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Sesame oil cake an inexpensive substrate for neutral protease production by penicillium chrysogenum ncim 737 in solid-state fermentation
 

Sesame oil cake an inexpensive substrate for neutral protease production by penicillium chrysogenum ncim 737 in solid-state fermentation

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    Sesame oil cake an inexpensive substrate for neutral protease production by penicillium chrysogenum ncim 737 in solid-state fermentation Sesame oil cake an inexpensive substrate for neutral protease production by penicillium chrysogenum ncim 737 in solid-state fermentation Document Transcript

    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 ISSN:2249-5347 IJSID International Journal of Science Innovations and Discoveries An International peer Review Journal for Science Research Article Available online through www.ijsidonline.info SESAME OIL CAKE-AN INEXPENSIVE SUBSTRATE FOR NEUTRAL PROTEASE PRODUCTION BY PENICILLIUM CHRYSOGENUM NCIM 737 IN SOLID-STATE FERMENTATION Center for Biotechnology, Dept of Chemical Engineering, Andhra University, AP, India B. Nagamani, M.V.V.Chandana Lakshmi*, V.Sridevi and P.Rajani Neutral protease production under solid-state fermentation was carried out byReceived: 03-07-2012 ABSTRACT using Penicillium chrysogenum NCIM 737. Among the six (green gram husk, black gramAccepted: 18-10-2012 husk, rice bran, coconut oil cake, sesame oil cake and paddy straw + rice bran (7:3)) agro-industrial waste materials evaluated, sesame oil cake supported maximum protease*Corresponding Author production. The physiological parameters such as fermentation time, fermentation temperature, pH, inoculum age, initial moisture content and the nutritional parameters namely carbon, organic and inorganic nitrogen sources were optimized for the production of protease. At 7 days of fermentation, 25°C, pH 7, 7-days old culture and at 45% initial moisture content, protease activity of 172.5 U/gds was obtained. Further the activity was raised to 197.5 U/gds by supplementing the substrate media with sucrose (1% w/w), peptone (1% w/w) and ammonium chloride (1% w/w).Address:Name: Keywords: Solid-state fermentation, Sesame oil cake, Penicillium chrysogenum NCIM 737,MVV. Chandana Lakshmi INTRODUCTIONPlace: Protease, Physiological parameters, Nutritional parameters.Vishakapatnam, AP, IndiaE-mail:mahantilakshmi@yahoo.com INTRODUCTION International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 436
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 Extra cellular enzymes are usually capable of digesting insoluble nutrient materials such as cellulose, protein, starch, INTRODUCTIONand the digested products are transported into the cell where they are used as nutrients for growth. Some extra cellularenzymes used in the food, dairy, pharmaceutical, textile industries, etc., are produced in large amounts by microbial synthesis1.Enzymes will probably play a key role for environmental friendly cleanup processes because of biodegradability and efficientprocessing in leather industry2. Proteolytic enzymes play an important role in the metabolism of almost all organisms (plants,animals, fungi, bacteria and viruses). Investigation of proteases is a central issue in enzymology due to both their immensephysiological importance and wide application in research and economical activities 3. Commercial application of microbialproteases is attractive due to the relative ease of large-scale production as compared to proteases from plants and animals4.Most of the enzymes market is related to hydrolytic type of enzymes such as proteases, lipases and the cellulases. Although useof enzymes has many advantages, the competitiveness of the enzymes compared to the chemicals is limited by their higherproduction costs. Hence, the use of a less expensive material as substrate is an interesting option 5. Traditionally, syntheticsubstrates were used for fermentations, which are now being largely replaced by agro- and agro-industrial by-products. Thesenot only provide a natural substrate for fungal growth and fermentation but they result in improved value of these agro-industrial residues6. Microbial proteases, which play a specific catalytic role in the hydrolysis of proteins, are indeed one of the mostimportant groups of industrial enzymes and account for about 60% of the total worldwide enzyme sales 7. Fungal proteasesoffer a distinct advantage over the bacterial enzymes in terms of ease of downstream processing 8. Fungal neutral proteases arethe most important component of commercial fungal protease preparations, which have applications in baking, foodprocessing, animal feeds and pharmaceutical industries 9, 10. Neutral proteases usually have low thermostability but there arereports of heat resistant neutral proteases, which can hydrolyze casein at fastest rate at 60 - 65°C11. Species of Penicillium are also the producers of neutral proteases9. Though most of filamentous fungi are capable ofproducing proteolytic enzymes, a demand for strain selection and fermentation media is an essential target in biotechindustry2. Protease producing strains can grow effectively in medium containing protein hydrolysates. Peptone is generallyused in culture media because of its rich amino acid and low-molecular weight peptide content10. Productions of cell andenzymes in Solid-State Fermentation (SSF) were remarkably influenced by the water content, so water is the limiting factor forfungal growth in SSF12, 13. SSF, generally known as the "bran process," was almost universally employed for the production offungal enzymes14. In recent developments, the organisms used in SSF produce high yields of pure enzymes, which are muchmore efficiently produced than in submerged fermentations. Fungi play a key role in SSF, for their hyphal development allowsthem to effectively colonise and penetrate the solid substrate12. The present study was undertaken for the production of neutral protease under SSF by P. chrysogenum NCIM 737using sesame oil cake as substrate, and to determine the effect of various physiological and nutritional parameters to enhancethe activity of protease. MATERIALS AND METHODS The substrates used in this study namely Green gram husk, Black gram husk, Rice bran, Coconut oil cake,SubstrateSesame oil cake (SOC) and Paddy straw + Rice bran (7:3) were obtained from local grocery shop in Visakhapatnam. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 437
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 The organisms used in the present study namely P. chrysogenum NCIM 737, Rhizopus oligosporus NCIM 1215 andMicroorganism and maintenance of cultureAcremonium chrysogenum NCIM 893 were obtained from National Collection of Industrial Microorganisms (NCIM), Pune,India. The cultures were maintained on potato dextrose agar slants and were sub-cultured every month. The inoculum was prepared by dispersing the spores from a week-old fungal slant culture in 0.1 % Tween-80 solutionInoculum preparationwith a sterile inoculation loop. Five grams of each substrate was taken in 250 mL Erlenmeyer flask separately were moistened with salt solutionSolid-State Fermentation[composition (%w/v) (g/100mL): ammonium nitrate 0.5, potassium dihydrogen orthophosphate 0.2, sodium chloride 0.1 andmagnesium sulfate 0.1] and sterilized at 121.5°C for 15 min, cooled and then inoculated with 1 mL of fungal spore suspensionand incubated at 25°C for 7 days. A solution of Tween-80 (0.1%) was added to 100 mL distilled water. 25 mL of water was added to 5 g of fermentedExtraction of crude enzymesubstrate and the substrate was homogenized on a rotary shaker at 180 rpm for 1h and then filtered. The solids were removedby centrifuging the homogenate at 8000 x g at 4ºC for 15 min and the resultant clear supernatant was used for analyticalstudies. To 200 µL of crude enzyme extract, 500 µL of casein (1%) and 300 µL of 0.2 mol/L phosphate buffer (pH 7.0) wereAssay for neutral proteaseadded. The reaction mixture was incubated at 60°C for 10 min and arrested by the addition of 1mL of 10% trichloro aceticacid16. The reaction mixture was centrifuged at 8000 x g at 40C for 15 min and to the supernatant, 5 mL of 0.4 mol/L Na 2CO3,1mL of 3-fold diluted Folin and Ciocalteau’s phenol reagent were added. The resulting solution was incubated at roomtemperature for 30 min and the absorbance of the blue color developed was read at 660 nm and its concentration wasdetermined using tyrosine standard curve. One unit of enzyme activity was defined as the amount of enzyme that liberatedone microgram of tyrosine from substrate (casein) per minute under assay conditions.Standard graph for tyrosine To a series of test tubes, 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1mL of standard solution of tyrosine (100 µg/mL) wasProceduretaken and water is added to each test tube to make the solution up to 1mL. Each test tube contains 10, 20, 40, 60, 80, 100µg/mL of tyrosine. To each test tube 5 mL of 0.5 M Na 2CO3 and 1mL of 3-fold diluted Folin and Ciocalteau’s Phenol reagentwere added and incubated for 30 min. The optical density of above solutions was measured at 660 nm (Fig. 1). Blank wasprepared with 1mL of water instead of tyrosine solution. Seven different substrates like green gram husk, black gram husk, rice bran, coconut oil cake, sesame oil cake, paddyScreening of substrates and fungal speciesstraw rice bran (7:3) were screened using three different fungal species namely Penicillium chrysogenum NCIM 737, Rhizopusoligosporus NCIM 1215 and Acremonium chrysogenum NCIM 893 for neutral protease production using SSF. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 438
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447OPTIMIZATION The protocol adopted for the optimization of process parameters was to evaluate the effect of an individual parameterOptimization of physiological parametersat a time and to incorporate it at the standard level before optimizing the next parameter.The production profile of neutral protease was studied by conducting the fermentation at different time intervals 1, 2, 3, 4, 5,Optimization of fermentation time6, 7, 8, 9, 10 days.The inoculated substrates were incubated at different temperatures ranging from 20 to 45°C, to determine the optimumOptimization of fermentation temperaturefermentation temperature for neutral protease production.Optimum pH for neutral protease production was determined by conducting the fermentation at different pH 4, 5, 6, 7, 8, 9 andOptimization of medium pH10. Different inoculum ages of 1-10 days were varied to determine the maximum production of neutral protease.Optimization of inoculum age Optimum initial moisture content for neutral protease production was determined by adjusting the initial moistureOptimization of initial moisture contentcontent of the fermentation substrate to varying levels of 30, 35, 40, 45, 50 and 55%.EFFECT OF NEUTIONAL PARAMETERS Influence of various carbon supplements on enzyme production was studied by adding different sugars namelyEffect of carbon supplementsxylose, maltose, galactose, sucrose and lactose at 1 % (w/w) to the fermentation media. Various organic nitrogen supplements - peptone, beef extract, yeast extract and malt extract at a concentration of 1 %Effect of organic nitrogen supplements(w/w) were added to the fermentation media to study its effect on protease production. Different sources of inorganic nitrogen - KNO3, NH4Cl, NH4NO3 and (NH4)2SO4 at 1 % (w/w) were added to theEffect of inorganic nitrogen supplementsfermentation medium to study its effect on enzyme production. The pH optimum of the neutral protease enzyme was determined by using buffer solutions of different pH (PhosphateCharacterization of the extracted enzymebuffer 7.0, Acetate buffer 5.0, Glycine-NaOH buffer 10.5) for enzyme assay. The buffers used were of the concentration 0.2mol/L. RESULTS AND DISCUSSION The three fungal species were inoculated individually in the six agro-industrial wastes. The results in the presentScreening of microorganisms and substratesstudy indicated that protease production varied with the type of agro-waste as shown in Table 1. The maximum activity of130.0 U/gds was obtained, when P.chrysogenum NCIM 737 was inoculated in the substrate, sesame oilcake. This could beattributed to solid materials dual role supply of nutrients to the microbial culture and anchorage for the growing cells. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 439
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 Table 1: Screening of microorganisms and substrates for the protease production Substrate protease activity(U/gds) protease activity(U/gds) protease activity(U/gds) P.chrysogenum R.oligosporous A. chrysogenum Green gram husk 50.00 47.50 5.00 Black gram husk 17.50 7.50 10.00 Rice bran 50.00 20.00 82.50 Coconut Oil cake 17.50 10.00 11.25 Sesame oil cake 130.00 102.50 2.50Paddy straw + Rice bran(7:3) 10.00 15.00 5.00 P.chrysogenum NCIM 737 proved to be the best strain for neutral protease production on sesame oilcake substrategiving maximum enzyme activity. Hence, this Penicillium strain was selected to optimize the physiological and nutritionalparameters to enhance the enzyme production under SSF. 1.2 1.0 optical density at 660 nm 0.8 0.6 0.4 0.2 0.0 0 20 40 60 80 100 Figure 1. Standard graph for tyrosine concentration of tyrosine (micro gram/ml) The enzyme production was gradually increased with the passage of time and highest enzyme activity of 135.0 U/gdsEffect of fermentation timewas obtained after 7 days of incubation as shown in Fig.2. The subsequent decrease in the enzyme may be due to theinactivation of the enzyme by other constituent proteases, the reduced availability of nutrients and production of toxicmetabolites11. Tremacoldi and Carmona, 2005 reported that the highest protease activity was obtained by Aspergillus clavatusafter 6 days of incubation for culture medium containing glucose and casein at 1% (w/v) as substrates 15. The enzyme production was carried out by P.chrysogenum NCIM 737 at 20-45°C temperature range. MaximumEffect of fermentation temperatureactivity of protease, 147.5 U/gds was obtained at a temperature of 25°C as shown in Fig. 3. Further increase in temperature,reduced the enzyme production. The reduction in enzyme activity may be due to the denaturation of the enzyme by losing itscatalytic properties at high temperature due to stretching breaking of weak hydrogen bonds with in the enzyme structure 11. Inearlier reports, Pushpa and Madhava Naidu, 2010 reported that the maximum production of protease from coffee by-productsusing Aspergillus oryzae was obtained at temperature 30°C16. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 440
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 180 Protease activity (U/gds) 140 100 60 20 -20 0 2 4 6 8 10 12 Fermentation time (days) Figure 2. Effect of fermentation time on the production of neutral protease by P.chrysogenum NCIM 737 180 Protease activity (U/gds) 140 100 60 20 -20 15 20 25 30 35 40 45 50 Fermentation temperature Figure 3. Optimization of fermentation temperature for neutral protease production by P.chrysogenum NCIM 737 Protease production by microbial strains depends on the extra cellular pH because culture pH strongly influencesEffect of pHmany enzymatic processes and transport of various components across the cell membranes, which in turn support cell growthand product production17,18. The enzyme synthesis was increased with increase of medium pH towards neutrality with amaximum activity of 162.5 U/gds as shown in Fig. 4. Similar results were also reported by Paranthaman et al., 2009 that themaximum production of neutral protease from rice mill waste using Aspergillus niger was obtained at pH 7.017. The effect of inoculum age on protease production was studied by conducting the fermentation with differentEffect of inoculum ageinoculum ages, 1-10 days. It was observed that 7-days old culture gave maximum production of protease, 165.0 U/gds asshown in Fig.5. Ikasari and Mitchell, 1994 reported that the 5-day old inoculum gave maximum protease yield with Rhizopusoligosporus ACM 145F19. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 441
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 200 Protease activity (U/gds) 160 120 80 40 0 2 4 6 8 10 12 pH Figure 4. Effect of pH on the production of protease production by P.chrysogenum NCIM 737 200 Protease activity (U/gds) 160 120 80 40 0 0 2 4 6 8 10 12 Inoculum age (days) Figure 5. Effect of inoculum age on the production of neutral protease by P.chrysogenum NCIM 737. Initial moisture content is a crucial factor affecting the formation of products through SSF. The optimum initial moistureEffect of initial moisture contentcontent for neutral protease production was determined by adjusting the initial moisture content of the fermentationsubstrate to varying levels of 30, 35, 40, 45, 50 & 55 %. From Fig. 6 it was observed that moisture level of 45 % was found tobe optimum for neutral protease production (172.5U/gds). Sumantha et al., 2006 reported that, the moisture content of 44.4% facilitated neutral protease production by Rhizopus microsporus NRRL 3671, on rice bran20. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 442
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 200 Protease activity (U/gds) 160 120 80 40 0 25 30 35 40 45 50 55 60 Initial moisture content (%) Figure 6. Effect of initial moisture content on the production of neutral protease by P.chrysogenum NCIM 737 Various carbon supplements namely xylose, maltose, galactose, sucrose and lactose of 1 % (w/w) were studiedEffect of carbon supplementsindividually. From Fig. 7 it was observed that all the carbon supplements to the substrate show influence on the enzymeproduction. Among the 5 different carbon sources, it was observed that sucrose showed the highest enzyme activity of181.25U/gds. Sumantha et al., 2006 reported that sucrose was the best carbon source in the carbohydrate deficient substratesand sucrose enhanced the protease production by Rhizopus microsporus NRRL 367120. 200 180 Protease activity (U/gds) 160 140 120 100 80 60 40 20 0 xylose maltose galactose sucrose lactose Carbon sources (1 % (w/w)) Figure 7. Effect of carbon sources on the production of neutral protease by P.chrysogenum NCIM 737. Different organic nitrogen supplements like peptone, beef extract, yeast extract and malt extract at a concentration ofEffect of organic nitrogen supplements1 % (w/w) were examined for the maximum protease activity. Among the 4 different organic nitrogen supplements, peptoneenhanced the production of protease i.e., maximum activity of 190.0 U/gds was obtained as shown in Fig. 8. According to International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 443
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447Kalaiarasi and Sunitha, 2009 also reported that peptone was the best organic nitrogen supplement for the maximumproduction of protease21. 195 190 Protease activity (U/gds) 185 180 175 170 165 160 155 150 145 peptone beef extract yeast extract malt extract Organic nitrogen sources (1% (w/w)) Figure 8. Effect of organic nitrogen sources on the production of neutral protease by P.chrysogenum NCIM 737 Various inorganic nitrogen supplements namely KNO3, NH4Cl, NH4NO3, and (NH4)2SO4 at a concentration of 1 % (w/w)Effect of inorganic nitrogen supplementswere studied to enhance the enzyme activity. From Fig. 9 it was observed that among the 4 different inorganic nitrogensupplements maximum protease activity of 197.5 U/gds was obtained using NH 4Cl. In the work of Sindhu et al., 2009,ammonium nitrate (0.5%) was the best inorganic nitrogen source for the maximum production of protease by Penicilliumgodlewskii SBSS 25 22. 200 195 Protease activity (U/gds) 190 185 180 175 170 165 160 155 150 KNO3 NH4Cl NH4NO3 (NH4)2SO4 Inorganic nitorgen source (1 %(w/w)) Figure 9. Effect of inorganic nitrogen sources on the production of neutral protease by P.chrysogenum NCIM 737 International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 444
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-447Enzyme characterization The enzyme showed the maximum specific activity at pH 7.0 (Fig. 10) indicating the instability of enzyme at other pHpHvalues. Sumantha et al., 2006 reported that the neutral protease from Rhizopus oligosporus NRRL 3671 also has optimum pH7.0. 200 Protease activity (U/gds) 160 120 80 40 0 4 5 6 7 8 9 10 11 pH Figure 10. pH curve of neutral protease of P.chrysogenum NCIM 737. The present study focused on the use of P. chrysogenum NCIM 737 to convert agro-industrial by-products to a value- CONCLUSIONadded product, an enzyme-protease. Neutral protease production under SSF was carried out by using P. chrysogenum NCIM737. At the optimum conditions of fermentation time 7 days, temperature 25 oC, pH 7.0, inoculum age 7 days, initial moisturecontent 45% the protease activity found was 172.5 U/gds. In addition to physiological parameters, the chemical parametersnamely, carbon (sucrose), organic nitrogen source (peptone) and inorganic nitrogen source (ammonium chloride) at aconcentration of 1% w/w enhanced the protease activity to 197.5 U/gds. Proteases have found a wide range of applications invarious industries such as food, pharmaceuticals, detergents etc. From the results, it could be inferred that neutral proteaseproduced through SSF of the sesame oil cake by P.chrysogenum NCIM 737 could possibly find useful application in foodindustries.1. Vania Sousa Andrade, Leonie Asfora Sarubbo, Kasutaka Fukushima, Makoto Miyaji, Kazuko Nishimura, Galba Maria de REFERENCES Campos-Takaki, “Production of extracellular protease By mucor circinelloides using D-glucose as carbon source / substrate”, Braz. J. Microbiol. 2002, 33, 106-110.2. Chellapandi, P. “Production and Preliminary Characterization of Alkaline Protease from Aspergillus flavus and Aspergillus terreus”, E-J. Chem., 2010, 7(2), 479-482. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 445
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-4473. Siddalingeshwara, K.G., Uday, J., Huchesh, C.H., Puttaraju , H.P., Karthic, J.,Sudipta, K.M., Pramod, T., Vishwanatha, T. “Screening and characterization of protease from Bacillus sp”, Int. J. Appl. Biol. Phar.Tech. 2010, 1, 575-581.4. Vamsi Krishna, K., Mayank Gupta, Nikhil Gupta, Hipal Gaudani, Soham Trivedi, Prasad Patil, Girish Gupta, Yogesh Khairnar, Amol Borasate, Dharmendra Mishra, “Optimization of growth and production of protease by Penicillium species using submerged fermentation”, Int. J. Microbiol. Res. 2009,1,14-18.5. Drouin, M., Lai, C. K., Tyagi, R.D., Surampalli, R.Y. “Bacillus licheniformis proteases as high value added products from fermentation of wastewater sludge: pre-treatment of sludge to increase the performance of the process”, Water Science & Technology 2007,599-605.6. Alagarsamy Sumantha, Chandran Sandhya, George Szakacs, Carlos R. Soccol Ashok Pandey, “Production and Partial Purification of a Neutral Metalloprotease by Fungal Mixed Substrate Fermentation”,Food Technol. Biotechnol. 2005, 43(4), 313–319.7. Wu, T.Y., Mohammad, A.W., Jahim, J. Md., Anuar, N. “Investigations on protease production by a wild-type Aspergillus terreus strain using diluted retentate of pre-filtered palm oil mill effluent (POME) as substrate”,Enzy. Microbial Tech., 2006, 39, 1223-1229.8. Mohamed Hajji, Ahmed Rebai, Neji Gharsallah, Moncef Nasri, “Optimization of alkaline protease production by Aspergillus clavatus ES1 in Mirabilis jalapa tuber powder using statistical experimental design”, Appl. Microbiol. Biotech. 2008, 79, 915-923.9. Alagarsamy Sumantha, Christian Larroche, Ashok Pandey, “Microbiology and Industrial Biotechnology of Food-Grade Proteases: A Perspective”, Food Technol. Biotechnol. 2006, 44(2), 211-220.10. Barnali Ray Basu, Ajit K. Banik, Manas Das, “Production and characterization of extracellular protease of mutant Aspergillus niger AB100 grown on fish scale”, World J. Microbiol. Biotechnol. 2008, 24, 449-455.11. Ikram-ul-haq, Hamid mukhtar, Hina umber, “Production of Protease by Penicillium chrysogenum Through Optimization of Environmental Conditions”, J. Agr. Soc. Sci. 2006, 2(1), 23-25.12. Rob te Biesebeke, George Ruijter, Yovita S.P. Rahardjo, Marisca J.Hoogschagen, Margreet Heerikhuisen, Ana Levin, Kenneth G.A. van Driel, Maarten A.I. Schutyser, Jan Dijksterhuis, Yang Zhu, Frans J. Weber, Willem M. de Vos, Kees A.M.J.J. van den Hondel, Arjen Rinzema, Peter J. Punt, “Aspergillus oryzae in solid-state and submerged fermentations Progress report on a multi-disciplinary project”,FEMS Yeast Res. 2002, 2, 245-248.13. 13. Hideki Narahara, Yosuke Koyama, Toshiomi Yoshida, Poonsuk Atthasampunna, Hisaharu Taguchi, “Control of water content in a solid state culture of Aspergillusoryzae”, J. Fermen. Tech. 1984, 62(5), 453-459.14. 14. Hwa L. Wang, Janet B. vespa, Hesseltine, C. W., “Acid Protease Production by Fungi Used in Soybean Food Fermentation”, Appl. Microbiol. 1974, 27(5), 906-911. Celia R. Tremacoldi and Eleonora Cano Carmona, “Production of extracellular alkaline proteases by Aspergillus clavatus”, World J. Microbiol. Biotech., 2005, 21,169-172.15. Pushpa S.Murthy, and Madhava Naidu, M., “Protease production by Aspergillus oryzae in solid state fermentation utilizing Coffee byproducts”, World Appl. Sci. J., 2010, 8(2), 199-205.16.17. Paranthaman, R., Alagusundaram, K. and Indhumathi, “Production of protease from rice mill wastes by Aspergillus niger in Solid State Fermentation”, World J. of Agri. Sci., 2009, 5(3), 308-312. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 446
    • B. Nagamani et al., IJSID, 2012, 2 (5), 436-44718. Prakasham, R.S., Subba Rao, Ch. and Sarma, P.N., Green gram husk—an inexpensive substrate for alkaline protease production by Bacillus sp. in solid-state fermentation. Biores. Tech., 2006, 97, 1449–1454.19. Ikasari, L., Mitchell, D.A. “Protease production by Rhizopus oligosporus in solid state fermentation”, World J. Microbiol. Biotechnol. 1994, 10, 320-324.20. Alagarsamy Sumantha, Paul Deepa, Chandran Sandhya, George Szakacs, Carlos Ricardo Soccol, Ashok Pandey, “Rice Bran as a Substrate for Proteolytic Enzyme Production”, Braz. Arch. Biol. Technol. 2006, 49(5), 843-851.21. Kalaiarasi, K., Sunitha, P. U. “Optimization of alkaline protease production from Pseudomonas fluorescens isolated from meat waste contaminated soil”, African JBiotechnol. 2009, 8(24), 7035-7041.22. Sindhu, R., Suprabha, G. N. and Shashidhar, S., “Optimization of process parameters for the production of alkaline protease from Penicillium godlewskii SBSS 25 and its application in detergent industry”, African Journal of MicrobiologyResearch, 2009, 3(2), pp. 515-522. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 447