African Journal of Microbiology Research Vol. 6(11), pp. 2748-2751, 23 March, 2012 Available online at http://www.academicjournals.org/AJMR DOI: 10.5897/AJMR11.1291 ISSN 1996-0808 ©2012 Academic Journals

Full Length Research Paper

Activation of phytase in Aspergillus niger LCWU 21 by surfactants

Arifa Tahir

Environmental Science Department, Lahore College for Women University, Lahore Pakistan. E-mail: [email protected].

Accepted 24 February, 2012

Present study describes the activation of enzyme phytase in Aspergillus niger LCWU 21 isolated from the soil by solid fermentation (SSF) using wheat bran as culture medium. The addition of surfactants showed stimulatory effect on enzyme formation. Effect of the addition of surfactants showed that the addition of Triton x-100 enhanced enzyme production (60.3 U/ml). It was found that concentration and time of addition of surfactant are critical for enzyme synthesis. The rate of phytase production was higher in the presence of Triton X-100. Addition of Triton X-100 at the time of inoculation gave maximum enzyme production. These results are encouraging for optimization under industrial conditions.

Key words: Aspergillus niger LCWU 21, surfactants, Triton X-100.

INTRODUCTION

Phytase may be produced directly in solid substrate MERCK and Fluka (Switzerland) chemical companies. fermentation (SSF) by filamentous fungi on selected feed ingredients, and the crude may be mixed in feed Organism rations as a value-added supplement. It has been known that many factors can affect enzyme production. Among Fungal culture Aspergillus niger previously isolated in our Lab from them is the addition of surfactants to the media (Rees soil was used in present study. The culture medium consisting of and Maguire, 1969; Amtual et al., 1988; Han and Wilfred, (g/L) malt extract 45.0, Glucose 5.0, yeast extract 5.0, and Agar 1988). Surfactants have been reported to have an effect 15.0 was used for the maintenance of isolated culture at 30°C and then kept in the refrigerator at 4°C. on the growth rate and enzyme production of fungi. Surfactants have been used in biotechnology for improving the yields of a number of produced Inoculum preparation by fermentation (Sukan et al., 1989). Reese and Maguire (1969) observed that the addition of surfactants to culture The spore inoculum for the biosynthesis of enzyme phytase by A. media, in general, increased the enzyme yield, but the niger LCWU21 was used. For the preparation of inoculum, A. niger LCWU21 was incubated for seven days at 30°C on pota to dextrose effects varied from organism to organism and from agar medium. With the help of inoculating needle, conidia formed enzyme to enzyme. Surfactants enhance extracellular on the agar surface were scraped off and then collected in 5 ml enzyme production by increasing cell membrane Tween 80 (1.0 g/L). The tube was shaken gently to break the permeability and change in lipid metabolism, which clumps of conidia. The conidial count was made using 6 resulted in a higher release of these enzymes (Reese haemocytometer (Neubauer Precicdor HBG, Germany). About 10 and Maguire, 1969). spores/ml were used as an inoculum.

Fermentation technique MATERIALS AND METHODS Air dried wheat bran 12.0 g was placed in 500ml cotton-plugged The substrate (wheat bran) used for fermentation studies was conical flasks respectively supplemented with 5ml salt solution purchased from local market of Lahore. All other chemical were of containing (g/1): NH 4NO 3, 23., MgSO 47H 2O 4.6; NaCl 4.6. The wet analytical grade and purchased from Sigma (USA), ACROS, substrate was sterilized at 121°C for 20 min. After cooling the Tahir 2749

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10 No Surfactant /Surfactant 80 No / 0.5% Tween 0.5% Triton X-100 0 1 2 3 4 5 6 7 8 9 1011121314 Time of incubation No Surfactant 0.5% Tween 80 0.5% Triton X-100

Figure 1. Effect of surfactants on the biosynthesis of enzyme. Each value is a mean average of three parallel replicates. Y-error bars indicate standard deviation among the replicates. Incubation temperature 30°C, Initial pH 6.0.

substrate was inoculated with spore suspension (4%) of the min. and was stopped by adding 5 ml of 10% trichloroacetic acid respective fungus at 10 6 cfu/m. The flasks were then incubated at (TCA). 30°C except otherwise stated.

Mycelial dry weight Analysis Glucose amine, after being released from biomass hydrolysis Phytic acid according to Sakurai et al. (1976) was measured by the method of Blix (1948). Phytic acid was extracted approximately from 2.0 g wet solid substrate culture samples by using 33 ml of 2.4% HCl under continuous shaking (200 rpm) for 1 h. After extraction, the suspension was centrifuged (6000 x g 15 min), the supernatant Unit definition collected and phytic acid was measured by the method of Haug and Lantzch (1983). One unit of enzyme activity is defined as the amount of enzyme required to release 1 mg of inorganic phosphorus from 1.0 ml of 2.2 mM phytic acid solution per hour at the given temperature and pH. Assay of enzyme phytase

Enzyme activity was determined in crude enzyme extracted from Statistical analysis solid substrate culture using 2% aqueous solution of CaCl 2.2H 2O and shaking the suspension for 1 h. on a rotary shaker at 200 rpm. The data on various parameters were tabulated and subjected to The wet solid substrate culture sample to extractant was 1:5 (w/v). statistical analysis using computer software Costat, cs 6204W. exe. At the end of extraction, the suspension was squeezed through a double layer cheese cloth and centrifuged (20,000 x g, 10 min). The clear supernatant was designated as a crude enzyme preparation. Phytase activity was assayed using sodium phytate as RESULTS AND DISCUSSION the substrate and measuring spectrophotometrically the inorganic phosphorus released as described by Harland and Harland (1980). The reaction mixture was consisted of 5.0 ml of 0.2 M acetate Two kinds of surfactants Tween-80 and Triton X- 100 buffer (pH 4.7), 1 ml of 2.25 mM phytic acid and 0.1 ml of crude were evaluated (Figure 1). The control culture without enzyme. The reaction was carried out in duplicate at 53°C for 10 any surfactant was also run in parallel. It was found that 2750 Afr. J. Microbiol. Res.

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54 0.5%0.5 0.7% 0.7 0.9% 0.9 1.1%1.1 1.3%1.3 1.5% 1.5 Concentration (%) Phytase u/ml/min Concentration

Figure 2. Effect of different concentration of Triton X-100 on the production of enzyme phytase by Aspergillus niger LCWU21.Each value is a mean average of three parallel replicates. Y-error bars indicate standard deviation among the replicates. Incubation temperature 30°C, Initial pH 6.0.

the solid-state cultures with surfactants produced more 2).The effect of the time of addition of Triton X-100 during enzyme than control. It might be due to fact that fermentation was also studied (Figure 3). The optimum surfactant act at the cell membrane to release the time for the addition of Triton X-100 however, was found enzyme; other enzyme should be set free simultaneously. to be at the time of inoculation (65 U/ml/min). The addition of Tween-80, sucrose monopalmitate or Further delay in its addition did not enhance the nonionic surfactants to fungal cultures increased the formation of phytase by mold. Earlier addition of the yields of enzymes such as , xylanase, , inducer reduced the growth and consequently lowers the benzyl esterase, and and purine nucleosidase. enzyme productivity. The inducer was also less effective The addition of surfactants to culture media, increase the for phytase formation when it was added to the culture enzyme yield, but the effects varies from organism to medium in later stages of growth after 6 h. The optimum organism and from enzyme to enzyme (Reese and concentration of Triton x-100 (0.9%) was also Maguire, 1969). Triton X-100 was the best surfactant and determined. The enzyme production was not increased at the enzyme production was maximum (60.3 U/ml/min). higher concentration. It might be due to fact that the The concentration of surfactant and its time of addition to surfactant in lower concentrations caused a more the basal medium are critical for the synthesis of enzyme abundant biomass growth, as in the case of phytase phytase. Effect of the addition of Triton X-100 at different production by Aspergillus carbonarius (Al-Asheh and levels (0.5 – 1.5% w/v) on enzyme formation by the Duvnjak, 1994), and thus influenced the production of the bacterium was investigated. enzyme. It was observed that Triton x-100 when added at The optimum level of Triton X-100 was found to the time of inoculation gave the maximum enzyme be0.9% w/v, the enzyme formation was decreased with production. The addition of Triton x-100 afterwards did the increase in the concentration of Triton X-100 (Figure not improve the enzyme formation. It may be due to Tahir 2751

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Figure 3. Effect of time addition of surfactants.

catabolite repression. Han YW, Wilfred AG (1988). Phytate hydrolysis in soybean and cottonseed meals by Aspergillus ficuum phytase, J. Agric. Food chem. 36: 259-262. Harland BF, Harland J (1980). Fermentative Reduction of Phytate in REFERENCES Rye, White, and Whole Wheat Breads. Cereal Chem., 57(3): 226- 229. Al-Asheh S, Duvnjak Z (1994). The effect of surfactants on the phytase Haug E, Lantzexh HJ (1983). Surfactants are stimulants of enzyme and the reduction of the phytic acid content in canola meal by Production. J. Bacteriol., 34: 1423-1426. Aspergillus carbonarius during solid state fermentation process. Reese ET, Maguire A (1969). The metal dependence of fungal phytase. Biotechnol. Lett., 16(2): 183-188. J. Appl. Microbiol., 17: 242-245. Amtual JS, Akhtar MW, Malik NN, Naz BA (1988). Purification of Sakurai Y, Lee TH, Shiota (1976). screening of fungi for phytase Phytase from Triticum aestivum. Enzyme Microb. Technol., 10: 626- production. J. Agric. Biol. Chem. 41: 619-624. 631.