Biotechnological
Communication
Biosci. Biotech. Res. Comm. 8(1): 78-83 (2015)
Effect of pH on the production of protease by Fusarium oxysporum using agroindustrial waste
Rupali R. Deshmukh and N. N. Vidhale*
Department of Microbiology, Shri Shivaji Science College, Amravati 444604, M.S., India
ABSTRACT
Agro industrial waste constitute valuable source for cultivation of microorganisms and production of enzyme. An attempt was made to study protease production at various pH by Fusarium oxysporum on agro industrial waste such as dal mill waste, oil mill waste, molasses, fruit waste and vegetable garbage. The maximum protease production (20.2 μg /ml) was obtained at pH 6 and 6.5 when dal mill waste was the substrate after 6th day of incubation. Simi- larly with oil mill waste protease production was (19.6 μg /ml and 20.6 μg /ml) after 6th and 7th day of incubation respectively when pH was 5.5 and 6. However with vegetable garbage, maximum protease was (21.8 μg /ml) at pH 5 after 120 hrs of incubation. Fruit waste showed production of enzyme maximum (12.3 μg /ml) at pH 5 and 6.5 after 96 hrs of incubation
KEY WORDS: AGRO INDUSTRIAL WASTE, FUSARIUM OXYSPORUM, PROTEASE.
INTRODUCTION
Enzymes are among the most important products obtained for human needs through microbial Sources. Protease enzyme constitutes two thirds of total enzymes used in various industries and this dominance in the industrial market need to increase production (Gupta et al., 2002).For production of enzyme for industrial use, isolation and characterization of strains using cheap carbon and nitrogen source is a continuous proc- ess (Parekh et al., 2002). This enzyme occurs widely in plants and animals, but commercially proteases are pro- duced exclusively from microorganisms. Molds of the
ARTICLE INFORMATION:
*Corresponding Author: vidhalenana@gmail.com Received 20th May, 2015
Accepted after revision 28th June, 2015 BBRC Print ISSN: 0974-6455
Online ISSN: 2321-4007 NAAS Journal Score : 3.48
©A Society of Science and Nature Publication, 2015. All rights reserved.
Online Contents Available at: http//www.bbrc.in/
genera Aspergillus, Penicillium and Rhizopus are espe- cially used for producing proteases (Sandhya et al., 2005 and Negi and Banerjee, 2010).
MATERIAL AND METHODS
SUBSTRATE PREPARATION
Agro industrial waste such as dal mill waste, oil mill waste, Molasses, fruit waste and vegetable garbage were collected and powdered to size about 2 mm in homog- enizer and then sieved through 20-40 mesh screens to
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obtain a particle having diameter between 0.42 to 0.85
mm.Each of such substrate was supplemented with 0.83
gm K2HPO4 and 0.16gm MgSO4, 1.5% agar-agar and 10 ml distilled water and autoclaved for 15 min. at 15 lb/ inch 2. Initial utilization of these substrates for produc- tion of protease by Fusarium oxysporum strain was studied under Solid state fermentation, incubated for 7 days at room temperature (25 to 300C). Initially 20 g of 50% moistened substrate was sterilized and thor- oughly mixed with 1ml spore suspension of 7 day old culture of Fusarium oxysporum. This substrate along with spore suspension was poured in sterilized Petri dish and allowed to incubate at room temperature for 7 days.
Deshmukh and Vidhale
min to remove all particulate matter. Protease activity was assayed as suggested by Keay and Wrildi (1970). To 1 ml of culture filtrate, 1ml of 2% casein solution was added and the mixture was incubated at 370C for 10 min. The reaction was terminated by adding 2ml 0.4 M TCA (Trichloro acetic acid), again incubated at 370C for 20 min. and filtered through Whatman filter paper no1. One ml of the filterate was added to 5 ml of sodium carbon- ate (0.4M) and 1ml Folin- Ciocalteus’s reagent and incu- bated at 370C for 30 min. The Absorbance was measured at 660 nm. in Spectrophotometer .
ENZYME ESTIMATION
After every 24 hrs of interval 1gm fermented substrate was harvested from petri plate and transferred to test tube containing 5ml phosphate buffer. The contents were homogenized and centrifuged at 2000 rpm for 30
EFFECT OF PH ON PRODUCTION OF PROTEASE BY FUSARIUM OXYSPORUM
All the five substrates were supplied with pH such as 5, 5.5, 6, 6.5 and 7 and subjected to SSF in Petri plates. Plates were incubated for 7 days. Enzyme activity was determined from 24 hrs to 7 days of incubation.
Table 4: Effect of pH on production of protease by Fusarium oxysporum in solid state fermentation using agro industrial waste as substrate.
Deshmukh and Vidhale
RESULTS AND DISCUSSION
All the five substrates were supplemented with different pH such as 5, 5.5, 6, 6.5, and 7 inoculated with Fusar- ium oxysporum and incubated for 7 days at room tem- perature under solid state fermentation. The production of protease was studied and mentioned in Table 4 and Fig.12, 13, 14, 15 and 16. From the result it has been found that the maximum protease production (20.2 μg /ml) was obtained at pH 6 and 6.5 when dal mill waste was the substrate after 6th day of incubation. Similarly when oil mill waste was the substrate maximum protease production (19.6 μg /ml and 20.6 μg /ml) was recorded after 6th and 7th day of incubation respectively when pH was 5.5 and 6. However maximum protease production was recorded after 4th and 5th day of incubation at all pH in case of molasses. As far as vegetable garbage as a substrate is concerned again all the five types of pH gave maximum production of protease on 6th day of incuba- tion at room temperature, however maximum protease was seen (21.8 μg /ml) at pH 5 after 120 hrs of incu- bation. On the contrary fruit waste substrate responded comparatively lesser as it showed production of enzyme
maximum (12.3 μg /ml) at pH 5 and 6.5 after 96 hrs of incubation when compared with other substrates. In general all the agro industrial waste when supplemented with pH 5.0, 5.5, 6.0 and 6.5 showed increase in protease production as compared to initial enzyme production without adjustment of pHs.
Studies dealing with the effect of initial pH on pro- tease production by other Sreptomyces strains as S. rimosus and Streptomyces sp. 594 in SSF conditions indicated pH values between 6.0 and 7.0 for optimal protease activity (Yang et al., 1999). Ali and Vidhale, (2013) observed maximum protease production with initial pH of substrate 7.0 where as Negi and Banerjee, (2010) achieved optimum protease yield at pH 5.5.Pro- duction of protease using Rhizopus microsporus var oligosporus was studies by Sarao et al., (2009) showed maximum enzyme activity at initial pH 5.5. Similarly Radha et al., (2012) showed production of acid protease at pH 5.0.Optimum pH for protease production by Fusar- ium oxysporum was recorded at 7.0 in the study of Ali and Vidhale, (2013).The optimum pH of protease enzyme production by B. Subtilis and A.niger was recorded pH7 and 8 respectively by Oyeleke et al., (2012).
FIGURE 12: Effect of pH on production of protease by Fusarium oxysporum in SSF using dal mill waste.
Deshmukh and Vidhale
FIGURE 13: Effect of pH on production of protease by Fusarium oxysporum in SSF using oil mill waste.
FIGURE 14: Effect of pH on production of protease by Fusarium oxysporum in SSF using molasses.
Deshmukh and Vidhale
FIGURE 15: Effect of pH on production of protease by Fusarium oxysporum in SSF using fruit waste.
FIGURE 16: Effect of pH on production of protease by Fusarium oxysporum in SSF using vegetable garbage.
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Radha S., Sridevi A., Himakiran Babu R., Nithya V. J., Prasad N. B. L. and Narasimha G. (2012). Medium Optimization for Acid protease production from Aspergillus sps under Solid
