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Acetone 50% 12.65 169.80 21.02 13.42 4.10

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Ammonium sulphate 50% 7.59 55.50 6.87 7.31 2.24

Table 2: Properties of free and immobilized extracellular partially p urified alkaline protease from Streptomyces avermectinus NRRL B-8165

Property Free enzyme Immobilized enzyme

Specific activity (U/mg protein) 13.42 9.54

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Optimum temperature (°C) 55 55

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Optimum pH 10 10

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Activation energy (Kcal/mole) 6.206 5.688

1/2Half life t (min) at

65 °C 165.0 808.6

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

70 °C 74.3 108.6

Deactivation rate constant(min ) at-1

65 °C 4.20 x 10 0.857 x 10-3 -3

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

70 °C 9.33 x 10 6.38 x 10-3 -3

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

mK (mg/ml) 0.59 4.55

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

m axV (U/mg protein) 16.95 12.50

Table 3: Effect of addition of different concentrations of some metal salt on the ac tivity of free and immobilized alkaline protease of

Streptomyces avermectinus NRRL B-8165.

Metal salt conc. (mM) Relative activity (%)

-------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------

Metal salts Free enzyme Immobilized enzyme

--------------------------------------------------------------- -------------------------------------------------------------------

0.1 mM 0.1 mM 0.1 mM 0.1mM 1 mM 10 mM

Control 100.00 100.00 100.00 100.00 100.00 100.00

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

2CaCl 100.41 106.48 90.97 111.10 119.66 90.25

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

2 2CoCl .6H O 94.80 90.03 73.42 100.00 93.68 89.07

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

2 2CuCl .2H O 93.44 100.81 99.74 100.25 100.89 100.50

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

3FeCl 86.72 86.13 84.59 100.55 98.07 90.78

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

4LiSO 92.72 96.83 101.45 96.62 104.75 123.11

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

2 2MgCl .6H O 98.02 98.99 98.08 104.50 112.4 159.57

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

2 2MnCl .4H O 89.78 95.80 116.35 101.08 107.28 134.29

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

KCl 92.35 112.01 114.95 96.46 102.97 119.74

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

NaCl 98.98 100.70 99.64 100.35 139.09 115.66

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

2ZnCl 97.93 100.97 102.50 99.64 98.86 100.86

*Control was carried out without any tested substances.

mLineweaver-Bürk plots of the free enzyme gave K(Michaelis constant) of 0.59mg/ml with casein, which

was 7.7-fold lower than that of the immobilized

maxenzyme (4.55mg/ml). The calculated value of V

(maximum reaction rate) of the free enzyme was16.95U/mg protein which was 1.36-fold higher than

that of the immobilized (12.5U/mg protein). So, the

maxV value of immobilized enzyme is lower than that


440

Fig. 1: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinus

NRRL B-8165 using covalent binding.


NRRL-B-8165 using ionic binding.


441

Fig. 3: Immobilization of extracellular partially purified alkaline protease produced by Streptomyces avermectinusNRRL B-8165 using physical adsorption.


NRRL B-8165 using entrapment in agar, agarose and calcium alginate.


442

Fig. 5: Thermal stability of immobilized extracellular partially purified alkaline protease produced by Streptomycesavermectinus NRRL B-8165.

Fig. 6: Thermal stability of immobilized extracellular partially purified alkaline protease produced by Streptomyces

avermectinus NRRL B-8165.

of the free one . In this respect, Wehidy[35,43 ] [49]

mfound K of free was 1.2-fold lower than the

m aximmobilized protease. While V of free and

immobilized was 7.67 and 7.27 U/mg protein,respectively.

The operational stability of immobilized enzyme is

one of the most important factors affecting theutilization of an important enzyme system. Therefore,

the operational stability of the immobilized alkaline

protease was evaluated in repeated batch process

(Fig. 7). The result indicated the durability of thecatalytic activity in repeated use for more than 15

cycles. The retained catalytic activity after being used

for 15 cycles was 50% of the initial activity. In thisrespect, it was reported by Chellapandian that the[13]

vermiculite-bound alkaline protease retained 78% of its

original activity after five repeated uses. Our result ishigher than that obtained by, Mittal et al. when[ 3 5]

reused immobilized dipeptidylpeptidase 6 cycles

(retained activity 30%).


443

Table 4: Detergent stability of crude extracellular alkaline protease produced by Streptomyces avermectinus NRRL B-8165.

Time (min)

--------------- Relative activity %

Detergent ---------------------------------------------------------------------------------------------------------------------------------------------------

0 15 30 45 60

Control 100.00 100.00 100.00 100.00 100.00

------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Ariel 100.00 100.00 97.05 86.68 73.46®

------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Persil color 100.00 100.00 100.00 98.66 82.03®

------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Tide 100.00 85.25 65.42 60.71 53.89®

------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

X.TRA 100.00 100.00 100.00 95.38 79.44®

Fig. 7: Operational stability of immobilized extracellular partially purified alkaline protease on sponge usingcovalent binding produced by Streptomyces avermectinus NRRL B-8165.

Photo 1&2: Decomposition of X-ray films by partially purified extracellular alkaline protease produced by

Streptomyces avermectinus NRRL B-8165. (1) control and (2) experiment.

The overall performance of the immobilized

alkaline protease is promising than the free enzyme.Thus it is suggested that Streptomyces avermectinus

NRRL B-8165 alkaline protease immobilized on sponge

by covalent binding is suitable for practical application.In our study, the enzyme compatibility with

commercial detergents was investigated. The enzyme

was most stable with Persil color up to 60 min where®

it lost only 17.97% of its activity. On the other hand,the lowest enzyme stability was observed with Tide .®

However, it lost 20.56% and 26.54% of its activity

after 60 min incubation with X-TRA and Ariel ,® ®

respectively at 40°C Table (4). In this respect, Hadj-Ali

et al. reported a detergent stable protease which[20]


444

Photo 3: Dehairing of buffalo hide by partially purified extracellular alkaline protease produced by Streptomycesavermectinus NRRL B-8165. (1) control, (2) after 12h and (3) after 24h.

retained 65-95% activity after 1h incubation withcommercial detergents at 40°C and Gupta et al.[19]

reported a protease which retained 30-40% activity

after 30min incubation with Ariel and Surf Exel but® ®

lost activity after that. On the other hand, it lost only

10-30% activity with other commercial detergents after

2h incubation at 40°C.In our studying the ability of the partially

purified enzyme to decompose the gelatin layer ofX-ray film in order to recover both silver and

polyethylene tetraphthalate film base within only 15

min (Photo 1&2). This result is similar to Masuiet al. and Karadzic et al. . They reported a[34] [23]

protease that decomposed the gelatin layer on X-

ray film from Bacillus sp. and Pseudomonasaeruginosa, respectively.

Also, studying the ability of the partially purified

enzyme to dehair buffalo hide was investigated(Photo 3). The enzyme removed intact hairs completely

from the hide after 24h incubation at 37°C. In this

respect, Zambare et al. reported an alkaline protease[51]

that was applied as a dehairing agent. The enzyme

acted on the epidermis and hair was removed entirely;

the intact hair can be used as a value-added byproduct.Using enzyme will reduce the time of the process and

yield better quality leather. However, Sivasubramanian

et al. reported that the use of protease in deharing of[46]

goat skins and cow hide revealed complete absence of

fine hair and epidermis and the pelts were whiter thanthat dehaired by chemical methods.

At last, it is worthy to note that the results of the

present work point out to the suitability of the strainStreptomyces avermectinus NRRL B-8165 as a new

promising producer of highly active extracellular

alkaline protea se using inexpensive materials.Furthermore, enzyme immobilization proved to be a

good technique for increasing enzyme thermostability

and its several reuse.

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Optimization, immobilization of extracellular alkaline ... · PDF fileThe enzyme was partially purified with 50% acetone and showed 4.1-fold purification. ... Streptomyces, application