B. Nagamani et al.

, IJSID, 2012, 2 (5), 436-447

ISSN:2249-5347

IJSID

International Journal of Science Innovations and Discoveries

Research Article

An International peer Review Journal for Science

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
Received: 03-07-2012 Accepted: 18-10-2012
*Corresponding Author

using Penicillium chrysogenum NCIM 737. Among the six (green gram husk, black gram agro-industrial waste materials evaluated, sesame oil cake supported maximum protease production. The physiological parameters such as fermentation time, fermentation

husk, rice bran, coconut oil cake, sesame oil cake and paddy straw + rice bran (7:3)) temperature, pH, inoculum age, initial moisture content and the nutritional parameters namely carbon, organic and inorganic nitrogen sources were optimized for the production 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). of protease. At 7 days of fermentation, 25C, pH 7, 7-days old culture and at 45% initial

Neutral protease production under solid-state fermentation was carried out by ABSTRACT

Address: Name: MVV. Chandana Lakshmi Place: Vishakapatnam, AP, India E-mail: mahantilakshmi@yahoo.com

Keywords: Solid-state fermentation, Sesame oil cake, Penicillium chrysogenum NCIM 737, INTRODUCTION Protease, Physiological parameters, Nutritional parameters.

INTRODUCTION

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B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 and the digested products are transported into the cell where they are used as nutrients for growth. Some extra cellular Enzymes will probably play a key role for environmental friendly cleanup processes because of biodegradability and efficient animals, fungi, bacteria and viruses). Investigation of proteases is a central issue in enzymology due to both their immense Extra cellular enzymes are usually capable of digesting insoluble nutrient materials such as cellulose, protein, starch, INTRODUCTION

enzymes used in the food, dairy, pharmaceutical, textile industries, etc., are produced in large amounts by microbial synthesis1. physiological importance and wide application in research and economical activities 3. Commercial application of microbial

processing in leather industry2. Proteolytic enzymes play an important role in the metabolism of almost all organisms (plants, proteases 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 use of enzymes has many advantages, the competitiveness of the enzymes compared to the chemicals is limited by their higher industrial residues6. production costs. Hence, the use of a less expensive material as substrate is an interesting option 5. Traditionally, synthetic Microbial proteases, which play a specific catalytic role in the hydrolysis of proteins, are indeed one of the most substrates were used for fermentations, which are now being largely replaced by agro- and agro-industrial by-products. These important groups of industrial enzymes and account for about 60% of the total worldwide enzyme sales 7. Fungal proteases offer a distinct advantage over the bacterial enzymes in terms of ease of downstream processing 8. Fungal neutral proteases are the most important component of commercial fungal protease preparations, which have applications in baking, food reports of heat resistant neutral proteases, which can hydrolyze casein at fastest rate at 60 - 65C11. processing, animal feeds and pharmaceutical industries 9, 10. Neutral proteases usually have low thermostability but there are

not only provide a natural substrate for fungal growth and fermentation but they result in improved value of these agro-

producing proteolytic enzymes, a demand for strain selection and fermentation media is an essential target in biotech used in culture media because of its rich amino acid and low-molecular weight peptide content10. Productions of cell and enzymes in Solid-State Fermentation (SSF) were remarkably influenced by the water content, so water is the limiting factor for them to effectively colonise and penetrate the solid substrate12. the activity of protease. Substrate fungal growth in SSF12, 13. SSF, generally known as the "bran process," was almost universally employed for the production of fungal enzymes14. In recent developments, the organisms used in SSF produce high yields of pure enzymes, which are much using sesame oil cake as substrate, and to determine the effect of various physiological and nutritional parameters to enhance MATERIALS AND METHODS

industry2. Protease producing strains can grow effectively in medium containing protein hydrolysates. Peptone is generally

Species of Penicillium are also the producers of neutral proteases9. Though most of filamentous fungi are capable of

more efficiently produced than in submerged fermentations. Fungi play a key role in SSF, for their hyphal development allows The present study was undertaken for the production of neutral protease under SSF by P. chrysogenum NCIM 737

Sesame oil cake (SOC) and Paddy straw + Rice bran (7:3) were obtained from local grocery shop in Visakhapatnam.

The substrates used in this study namely Green gram husk, Black gram husk, Rice bran, Coconut oil cake,

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B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 Microorganism and maintenance of culture Acremonium 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. Inoculum preparation with a sterile inoculation loop. Solid-State Fermentation The organisms used in the present study namely P. chrysogenum NCIM 737, Rhizopus oligosporus NCIM 1215 and The inoculum was prepared by dispersing the spores from a week-old fungal slant culture in 0.1 % Tween-80 solution Five grams of each substrate was taken in 250 mL Erlenmeyer flask separately were moistened with salt solution

[composition (%w/v) (g/100mL): ammonium nitrate 0.5, potassium dihydrogen orthophosphate 0.2, sodium chloride 0.1 and and incubated at 25C for 7 days. Extraction of crude enzyme

magnesium sulfate 0.1] and sterilized at 121.5C for 15 min, cooled and then inoculated with 1 mL of fungal spore suspension substrate and the substrate was homogenized on a rotary shaker at 180 rpm for 1h and then filtered. The solids were removed by centrifuging the homogenate at 8000 x g at 4C for 15 min and the resultant clear supernatant was used for analytical studies. Assay for neutral protease added. The reaction mixture was incubated at 60C for 10 min and arrested by the addition of 1mL of 10% trichloro acetic To 200 L of crude enzyme extract, 500 L of casein (1%) and 300 L of 0.2 mol/L phosphate buffer (pH 7.0) were A solution of Tween-80 (0.1%) was added to 100 mL distilled water. 25 mL of water was added to 5 g of fermented

acid16. 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 Ciocalteaus phenol reagent were added. The resulting solution was incubated at room determined using tyrosine standard curve. One unit of enzyme activity was defined as the amount of enzyme that liberated one microgram of tyrosine from substrate (casein) per minute under assay conditions. Standard graph for tyrosine Procedure To a series of test tubes, 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1mL of standard solution of tyrosine (100 g/mL) was temperature for 30 min and the absorbance of the blue color developed was read at 660 nm and its concentration was

taken and water is added to each test tube to make the solution up to 1mL. Each test tube contains 10, 20, 40, 60, 80, 100 g/mL of tyrosine. To each test tube 5 mL of 0.5 M Na 2CO3 and 1mL of 3-fold diluted Folin and Ciocalteaus Phenol reagent were added and incubated for 30 min. The optical density of above solutions was measured at 660 nm (Fig. 1). Blank was prepared with 1mL of water instead of tyrosine solution. Screening of substrates and fungal species Seven different substrates like green gram husk, black gram husk, rice bran, coconut oil cake, sesame oil cake, paddy

straw rice bran (7:3) were screened using three different fungal species namely Penicillium chrysogenum NCIM 737, Rhizopus oligosporus NCIM 1215 and Acremonium chrysogenum NCIM 893 for neutral protease production using SSF.

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B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 OPTIMIZATION Optimization of physiological parameters at a time and to incorporate it at the standard level before optimizing the next parameter. Optimization of fermentation time 6, 7, 8, 9, 10 days. The protocol adopted for the optimization of process parameters was to evaluate the effect of an individual 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 temperature fermentation temperature for neutral protease production. Optimization of medium pH 10.

The inoculated substrates were incubated at different temperatures ranging from 20 to 45C, to determine the optimum Optimum pH for neutral protease production was determined by conducting the fermentation at different pH 4, 5, 6, 7, 8, 9 and Optimization of inoculum age Different inoculum ages of 1-10 days were varied to determine the maximum production of neutral protease.

Optimization of initial moisture content

content of the fermentation substrate to varying levels of 30, 35, 40, 45, 50 and 55%. EFFECT OF NEUTIONAL PARAMETERS Effect of carbon supplements xylose, maltose, galactose, sucrose and lactose at Effect of organic nitrogen supplements

Optimum initial moisture content for neutral protease production was determined by adjusting the initial moisture Influence of various carbon supplements on enzyme production was studied by adding different sugars namely 1 % (w/w) to the fermentation media.

(w/w) were added to the fermentation media to study its effect on protease production. Effect of inorganic nitrogen supplements fermentation medium to study its effect on enzyme production. Characterization of the extracted enzyme mol/L. buffer 7.0, Acetate buffer 5.0, Glycine-NaOH

Various organic nitrogen supplements - peptone, beef extract, yeast extract and malt extract at a concentration of 1 %

Different sources of inorganic nitrogen - KNO3, NH4Cl, NH4NO3 and (NH4)2SO4 at 1 % (w/w) were added to the The pH optimum of the neutral protease enzyme was determined by using buffer solutions of different pH (Phosphate RESULTS AND DISCUSSION The three fungal species were inoculated individually in the six agro-industrial wastes. The results in the present buffer 10.5) for enzyme assay. The buffers used were of the concentration 0.2

Screening of microorganisms and substrates

study indicated that protease production varied with the type of agro-waste as shown in Table 1. The maximum activity of attributed 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

130.0 U/gds was obtained, when P.chrysogenum NCIM 737 was inoculated in the substrate, sesame oilcake. This could be

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giving maximum enzyme activity. Hence, this Penicillium strain was selected to optimize the physiological and nutritional parameters to enhance the enzyme production under SSF.
1.2

P.chrysogenum R.oligosporous A. chrysogenum protease activity(U/gds) protease activity(U/gds) protease activity(U/gds) 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 2.50 Paddy 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 substrate

Substrate

Table 1: Screening of microorganisms and substrates for the protease production

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1.0

optical density at 660 nm

0.8

0.6

0.4

0.2

0.0 0 20 40 60 80 100

concentration of tyrosine (micro gram/ml)

Effect of fermentation time

was obtained after 7 days of incubation as shown in Fig.2. The subsequent decrease in the enzyme may be due to the after 6 days of incubation for culture medium containing glucose and casein at 1% (w/v) as substrates 15. Effect of fermentation temperature

inactivation of the enzyme by other constituent proteases, the reduced availability of nutrients and production of toxic metabolites11. Tremacoldi and Carmona, 2005 reported that the highest protease activity was obtained by Aspergillus clavatus activity of protease, 147.5 U/gds was obtained at a temperature of 25C as shown in Fig. 3. Further increase in temperature, using Aspergillus oryzae was obtained at temperature 30C16. The enzyme production was carried out by P.chrysogenum NCIM 737 at 20-45C temperature range. Maximum

The enzyme production was gradually increased with the passage of time and highest enzyme activity of 135.0 U/gds

Figure 1. Standard graph for tyrosine

reduced the enzyme production. The reduction in enzyme activity may be due to the denaturation of the enzyme by losing its

catalytic properties at high temperature due to stretching breaking of weak hydrogen bonds with in the enzyme structure 11. In earlier reports, Pushpa and Madhava Naidu, 2010 reported that the maximum production of protease from coffee by-products International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012

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180

Protease activity (U/gds)

140 100 60 20 -20 0 2 4 6 8 10 12

Figure 2. Effect of fermentation time on the production of neutral protease by P.chrysogenum NCIM 737
180

Fermentation time (days)

Protease activity (U/gds)

140 100 60 20 -20 15 20 25 30 35 40 45 50

Fermentation temperature

Effect of pH

many enzymatic processes and transport of various components across the cell membranes, which in turn support cell growth maximum production of neutral protease from rice mill waste using Aspergillus niger was obtained at pH 7.017. Effect of inoculum age

Figure 3. Optimization of fermentation temperature for neutral protease production by P.chrysogenum NCIM 737

and product production17,18. The enzyme synthesis was increased with increase of medium pH towards neutrality with a

maximum activity of 162.5 U/gds as shown in Fig. 4. Similar results were also reported by Paranthaman et al., 2009 that the inoculum ages, 1-10 days. It was observed that 7-days old culture gave maximum production of protease, 165.0 U/gds as oligosporus ACM 145F19. International Journal of Science Innovations and Discoveries, Volume 2, Issue 5, September-October 2012 The effect of inoculum age on protease production was studied by conducting the fermentation with different

Protease production by microbial strains depends on the extra cellular pH because culture pH strongly influences

shown in Fig.5. Ikasari and Mitchell, 1994 reported that the 5-day old inoculum gave maximum protease yield with Rhizopus

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200

Protease activity (U/gds)

160 120 80 40 0 2 4 6 pH 8 10 12

Figure 4. Effect of pH on the production of protease production by P.chrysogenum NCIM 737

200 160 120 80 40 0 0 2 4 6 8 10 12 Inoculum age (days)

Effect of initial moisture content

content for neutral protease production was determined by adjusting the initial moisture content of the fermentation % facilitated neutral protease production by Rhizopus microsporus NRRL 3671, on rice bran20.

substrate to varying levels of 30, 35, 40, 45, 50 & 55 %. From Fig. 6 it was observed that moisture level of 45 % was found to be optimum for neutral protease production (172.5U/gds). Sumantha et al., 2006 reported that, the moisture content of 44.4

Initial moisture content is a crucial factor affecting the formation of products through SSF. The optimum initial moisture

Figure 5. Effect of inoculum age on the production of neutral protease by P.chrysogenum NCIM 737.

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200

Protease activity (U/gds)

160 120 80 40 0 25 30 35 40 45 50 55 60 Initial moisture content (%)

Effect of carbon supplements

individually. From Fig. 7 it was observed that all the carbon supplements to the substrate show influence on the enzyme production. Among the 5 different carbon sources, it was observed that sucrose showed the highest enzyme activity of 181.25U/gds. Sumantha et al., 2006 reported that sucrose was the best carbon source in the carbohydrate deficient substrates and sucrose enhanced the protease production by Rhizopus microsporus NRRL 367120.
200 180 160 140 120 100 80 60 40 20 0 xylose maltose galactose sucrose lactose Carbon sources (1 % (w/w))

Various carbon supplements namely xylose, maltose, galactose, sucrose and lactose of 1 % (w/w) were studied

Figure 6. Effect of initial moisture content on the production of neutral protease by P.chrysogenum NCIM 737

Effect of organic nitrogen supplements

1 % (w/w) were examined for the maximum protease activity. Among the 4 different organic nitrogen supplements, peptone enhanced 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

Different organic nitrogen supplements like peptone, beef extract, yeast extract and malt extract at a concentration of

Figure 7. Effect of carbon sources on the production of neutral protease by P.chrysogenum NCIM 737.

Protease activity (U/gds)

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Kalaiarasi and Sunitha, 2009 also reported that peptone was the best organic nitrogen supplement for the maximum production of protease21.
195
Protease activity (U/gds)

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190 185 180 175 170 165 160 155 150 145 peptone beef extract yeast extract malt extract Organic nitrogen sources (1% (w/w))

Effect of inorganic nitrogen supplements

were studied to enhance the enzyme activity. From Fig. 9 it was observed that among the 4 different inorganic nitrogen godlewskii SBSS 25 22.

Figure 8. Effect of organic nitrogen sources on the production of neutral protease by P.chrysogenum NCIM 737

supplements 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 Penicillium

Various inorganic nitrogen supplements namely KNO3, NH4Cl, NH4NO3, and (NH4)2SO4 at a concentration of 1 % (w/w)

200
Protease activity (U/gds)

195 190 185 180 175 170 165 160 155 150 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

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B. Nagamani et al., IJSID, 2012, 2 (5), 436-447 Enzyme characterization pH 7.0. values. Sumantha et al., 2006 reported that the neutral protease from Rhizopus oligosporus NRRL 3671 also has optimum pH The enzyme showed the maximum specific activity at pH 7.0 (Fig. 10) indicating the instability of enzyme at other pH

200
Protease activity (U/gds)

160 120 80 40 0 4 5 6 7 pH
Figure 10. pH curve of neutral protease of P.chrysogenum NCIM 737. CONCLUSION

10

11

added product, an enzyme-protease. Neutral protease production under SSF was carried out by using P. chrysogenum NCIM 737. At the optimum conditions of fermentation time 7 days, temperature 25 oC, pH 7.0, inoculum age 7 days, initial moisture namely, carbon (sucrose), organic nitrogen source (peptone) and inorganic nitrogen source (ammonium chloride) at a various industries such as food, pharmaceuticals, detergents etc. From the results, it could be inferred that neutral protease industries. 1. 2. REFERENCES concentration of 1% w/w enhanced the protease activity to 197.5 U/gds. Proteases have found a wide range of applications in content 45% the protease activity found was 172.5 U/gds. In addition to physiological parameters, the chemical parameters produced through SSF of the sesame oil cake by P.chrysogenum NCIM 737 could possibly find useful application in food Vania Sousa Andrade, Leonie Asfora Sarubbo, Kasutaka Fukushima, Makoto Miyaji, Kazuko Nishimura, Galba Maria de substrate, Braz. J. Microbiol. 2002, 33, 106-110. terreus, E-J. Chem., 2010, 7(2), 479-482. Campos-Takaki, Production of extracellular protease By mucor circinelloides using D-glucose as carbon source /

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protease from Penicillium godlewskii SBSS 25 and its application in detergent industry, African Journal of

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