OPTIMIZATION OF SOME PHYSICAL AND NUTRITIONAL PARAMETERS FOR THE PRODUCTION OF HYALURONIDASE BY STREPTOCOCCUS EQUI SED 9

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1 Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 64 No. 6 pp. 517ñ522, 2007 ISSN Polish Pharmaceutical Society OPTIMIZATION OF SOME PHYSICAL AND NUTRITIONAL PARAMETERS FOR THE PRODUCTION OF HYALURONIDASE BY STREPTOCOCCUS EQUI SED 9 SABUJ SAHOO *, PRASANA K. PANDA, SATYA R. MISHRA, ANINDITA NAYAK, POLURI ELLAIAH a and SASHI K. DASH b University Department of Pharmaceutical Sciences, Utkal University, Bhubaneswar , Orissa, India. a Pharmaceutical Biotechnology Division, Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam , Andhra Pradesh, India b P.G. Department of Microbiology, Orissa University of Agriculture and Technology, Bhubaneswar , Orissa, India. Abstract: The effects of some physical and nutritional parameters were studied for the optimum production of an extracellular enzyme hyaluronidase employing Streptococcus equi SED 9 by submerged fermentation. The effects of initial ph, incubation temperature and time, inoculum level and age of inoculum were studied. The maximum enzymatic activity was obtained with an initial ph 5.5, incubation temperature 37 O C, incubation time for 48 h and inoculum level 10% with inoculum age 48 h. The effects of various carbon and inorganic nitrogen sources, vitamins, amino acids and growth hormones were studied. The results indicated that dextrose, ammonium sulfate, nicotinic acid, L-cysteine and kinetin showed the highest enzymatic activity among them. Keywords: Streptococcus equi SED 9, hyaluronidase, nutritional parameters, submerged fermentation Hyaluronidase (Hyase) enzyme acts as an adjuvant, accelerates and increases absorption and dispersion of injected drugs, fluids (1), resorption of radiopaque agents (2) and facilitates diffusion of antiviral drugs, dyes and toxins (3). Bacterial hyaluronate lyases are considered as virulence factors that facilitate the spreading of bacteria in host tissues by degradation of hyaluronan (4). Hyases, especially bovine testicular hyaluronidase (BTH) preparations, are widely used in many fields like orthopaedics, surgery, dentistry, ophthalmology (vitrectomy), internal medicine, oncology, dermatology and gynecology (5) and fertilization (6). The present work was undertaken to optimize enzyme production parameters including effect of ph (7), temperature, incubation period, inoculum level and age of inoculum employing Streptococcus equi. The effects of various carbon and inorganic nitrogen sources, vitamins, amino acids and growth hormones were studied. EXPERIMENTAL Material and methods Instruments: Spectrophotometric analysis was performed using Systronics, model-118 (India). Centrifugation was carried out using Remi C-24, shaking was done using rotary shaker (Ilshin Lab Co., Korea, model BBT-1). Chemicals: Hyaluronic acid sodium salt and bovine serum albumin fraction-v were procured from Sigma Aldrich, USA and Sisco Research Labs. Pvt. Ltd. Mumbai (India), respectively. All nutrient medium, carbohydrates, vitamins and growth hormones were procured from HiMedia Labs. Pvt. Ltd. Mumbai. Amino acids and other chemicals of analytical grade were procured from s.d. Fine-Chem, Ltd. Mumbai. A pathological isolate from Orissa University of Agriculture and Technology, Bhubaneswar, Orissa identified as Streptococcus equi SED 9 producing an extracellular hyaluronidase enzyme within 48 h was used for subsequent experiments. The slants were maintained for subsequent experiments at 2% nutrient agar slants at 4 O C. The culture was grown in nutrient agar plates, incubated at 37 O C for 48 h. Small quantities of this culture were transferred onto nutrient agar slants and incubated at 37 O C for 24 h. The culture was grown in nutrient agar plates, incubated at 37 O for 48 h. The culture was rejuvenated in medium containing composition * Correspondence: sabujbiotech@rediffmail.com 517

2 518 SABUJ SAHOO et al. (g/l) of peptic digest of animal tissue, 5; sodium chloride, 4; beef extract, 1.5; yeast extract, 1.5; casein enzyme hydrolysate type-1, 4; Na 2 HPO 4, 3; magnesium sulfate, 3; sodium citrate, 1; hyaluronic acid (HA), % with ph 5.5. The growth content of each slant was suspended in 5 ml of sterile water and the optical density (OD) of the pooled suspension was measured at 600 nm resulting OD (equivalent to cfu / ml) that constitutes the inoculum. A 10% level of inoculum was transferred into 250 ml Erlenmeyer flask containing 50 ml of modified nutrient broth. After inoculation, the flasks were incubated at 37 O C on a rotary shaker at 150 rpm for 48 h. During fermentation, the microbial growth and hyase production were monitored. The microbial growth was monitered by measuring OD at 675 nm with UV- Visible spectrophotometer. At the end of fermentation 5 ml broth was aseptically withdrawn and centrifuged at 8000 g for 30 minutes at 4 O C. The clear supernatant was subjected to enzyme assay. Hyase activity was measured spectrophotometrically by turbidity reduction assay (8) using HA sodium salt as a substrate. The enzymatic assay is based on Dorfmans method (9) in which the enzymatic reduction in turbidity, resulting when 1 ml of HA at 70 µg/ml was incubated with 1 ml of enzyme sample solution in the presence of 0.05 M sodium phosphate buffer with 0.05 M NaCl (ph 7.0). The mixture was allowed to stand for 30 min. To the above incubated mixture, 2.5 ml of acidified protein solution (1% w/v) bovine serum albumin fraction-v (BSA) in 0.5 M sodium acetate buffer, (ph 3.1) was added and incubated at 37 O C for 10 min and reduction in turbidity was read by measuring the absorbance at 600 nm. One unit of enzyme activity was defined as the amount of enzyme that reduced the absorbance by 0.1 at 600 nm (A) in 30 min at 37 O C, ph 7.0 under assay conditions similar to that caused by one unit of an international standard. Optimization of physical parameters Influence of initial ph To investigate the influence of initial ph on enzyme production, the production medium was adjusted to various levels of ph ( ). Fermentation was conducted and samples were assayed for enzymatic activity which is shown in Figure 1. Effect of initial temperature and incubation period on enzyme production and cell growth To study the effect of initial temperature and incubation period on enzyme production and cell growth, the production medium was inoculated and incubated at various temperatures ranging from 20 O C to 55 O C for 96 h. The samples were withdrawn at regular intervals of 12 h and assayed for biomass (mg/ml) and enzymatic activity. The optimal temperature and incubation period obtained at this level was used for further studies as shown in Figure 2 and time course profiles of hyase production, cell mass (mg/ml) and ph by S. equi is given in Figure 3. Effect of inoculum size and age on enzyme production and cell mass The flasks with the basal production medium were inoculated with inoculum age of 24 h level at 0.1, 1, 2, 4, 6, 10 and 12 % level and incubated at 37 O C for 48 h and 5 ml samples were withdrawn at 12 h intervals and examined for biomass (mg/ml) Figure 1. Effect of initial ph on hyase production.

3 Optimization of some physical and nutritional parameters and enzyme activity which is indicated in Figure 4. The optimal level of inoculum obtained was used in further experiments. Optimization of nutritional parameters Effect of carbon sources Various carbohydrates such as glucose, lactose, sucrose, mannitol, dextrin, cellobiose, melibiose, starch, sodium CMC, trehalose, dextrose, rhamnose, sodium alginate, adonitol and arabinose were studied by adding at a concentration of 5 mg/ml to the basal production medium (10). After fermentation enzyme activity was assayed and shown in Figure 5. Effect of inorganic nitrogen sources Various inorganic nitrogen sources: ammonium acetate, ammonium bicarbonate, ammonium chloride, ammonium sulfate, sodium nitrite and sodium nitrate were added (5 mg/ml) to the basal production medium. A control was also run. After fermentation enzyme activity was assayed (Figure 6). Effect of vitamins Water soluble vitamins viz. B 1, B 2, C, biotin, pyridoxine, folic acid, nicotinic acid, calcium pantothenate, and inositol were added at a concentration of 5 µg/ml to the production medium that supports the best enzyme production. A control was runned containing distilled water instead of vitamins. The effect of vitamins on hyase activity was investigated (Figure 7.) Effect of amino acids The amino acids: L-arginine, L-tyrosine, L- cysteine, L-tryptophan, Lñlysine, L-ornithine, L- alanine, L-aspartic acid, L-glutamic acid, L-proline, glycine and L-histidine were dissolved in distilled water and added aseptically to the sterile basal production medium at a concentration of 25 µg/ml. A control was run with distilled water. The results are presented in Figure 8. Effect of growth hormones Effect of plant growth hormones including kinetin, gibberelic acid, indole-3-acetic acid and NAA (α-naphthalene acetic acid) were added at a concentration of 10 µg/ml each to production medium along with control and assayed for enzymatic activity (Figure 9). Figure 2. Effect of initial temperature on hyase production by S. equi. Figure 3. Time course profiles of hyase production, cell mass (mg/ml) and ph by S. equi. ( ) enzyme activity, ( ) cell mass, ( ) ph

4 520 SABUJ SAHOO et al. RESULTS A pathological isolate Streptococcus equi SED 9 was used for production of an extracellular enzyme hyase within 48 h by submerged fermentation. The effects of some physical and nutritional parameters were studied for the optimum production of the enzyme. The effect of initial ph and initial temperature on hyase production at different initial ph and temperature values is shown in Figures 1 and 2, respectively. The result of incubation period on the fermentation cycle is given in Figure 3. The effect of inoculum level on hyase production is indi- Figure 4. Influence of inoculum level on hyase production by S. mitis. ( ) 0.1 %, ( ) 1 %, ( ) 2 %, ( ) 4 %, ( ) 6 %, (o) 10 %, ( ) 12 % Figure 5. Influence of carbon sources on hyase production. 1. glucose, 2. lactose, 3. sucrose, 4. mannitol, 5. dextrin, 6. cellobiose, 7. melibiose, 8. starch, 9. sodium CMC, 10. trehalose, 11. dextrose, 12. rhamnose, 13. sodium alginate, 14. adonitol and 15. arabinose Figure 6. Influence of inorganic nitrogen sources on hyase production. 1. ammonium acetate, 2. ammonium bicarbonate, 3. ammonium chloride, 4. ammonium sulfate, 5. sodium nitrite and 6. sodium nitrate, respectively

5 Optimization of some physical and nutritional parameters Figure 7. Influence of vitamins on hyase production. 1. vitamin B 1, 2. vitamin B 2, 3. vitamin C, 4. biotin, 5. pyridoxine, 6. folic acid, 7. nicotinic acid, 8. calcium pantothenate and 9. inositol Figure 8. Influence of amino acids on hyase production. 1. L-arginine, 2. L-tyrosine, 3. L-cysteine, 4. L-tryptophan, 5. Lñlysine, 6. L-ornithine, 7. L-alanine, 8. L-aspartic acid, 9. L-glutamic acid, 10. L-proline, 11. L-glycine and 12. L-histidine, 13. control cated in Figure 4. The effect of various nutritional parameters including carbohydrates, inorganic nitrogen sources, vitamins, amino acids and growth hormones on hyase production is reported in Figure 5, 6, 7, 8 and 9, respectively. DISCUSSION AND CONCLUSION Figure 9. Influence of growth hormones on hyase production. K- kinetin, GA- gibberelic acid, IAA- indole-3-acetic acid and NAA- α-naphthalene acetic acid The effect of initial ph was studied at different ph values to achieve optimum enzyme production. The highest enzyme yield was observed at ph 5.5 (165 U/mL) while the lowest was recorded at ph 9.0 (31 U/mL). The enzyme was found to be active from ph range 5.5 to 7.4, with a gradual decrease in yield

6 522 SABUJ SAHOO et al. in the above ph range, above and below this range, the activity decreased sharply. The results of incubation temperature on enzyme production indicated the optimum activity (167 U/mL) at 37 O C with a gradual decrease in activity later. The time course profile of hyase production showed optimum enzyme activity (169 U/mL) and cell mass (3.55 mg/ml) was reported at 48 h with the change in medium ph from 5.5 to The highest enzyme activity (168 U/mL) and cell mass (3.49 mg/ml) was recorded with 10 % inoculum whereas the lowest yield (57 U/mL) and cell mass (0.73 mg/ml) was observed with 0.1 % level. There was a gradual decrease in yield beyond 10 % inoculum. The results showed maximum enzyme production (271, 255, 243 and 225 U/mL) with dextrose, trehalose, glucose and sucrose, respectively. Other carbohydrates decreased the enzyme yield. Ammonium sulfate showed the highest enzyme production (258 U/mL) followed by ammonium chloride. The lowest yield was observed with sodium nitrate (121 U/mL). The effect of various vitamins added at 5 µg/ml to the production medium showed that vitamin B 1, biotin, folic acid and nicotinic acid stimulated enzymatic activity with the highest activity (260 U/mL) exhibited by nicotinic acid whereas vitamin C showed strong inhibitory activity on enzyme production. Vitamin B 2, pyridoxine, calcium pantothenate and inositol added into the production medium showed no stimulatory effect on enzyme production. The effect of various amino acids added to the production medium at 25 µg/ml showed that among amino acids under test L-cysteine, followed by L- tyrosine, L-ornithine, glycine and L-tryptophan stimulated hyase production while remaining amino acids showed no stimulatory effect on hyase production when compared with control (165 U/mL). L-cysteine and L-alanine exhibited the highest and lowest enzyme production (233 and 103 U/mL), respectively, as indicated in Figure 8. The effect of growth hormones added to the production medium at 10 µg/ml showed better activity (252 U/mL) with kinetin followed by gibberelic acid, indole-3-acetic acid and almost no stimulatory effect with NAA (α-naphthalene acetic acid) (148 U/mL) when assayed for enzymatic activity. In conclusion, an extracellular enzyme hyase from a pathological isolate Streptococcus equi SED 9 was produced by submerged fermentation and some physical and nutritional parameters were studied. The data incorporated in this article may help biotechnologists for further investigations including its stability, purification and scale up studies. Acknowledgement The authors are thankful to A.I.C.T.E., New Delhi, India for sanction of RPS project to one of the authors, Prof. P. K. Panda, U.D.P.S., Utkal University for carrying out the research work. The authors are also thankful to the HOD, P.G. Dept. of Microbiology, and HOD, Agril. Biotechnology, Orissa University of Agriculture and Technology, Bhubaneswar, Orissa, for providing research facilities. REFERENCES 1. Csoka T.B., Frost G.I., Stern R.: Trends Glycosci. Glycotechnol. 8, 419 (1996). 2. Law R.O., Rowen D.: J. Physiol. 311,341 (1981). 3. Duran-Raynals F.: J. Exp. Med. 58, 161 (1933). 4. Akhtar M.S., Bhakuni V.: Current Sci. 86, 285 (2004). 5. Farr C., Menzel J., Seeberger J., Schweigle B.: Wien. Med. Wochenschr. 147, 347 (1997). 6. Primakoff P., Lathrop W., Woolman L., Cowan A., Myles D.G.: Nature 335, 543 (1988). 7. McDermid A.S., McKee A.S., Marsh P.D.: Infect. Immun. 56, 1096 (1988). 8. Tam Y.C., Chan E.C.S.: J. Microbiol. Methods 1: 255 (1983). 9. Dorfman A.: Methods in Enzymology. Vol. I, p. 166, Academic Press, New York Rogers H.J.: J. Pathol. Bacteriol. 39, 436 (1944). Received: