Dr.N.G.P Arts and Science College, Coimbatore-48  India.

Article Publishing History

INTRODUCTION

Fruit and vegetable waste is the ever growing global question. Several techniques have been developed to facilitate the waste into possible sources of energy and helps in reducing the environmental pollution. Globally, the overall production of fruits and vegetables is about 675 million metric tons annually and out of the which 1.3 billion ton wastage is produced. India alone produces 86.602 million metric tons of fruits and vegetables and constitute about 5.6 million ton of waste annually.

Manyof the garbage are removed in ecologically unsustainable manner or the  minimum quantity of waste was enzymatically decomposed with microorganisms due to their ability to secrete enzymes (Elvira C et al., 1998 Chao et al., 2017).   ^.Papaya is a popular fruit consumed worldwide and its well known for food and nutritional values. It is used in food industries, detergent industries, chewing gum industries etc. As a result, the  industries generate huge amounts of papaya peel (PP) and seeds as by-products, which are typically considered a waste, and thus discarded, but that can be converted into many value-added products (Pathak et al., 2019).

Now a days enzymes play a key role in industrial areas. The two major enzymes from papaya include lipase and protease. Commercial proteases account for nearly 60% of the total industrial enzyme market (Katsuhisa et al., 2007). Bacillus sp is the most important group of bacteria that are involved in the the enzyme industry and this bacterium is also known to  produce proteolytic enzymes quite effectively (Patel 1985).Although many other classes of enzymes are currently in industrial use, the focus of this paper is on alkaline proteases from bacterial species. This is because the organisms producing enzymes capable of catalysing the reactions at the extremes of pH above neutrality (Arunachalamand  & Saritha  2009).

The genus Bacillus is vital for commercially important alkaline protease which is active at alkaline pH ranging between 9 and 11 .The detergent industry consumes alkaline proteases most abundantly (Moon, & Parulekar 1991). The largest application of protease is in laundry detergents and leather industry, where they remove protein based stains from clothes and dehairing purpose, respectively. As the modern world focuses on ecofriendly products and product output, subsequently more chemical processes are being replaced by enzymatic methods (Manavalan et al., 2020  Bektas et al 2023).

Protease is one of the important enzymes used in textile industry. The presently available proteases are not sufficient to meet industrial demands. Hence; there is continuous search for new proteases with novel characteristics for industrial application from diverse bacteria isolates. Microbes from varied habitats have been examined by many researchers to obtain the industrially suitable proteases. This  present study was to discuss about the isolation of alkaline protease  from the fruit waste papaya peel and discuss the stain removal  (detergent activity) against the different stains as made.

MATERIAL AND METHODS

Isolation and characterization of microorganism:  Protease producing isolates were analysed from the papaya peel using a skim milk agar plates after incubation. Among the isolates, Bacillus species exhibited a prominent clear zone. Pure culture was obtained for the best three isolates of clear zone after the incubation period of 24-48 hrs at 37⁰C. This  was selected for the study of protease production. This was maintained at 4⁰C in skim milk agar plates^[10]. The morphological and physiological properties of the isolate were investigated according to Bergey’s manual of determinative bacteriology (Towatana 1999 & Becerra et al., 2016).

The three bacterial isolates were inoculated separately to three conical flasks containing 100 ml nutrient broth and incubated over 2-3 days period at 37⁰ C in a rotary shaker. Nutrient  broth without bacterial inoculation kept as control. The contents of the flasks were collected in a centrifuge tube and it was centrifuged at 10000 rpm for 10 minutes at 4⁰ C in order to get the cell free supernatant containing crude enzyme.

Quantitative analysis of protease enzyme activity: Protease activity in the culture supernatant was determined a by using casein as a substrate.  A mixture of 500 μl of 1% (w/v) of casein in 50 mM phosphate buffer, pH 7 and 200 μl crude enzyme extract were incubated in a water bath at 40°C for 20 minutes. After 20 minutes, the enzyme reaction was terminated by the addition of 1 ml of 10% (w/v) trichloroacetic acid (TCA) and was kept at room temperature for 15 minutes. Then, the reaction mixture was centrifuged to separate the unreacted casein at 10,000 rpm for 5 minutes. The supernatant mixed with 2.5 ml of 0.44M Na2CO3. 1 ml of 3-fold diluted FollinCiocalteus phenol reagent was added (Lu et al., 2018).

The resulting solution was incubated at room temperature in the dark for 30 minutes and absorbance of the blue colour developed was measured at 660 nm against a reagent blank using a tyrosine standard (Sony & Potty 2016). This was modified as Lowry’s method (Lowry et al., 1951).  One unit of protease is defined as the amount of enzyme that releases 1 μg of tyrosine per ml per minute under the standard conditions of supernatant solution.

Enzyme activity was calculated by the formula

Effect of pH on enzyme activity: The culture was incubated at 37⁰C for 48hrs and the pH was adjusted using different buffers ranging from 3-9. The OD values of enzyme activities  were taken in spectrophotometer at 600nm (Badhe et al., 2016).

Effect of Temperature on enzyme activity: To study the optimum temperature where an enzyme shows its maximum activity were exposed to different temperature ranging from 4⁰C – 100⁰C. After incubation the OD values were taken in spectrophotometer at 600nm (Olajuyigbe et al., 1980).

Stain removal activity with crude enzyme: The three sample broths were taken for the washing activity. Application of protease enzyme by isolated organism as a detergent additive was studied (Kalapana et al., 2008). The washing test is performed with the three prepared broth samples for three different stains.

Table 1. Activity performed with different dilutions against different stains

Stains	Tap water (C)	Broth 10-5	Broth 10-7	Broth 10-9	Detergent    + Broth	Detergent
Blood stain	20ml	20ml	20ml	20ml	0.2g in 20ml of tap water, 10-5,10-7,10-9	0.2g  in 20ml tap water
Banana stain	20ml	20ml	20ml	20ml	0.2g in 20ml of tap water, 10-5,10-7,10-9	0.2g  in 20ml tap water
Tea stain	20ml	20ml	20ml	20ml	0.2g in 20ml of tap water,  10-5,10-7,10-9	0.2g  in 20ml tap water

Partial Purification Method:  Based on this washing test the best stain removal test sample medium 10^-5was  identified and done partial purification for the sample  by saturated  Ammounium sulphate method (Mark et al., 2002).

Characterization of Proteins Using SDS-PAGE analysis: Electrophoresis is the process of migration of charged molecules in response to an electric field.Proteins have a net charge at any pH other than their isoelectric point (pI), thus when placed in an electric field, proteins will migrate towards the electrode of the opposite charge. This principle is used to separate molecules of differing charges (Sanbrook et al., 1989).

Stain removal capability after partial purification:  The washing test activity is done again after the partial purification method to identify the capacity of the enzyme in stain removal ability. The three different stains were tested against the sample 10^-5 medium. The stain removal of the enzyme was studied along with the detergent powder. The stain removal was studied under the following ways in table 2

Table 2. Activity performed with effective broth dilution against different stains

Stains	Tap water	Broth 10-5	Detergent + Broth	Detergent
Blood stains	20ml	20ml	0.2g in 10ml tap water and 10ml broth	0.2g  in 20ml tap water
Banana stain	20ml	20ml	0.2g in 10ml tap water and 10ml broth	0.2g  in 20ml tap water
Tea stain	20ml	20ml	0.2g in 10ml tap water and 10ml broth	0.2g  in 20ml tap water

RESULTS AND DISCUSSION

Isolation and characterization of microorganism: Out of 86 isolates screened the best hydrolysis zone were obtained for three isolates after 48hrs of incubation at 37⁰C . The zone of isolation of three three isolates were named as S_1,S_2,S₃. The zone of hydrolysis on skim milk agar is shown in Fig.1 Their morphological and physiological study for casein hydrolyse study as as shown in table 2 and 3.

Estimation of  Protease activity:  The maximum enzyme activity was given by the sample test 1 which showed its higher activity at 0.45nm. The enzyme acidity gradually increased as the substrate concentration also increased and also there was a gradual decrease in the test 2 and 3 with its substrate concentration. Table 4 shows the test samples enzyme activity.

Optimization of growth using Temperature and pH: The enzyme showed the best activity between temperature range 25⁰C and 45⁰C with maximum activity at 37⁰C. As the graph shown in (Fig 2) the enzyme was constant and stable at 37⁰C. At 45⁰C and above it started to decrease its activity and get completely inactivated by 120 min at the further temperatures. And in the same way the enzyme showed the reasonable activity between the pH 8 and 11 with maximum activity at 10 as shown in (Fig 3) which shows enzyme was stable at 10. At above pH 11 the enzyme started to reduce its activity after few minutes and get inactivated by 90min at further pH.

Stain removal activity with crude enzyme: A good detergent additive protease enzyme must have a cabaple to remove the protein stains by cleaving the protein bond breakage. Therefore, the effects of various oxidizing agents and surfactants on the protease enzyme were studied.stability of enzyme towards these stains were also analysed with the incubation period of 60min and 120mins (Kumar; Takagi 1999 & Joo 2005). protein stability has commercially wide applications in detergent industry has been reported in many studies (Kumar & Bhalla 2004) Oxidizing agents are major ingredientsof modern bleach based detergents. Alkaline protease from bacillus shows extreme stability towards oxidizing agents (Nadeem et al., 2013).

By consisdering these facts the washing test were perfomed for the blood stain, banana stain,tea stain with crude enzyme of three isolates were washed after the incubation period of 2hrs .The best results were obtained from the sample 1 isolate  as shown in  (Fig 2,3 and 4).

Partial  purification and molecular weight determination: To obtain proteins from cell free culture filtrate the sample 1 isolate  were partially purified with ammonium sulphate at 75%(w/v)saturation by adding  powdered Ammonium sulphate slowly with continuous stirring . The yield of protein was 2.30mg/mL, with a total activity of 415.26U/mL per min. This yield of partially purified protein was undergone for molecular weight determination by SDS -PAGE analysis and the result was shown in the( Fig 5)

Washing test with partially purified enzyme: The enzyme with extreme stability towards oxidizing agent is commercially significant as the peroxidise are common ingredients of bleach based detergents. The enzymes prepared are mostly stored under low temperatures which will prevent its denaturation  if the enzymes can withstand room temperature it is very useful  in lifetime of a detergent product. So by consisdering these issues the partially purified enzyme sample 1 isolate  has withstand its room temperature and also removed the  blood stain, banana stain, tea stain. Thus this reveals its use in detergent industry. The washing test performed  is shown in ( Fig 6,7, and 8.)

Isolation and characterization:

Growth of Bacillus sp., in different dilutions

Table 3. shows the results of Morphological study

SI.No	Gram staining	Negative staining	Spore staining
S1	Positive	Short chains	Endo spores
S2	Positive	Short chains	Endo spores
S3	Positive	Short chains	Endo spores

Table 4. shows the results of Physiological stud
Casein hydrolyse test

Sample	Growth	Zone of inhibition(mm)
S1	Yes	15mm
S2	Yes	11mm
S3	Yes	9mm

Table 5. Estimation of protein by Lowry’s method

Test tube	Tyrosine   Vol. Conc.	Alkaline reagent	Cond.	Dis. water	Folins phenol	Cond.	OD at 660nm
B	–             –	2	Incubation	2.00	0.2	Keep	0.00
S1	0.05     5	2	in	1.95	0.2	in	0.25
S2	0.10     10	2	dark	1.90	0.2	dark	0.33
S3	0.15     15	2	room	1.85	0.2	room	0.40
S4	0.20     20	2	for	1.80	0.2	for	0.53
S5	0.25    25	2	10 mins	1.75	0.2	30mins	0.60
T1	1.00	2		1.00	0.2		0.45
T2	1.00	2		1.00	0.2		0.37
T3	1.00	2		1.00.	0.2		0.33

Graph 1: shows the growth rate in pH  values

Graph 2: shows the growth rate in Temperature values

Washing test with crude enzyme:
Blood stain

Figure 2(a): Stained blood clothes in S1 sample.

Figure 2(b) : After 2 hrs the washing activity shows best in S1 sample.

Banana stain:

Figure  3(a) :  Stained banana clothes in S1 sample.

Figure 3(b) : After 2 hrs the washing activity shows best

Tea stain:

Figure 4 (a) : Stained tea clothes in S1 sample

Figure 4(b) : After 2 hrs the washing activity shows best.

Partial  purification and molecular weight determination

Lane -1 : Marker, Lane -2 : Crude lane, Lane -3 : Partial Purified lane    Marker : 10kda , 50kda, 75kda, 100kda

Figure (5) : SDS-PAGE analysis of isolated protease from papaya peel waste.

1. Molecular weight of crude proteins :

1^st band – 82kda, 2^nd band- 68kda, 3^rd band- 12kda

2. Molecular weight of partially purified proteins :

 

1^stband -81kda, 2^nd band- 47kda,  3^rd band – 10kda

Washing test with partially purified enzyme :

Blood stain

Figure (6a) Blood stained clothes purified protein

Figure 6(b) : Washing activity after 2hrs  proved best partially results  in   10^-5

Banana stain

Figure (7a): Banana stain clothes in partially purified protein

Figure (7b): Washing activity after 2hrs proved best results  in 10^-5

Tea stain :

Figure (8a): Banana stain clothes in partially purified protein

Figure (8b): Washing activity after 2hrs proved best results   in 10^-5

CONCLUSION

Papaya fruit waste was used to extract protease enzyme to create a useful detergent for stain removal. The enzyme’s activity was tested at different pH and temperature, and the best results were obtained from three samples. Washing activity was performed with three different stains, and the best sample was identifies as 10 – 5 broth, showing the best stain removal activity after 2 hours.The sample was partially purifies and tested again, confirming the presence of protein by SDS – PAGE analysis with a specific molecular weight The study suggests that alkaline protease enzyme from papaya waste is effective in removing protein stains and could help manage solid waste while being produced at lower temperature.

ACKNOWLEDGEMENTS

The authors acknowledge the use of instrumentation and infrastructure facilities provided  by DST-FIST  and DBT-  under Star College Scheme, Ministry of Science and Technology, Goverment of  India  for the successful completion of  the project. We thank the host institution  Dr. N.G.P Arts and Science College and its  management for rendering all the facilities and support.

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