enhanced production of protease by mutagenized ...

Sci.Int.(Lahore),22(2), 119-123,2010

ISSN 1013-5316; CODEN: SINTE 8

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ENHANCED PRODUCTION OF PROTEASE BY MUTAGENIZED STRAIN OF ASPERGILLUS ORYZAE IN SOLID SUBSTRATE FERMENTATION OF RICE BRAN Muhammad Yousaf1, Muhammad Irfan1*, Zia ulla Khokhar,2 Qurat-ul-Ain Syed,1 Shahjahan Baig1 and Amber Iqbal2 1

Food & Biotechnology Research Center, Pakistan Council of Scientific and Industrial Research Laboratories Complex Ferozpur Road Lahore, Pakistan. 2 Department of Chemistry, Government Islamia Post Graduate College Gujranawala , Pakistan 1* (Corresponding Author E-mail: [email protected])

ABSTRACT: Neutral protease activity of parent strain of Aspergellus oryzae was enhanced by UV and chemical mutagenization with ethyl methane sulphonate (EMS). After screening, a hyper producing strain was isolated and found effective for the production of neutral protease as compared to the parent strain of Aspergellus oryzae. Solid substrate fermentation was carried out in 250ml conical flask with 45 % initial moisture contents at a temperature of 300C for 72 hours. Under the optimum conditions maximum yield of neutral protease obtained was 662.61 ± 0.36 U/gds. Almost all the organic nitrogen supplements favored the enzyme production while sucrose proved as a best carbon source. INTRODUCTION Proteases are proteolytic (protein-digesting) enzymes that are mainly classified on the basis of the pH optimum as acidic, neutral, and alkaline proteases and catalyze hydrolytic reactions in which protein molecules are degraded to peptides and amino acids. Proteases constitute a large and complex group of enzymes which differ in properties such as specificity, active site and catalytic mechanism, pH, temperature optima and stability profile [1]. These biocatalysts find wide application in industries such as baking, brewing, leather, textile, laundry and detergent. One of the more recent application of these proteases are ecofriendly nature and hence their suitability to act as foodprocessing aids, wherein these enzymes are used for the extraction of plant oils thus largely replacing hazardous organic solvent such as hexane which has been traditionally used for such purposes. Proteolytic enzymes are ubiquitous in occurrence, being found in all organisms, and are essential for cell growth and differentiation. Proteolytic enzymes from microorganisms may be located within cell (intracellular), cell wall associated (periplasmic), or excreted into the media (extra cellular)[2]. Extra cellular enzymes are usually capable of digesting insoluble nutrient materials such as cellulose, protein and starch, and the digested products are transported into the cell where they are used as nutrients for growth [3]. Microorganisms are the most important sources for enzyme production. Selection of right organism plays a key role in high yield of desired enzyme. The Aspergillus species produce a large variety of extra cellular enzymes, of which amylases and proteases are of significant industrial importance [4]. Protease production had been carried out under both submerged and solid state fermentation using various substrates such wheat bran, mango peel, banana peel [5] , rice bran [6] and steamed rice [7] . In the present studies neutral protease is produced by submerged fermentation using rice bran as a substrate by Aspergellus oryzae. The effect of different cultivation conditions including incubation time, initial moisture contents, effect of incubation temperature, effect of nitrogen and carbon source on neutral protease were studied.

MATERIALS AND METHODS Microorganism The strain of Aspergellus oryzae was obtained from the Microbiology Lab. PCSIR Laboratories complex Lahore and maintained on potato Dextrose Agar (PDA) medium. All media, otherwise stated, were sterilized at 1210C for 15 minutes. The culture was incubated for 5-7 days at 300C for maximum sporulation and then stored in the refrigerator. Subculturing was made after every fortnight. UV mutagenesis Spore suspension was prepared in serial dilution method from 5days old culture slant one ml of 106 dilution was poured in petri plate and placed under UV lamp (240nm) at a 10cm distance up to 60 minutes with regular interval of five minutes. After irradiation of spores, were cultured on PDA plates incubated at 30 ± 1°C for five to seven days until sporulation of fungal culture. Mutagenised colonies were screened by analyzing enzyme activity after fermentation batch. Best protease producing strains were selected for further study. Chemical mutagenesis Chemical mutagenesis was carried out with Ethyl Methane Sulfonate for varying time period (60 minutes with regular interval of five minutes). One milliliter of spore suspension was centrifuged at 10,000 rpm for 3 minutes. Supernatant was discarded and pellet was dissolved in EMS (4µl/ml) and incubated at 30oC for different time intervals. After incubation at varying time intervals spores were washed with distilled water to completely remove EMS traces. After treatment the pellet was serially diluted and cultured. Mutagenised colonies were screened by analyzing enzyme activity after fermentation batch. Best protease producing strains were selected for further study. Inoculum Preparation The inoculum medium containing (g/L) glucose 16.5, corn steep liquor 4.2, NH4NO3 2.1, MgSO4. 7H2O 1.0, KH2PO4 1.0 and citric acid 0.22 was used for inoculum development. The pH of the medium was adjusted at 7.0 and sterilized at 1210C for 15 minutes. 50 ml of medium was taken in 250 ml conical flask, was aseptically inoculated by spore suspension of Aspergillus oryzae made from 7 days old culture slant in 0.1 % tween 80 solution and incubated on a rotary shaker (150 r.p.m) at 30 ± 10C for 48 hours.


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Sci.Int.(Lahore),22(2), 119-123,2010

Table: 1. Screening of mutants after UV and Chemical treatment. Mutants (UV)

Time period (min)

Enzyme Production (U/gds)

Mutants (Chemical)

Enzyme Production (U/gds)

Control

0

164. 12 ± 0.10

Control

164. 12 ± 0.10

AMU1

5

259.2 ± 0.43

AMC1

329.2 ± 0.33

AMU2

10

541. 42 ± 1.13

AMC2

461. 22 ± 1.03

AMU3

15

446.1 ± 0.62

AMC3

662.61 ± 0.36

AMU4

20

314.1± 0.02

AMC4

384.21± 0.41

AMU5

25

310.8 ± 0.47

AMC5

371.4 ± 0.86

AMU6

30

264.3 ± 0.87

AMC6

432 ± 0.67

AMU7

35

210.6 ± 1.24

AMC7

322.4 ± 0.98

AMU8

40

194.3 ± 0.78

AMC8

246.4 ± 1.147

AMU9

45

162.3 ± 0.34

AMC9

243.7 ± 1.126

AMU10

50

150.8 ± 0.41

AMC10

198.6 ± 0.67

AMU11

55

1.33.5 ± 0.39

AMC11

176.5 ± 0.14

AMU12

60

104.4 ± 0.75

AMC12

140.5 ± 0.11

Solid State Fermentation 10 g of rice bran in 250 ml cotton plugged conical flask was moistened with 4.5 ml phosphate buffer (pH 7.0) to achieve the desired moisture content. The flask was autoclaved at 1210C for 15 minutes, cooled and sterilized rice bran was inoculated with 2 ml of inoculum and incubated at 30 ± 10C for 72 hours. Extraction of Enzyme 40 ml of tween 80 (0.1% w/v) solution was added in the fermented rice bran and substrate was homogenized on a rotary shaker at 100 r.p.m for 30 minutes. The solids were removed by centrifugation the fermented rice bran in refrigerated centrifuge at 40C for 10 minutes. Assay for Neutral Protease Activity Protease activity was assayed using casein as substrate [8].1.0 ml of 0.6 % casein solution in 50 mM phosphate buffer (pH 7.0) was mixed with 0.2 ml of crude enzyme extract. Mixture was incubated at 400 C for 10 minutes. 4.0 ml of 6% HClO4 was added to terminate the reaction. The insoluble part of the mixture was filtered trough Whatman filter paper No. 2 then 4.0 ml of 0.8 mM Na2CO3 and 1.0 ml of folin ciocalteau phenol reagent was added to 1.0 ml filtrate. The absorbance at 655 nm was measured by spectrophometer. The blank was prepared by reversing the sequence of addition of enzyme solution and HClO4. Unit of Enzyme Activity One unit of enzyme activity was defined as the amount of enzyme that liberate 1µg of tyrosine from substrate (casein) per minute under the assay conditions and reported in term of protease activity per gram dry fermented substrate. RESULTS AND DISCUSSION The process parameters for the production of neutral protease by Aspergillus oryzae grown on rice bran as substrate were carried on by single parameter mode. Aspergillus species are used in the commercial production of industrially valuable enzymes and other products [9-11].

Mutagenesis of Aspergillus oryzae For enhanced production of protease, 5-7 days old culture slants were UV irradiated for various time period and it was noted that strain AMU2 gave maximum protease activity (541.42 ± 1.13) was obtained by 10 min of exposure to UV light. As the exposure time increased the enzyme production was decreased (strain AMU12104.4 ± 0.75). When the strains were subjected to chemical mutation protease activity was further enhanced up to 662.61 ± 0.36 U/gds (strain AMC3) with chemical dose of 4 µl/ml with incubation time of 15 minutes. Djamel et al [12] selected best proteolytic producer strains by combined treatment of spores with UV and ethyleneimine reporting 2min of exposure time to UV rays. Rakariyatham et al [13] reported that EMS mutant can produced a stable protease. Meraz et al. [14] obtained a best mutant strain of Bacillus sp which was 8 fold higher in enzyme production than wild with concentration of 100µg/ml at incubation period of 60 min. Table: 1. Screening of mutants after UV and Chemical treatment.

Effect of Initial Moisture Contents of Medium Initial moisture contents of substrate is an important factor affecting the enzyme production in solid substrate fermentation. The exact moisture level of the solid substrate plays the critical role for growth of organism and enzyme production because of the possible efficient solute [15,16] and oxygen transfer [17]. Figure 1 shows the effect of different moisture levels in rice bran on the enzyme production. Enzyme synthesis gradually increased with increasing the moisture contents and maximum activity (564 ± 1.43 U/gds) was achieved when substrate moisture was 45% (v/w). Further increase of moisture contents resulted in decrease of enzyme activity. The order of higher enzyme production was observed in strains AMC3, AMU2 and than parent respectively. Sumantha et al [18] produced a neutral metalloprotease by fungal mixed substrate fermentation and

Sci.Int.(Lahore),22(2), 119-123,2010


reported 50% of optimized moisture level yielding highest protease activity (7.46U/g).

Parent Strain Enzyme Production (U/g)

600

AMU2 AMC3

500 400 300

Parent Strain

600 Enzyme Production (U/g)

700

121

AMU2

500

AMC3

400 300 200 100 0

200

0

100

24

48

72

96

120

144

Fermentation Period (hrs)

0 30

35

40

45

50

55

60

Moisture Content (%)

Fig. 1. Effect of initial moisture level of medium on neutral protease production.

Effect of Incubation Period The production profile of neutral protease was studied by conducting fermentation for different time intervals is shown in Figure 2. In solid substrate fermentation incubation period is very critical and depends upon the characteristic of culture, growth rate and production of enzyme. There is a regular increase in enzyme formation and it becomes maximum i.e. 487 ± 0.985 U/gds after 72 hours incubation using chemically treated strain (AMC3) which was higher than UV- treated strain AMU2 (396 ± 1.35 U/gds) and parent strain(195 ± 0.78 U/gds) respectively. Murthy and Naidu [19] reported 96hr of fermentation period was optimum (7142 U/gds) for protease production by Aspergillus oryzae in solid state fermentation. The activity decreased gradually on further increase of incubation period and is caused probably by the inactivation of enzyme by other constituent proteases and interaction with other components of the medium [20]. Effect of Incubation Temperature The influence of temperature on protease production in solid substrate fermentation is related to the growth of organism, moisture level of substrate and rate of diffusion of gases during fermentation [21]. To determine the optimum initial temperature for neutral protease production, solid substrate fermentation was carried out at different temperatures. The results of Figure 3 shows the results obtained from different strains i.e. chemically treated, UV-treated and parent strain. It was observed that enzyme yield was maximum i.e. 468 ± 1.68 U/gds in case of chemically treated strain at 300C. Both

Fig. 2. Effect of incubation period on protease production.

UV-treated and parent strains produced less enzyme production than chemically treated strain. At lower and higher temperature protease synthesis was inhibited. It has also been reported that metabolic heat generated during microbial cultivation in solid substrate fermentation exerts harmful effect on microbial activity [22] and thus the initial set temperature is very critical. 600 Enzyme Production (U/g)

25

Parent Strain AMU2

500

AMC3

400 300 200 100 0 15 20 25 30

35 40 45 50

Incubation temperature (oC)

. Fig. 3. Effect of incubation temperature on neutral protease production. Effect of Nitrogen Source The nutritional requirements of Aspergillus oryzye are reported to be complex for growth and enzyme synthesis as the structural macromolecule of agroresidues provide an inert matrix in the solid substrate fermentation [20]. Different organic nitrogen sources in complex form such as corn steep liquor, casein, peptone, urea, tryptone, yeast extract and beef extract were supplemented at rate of 1% (w/w) where as inorganic nitrogen sources like NaNO3, NH4H2PO4, NH4Cl, (NH4)2HPO4, NH4NO3, (NH4)2SO4, and NH4HCO3 were added at a rate of 0.5% w/w to the medium for the biosynthesis of enzyme as shown in Table 2.


122

Table 2. Effect of different nitrogen sources on production of neutral protease from A.oryzae using rice bran as substrate in solid state fermentation.

Inorganic Nitrogen Source Control NaNO3 NH4H2PO4 NH4Cl (NH4)2HPO4 NH4NO3 (NH4)2SO4 NH4HCO3

Enzyme Production (U/gds) Parent Strain

AMU2

600

AMC3

175 ± 0.13 234 ± 0.43 182 ± 0.74 257 ± 0.13 248 ± 1.40 386 ± 0.34 242 ± 1.34 346 ± 0.87 198 ± 1.02 247 ± 1.16 24 ± 0.94 146 ± 0.44 209 ± 0.48 197 ± 0.02 215 ± 1.61 266± 0.64 Enzyme Production (U/gds) Parent Strain AMU2

AMC3

175 ± 0.13 172 ± 0.25 165 ± 0.46 132 ± 0.73 112 ± 1.03 118 ± 1.30 145 ± 0.94 188 ± 0.73

342 ± 0.21 395 ± 0.71 376 ± 0.54 334 ± 0.44 253 ± 0.38 276 ± 0.67 391 ± 0.79 463 ± 0.41

234 ± 0.43 297 ± 0.11 289 ± 0.07 226 ± 0.64 204 ± 0.77 194 ± 0.39 224 ± 0.41 387± 0.81

342 ± 0.21 366 ± 0.11 438 ± 0.71 416 ± 0.94 347 ± 0.07 278 ± 1.3 335 ± 1.84 379 ± 0.58

Enzyme Production (U/g)

Control Corn Steep Liquor Casein Peptone Urea Tryptone Yeast Extract Beef Extract

carbohydrate deficient rice bran substrate which only contains 1.3% of reducing sugar [6].

Parent Strain

AMU2

AMC3

500 400 300 200 100 0

Almost all the organic nitrogen sources enhanced the enzyme production as compared to control i.e. 175 U/gds. Casein and peptone enhanced enzyme yield upto 248 ± 1.40 U/gds and 242 ± 1.34 U/gds respectively and proved to be good inducer for neutral protease synthesis. Inducing effect of casein for protease production has also been reported by other researchers [23]. Syed et al [24] also reported similar findings. Corn steep solids is also a good organic nitrogen supplement which enhances the enzyme production [18]. Among the various inorganic nitrogen sources supplemented to the fermentation medium, NaNO3, NH4H2PO4 and NH4HCO3 enhanced the enzyme production. These compounds have less molar concentration of nitrogen as compared to the other inorganic nitrogen compounds. The maximum protease production by NH4HCO3 is not related to nitrogen present in but may be due to the carbonate. Carbonate as a constituent of the extraction buffer enhanced protease recovery from rice bran fermented by Aspergillus niger [25]. In another study among the various inorganic sources, NH4NO3 showed the optimum protease production [18]. Effect of Carbon Source Different carbon sources have different influences on extracellular enzyme production by different strains [26,27]. Fig. 4 shows the effect of various carbon sources on enzyme production. Different sugars such as glucose, fructose, galactose, maltose, sucrose, xylose, lactose and sorbitol were added at rate of 1% w/w to the fermentation media. Almost all the sugars supplemented as carbon sources enhanced the protease production. Sucrose proved to be best carbon source increasing enzyme yield to 536 ± 1.87 U/gds as compared to control i.e.180 ± 0.58 U/gds. Some reports described that sugars induce the protease production in different species of Aspergillus oryzae and Penicilium [28]. Sucrose probably provided the much required carbon in the

co nt G rol lu co Fr se uc G tos al e ac to s M e al to Su se cr os Xy e lo La se ct os So e rb ito l

Organic Nitrogen Source

Sci.Int.(Lahore),22(2), 119-123,2010

Different Carbon sources

Fig.4. Effect of carbon source on neutral protease production.

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