Production And Properties of Bacterial Alkaline Proteases Obtained from Alkaline Habitats of Sagar Region

Abstract

The aim of present study was to investigate the alkaline protease producing bacteria from various habitats of Sagar region for the suitability of enzyme for industrial application. Soil samples were collected from different habitats including detergent factory (DF, pH 10.0), chicken market (CM, pH 10.0), and paper factory (PF, pH 10.0). Thirty bacterial strains were identified on the basis of Gram’s staining and screened for alkaline protease production on CPYA agar medium. Selected bacterial sp. was further studied for extracellular alkaline protease production in the shake flask conditions. Bacterial isolate from detergent factory (DF-10) showed maximum protease activity 18.10 U/ml and CM-6 produced 13.26 U/ml when haemoglobin was used as substrate. The protease enzyme showed optimal activity at 37°C and pH 10.0. Wash performance test of alkaline protease with detergent showed the suitability of Bacillus sp DF-10 strain for detergent industry.

Keywords

Introduction

Alkaline proteases (EC 3.4.21-24 and 99) are a type of serine proteases, contain (serine and histidine) catalytic amino acid in their active sites and perform hydrolytic reactions. Most alkaline proteases show high catalytic activity and stability in high alkaline range (pH 9.0-12.0) are useful for biotechnological and industrial applications. Alkaline proteases are effectively utilized in the detergent, feed production, waste management, leather, and pharmaceutical industries
(Eatemadi et al. 2017; Zhou et al., 2018; Sharma et al. 2021). Efficiently alkaline proteases are used as an essential component for the detergent industry. They are widely used as a supplementary component in commercial detergents to enhance cleaning efficiency by removing blood and food stains on cloth. Bacillus sp. is considered as an excellent source of protease producers because they are the largest groups of hydrolytic enzymes share about 60% of total worldwide market of industrial enzymes (Saggu and Mishra, 2017). Alkaline proteases are produced by a variety of bacterial species, however, Bacillus spp are capable to produce extracellular protease in large amount. Various Bacillus sp. are suitable candidate for the production of extracellular  protease production viz. Bacillus subtilis (Sun et al., 2023), Bacillus stearothermophilus (Karray et al., 2021), Bacillus licheniformis (Zhou et al., 2018), and Bacillus infantis (Saggu and Mishra, 2017).  In the present study, we have isolated new bacterial strains from Sagar region capable to produce alkaline protease. We have characterized the various biochemical properties of the alkaline protease and evaluated its ability in stain removal and suitability in detergent.

Materials and methods

Isolation and screening of alkaline protease producing bacteria

Soil samples were collected from different habitats including, detergent factory (DF) (pH 10.0), chicken market (CM) (pH 10.0), and paper factory (PF) (pH 10.0).  One gram of soil sample was used for the isolation of protease producing microorganism. Isolation was carried out using serial dilution agar plate technique (Waksman, 1972). Isolated bacterial colony was spread over the casein peptone yeast extract agar (CPYA) medium containing plates. The CPYA medium (g/l) (pH 10.0; Casein 5.0, Peptone 5.0, Yeast extract 2 .0, NaCl 5.0, MgSO₄.7H₂O 0.2, CaCl₂   0.1, K_­2HPO4 1.0, Na₂CO₃ 6.0) was used for isolation. The casein was solubilised separately by dissolution in 0.01 N NaOH prior to autoclaving. The pH of the casein medium was adjusted after autoclaving to 10.0 by addition of sterile sodium carbonate. Sodium carbonate was separately autoclaved and added to the rest of medium after cooling to avoid precipitation of casein. These plates were incubated at 37°C for 24-48 h. Colonies forming transparent zones, because of partial hydrolysis of casein, were selected for further study. Pure cultures of selected isolates were streaked on CPYA for further study and nutrient agar slants for storage at 4°C.

Solid medium and shake flask studies for screening of isolates                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Qualitative screening method was used by employing solid medium to detect comprehensive range of enzyme activities of microorganisms. For alkaline protease production it was attained by single streaking of pure isolates on CPYA medium plate and after incubation at 37ºC for 24 hours, plates were flooded with 10% TCA (w/v) solution for 30 minutes. Zone of proteolysis was observed as transparent zone of hydrolysis around the colony.

Production of alkaline protease

For the study of production of extracellular alkaline protease in liquid cultures medium, selected proteolytic isolates were grown on medium supplemented with casein at submerged condition. The liquid medium had following chemical composition in g/l - casein, 5.0; peptone, 5.0; yeast extract, 2.0; NaCl, 5.0; MgS0₄.7H₂O, 0.2; CaCl₂, 0.1; Na₂CO₃, 06.0; (pH 10.0). Culture filtrate was harvested and cells were separated by centrifugation at 10000 rpm for 10 minutes. The resulting cell free culture filtrate was used as a crude enzyme source for further study.

Protease assay

Assay of proteolytic activity in culture filtrate

Protease production by alkalophilic isolates was examined by radial diffusion assay method for this casein agar medium was used. The composition of the medium was as follows
(A) 5 g casein dissolve in 0.01 N NaOH solution (400ml), (B) 20 g agar dissolved in 595 ml distilled water, (C) 6.0 g Na₂CO₃ dissolved in 20 ml distilled water. All three constituents of the medium i.e. A, B, and C were autoclaved separately to avoid coagulation of casein due to pressure after that all medium ingredients were mixed. Sodium carbonate was used to maintain pH 10.0 of the medium. Culture filtrate was poured in agar well prepare using corkborer. These plates were than incubated at 37°C for 24 h. Plates were observed for transparent zone of hydrolysis around the agar well was calculated. Relative enzyme activity = Diameter of zone of enzyme activity (mm)/ Diameter of well (mm) One unit of alkaline protease was defined as the amount of enzyme that catalyses the release of
1 mg of tyrosine/ml/min from substrate used (haemoglobin and casein) under standard assay condition.

Quantification of protease activity using haemoglobin as substrate

Quantitative analysis of alkaline protease activity in the supernatant was determined using haemoglobin (1% w/v dissolved in 0.1 N NaOH) as substrate. Commercially available Folin Ciocalteau's reagent was used for analysis of protein in culture filtrate. Two tubes were labelled as enzyme blank and reaction mixture for a sample.  The aliquots of 1ml haemoglobin solution were equilibrated at 50ºC for 15 min in tubes.  The assay was initiated by addition of 0.2 ml of enzyme sample (crude culture filtrate) in reaction blank only.  The tubes were incubated at the same temperature for 60 minutes. At the end of incubation period the reaction was terminated by adding 2 ml of TCA solution. The reaction mixture was allowed to stand for 5 minutes and centrifuged at 7000 rpm for 12 minutes.  In enzyme blank 0.2 ml of culture filtrate was added after addition of TCA solution. To the above solution 0.5ml of phenol reagent is added with vigorous mixing and tubes were incubated for 30 minutes in dark. The colour developed was measured at 750 nm against reagent blank. The protease activity was calculated under standard assay condition (Lowry et. al. 1951).

Wash performance test of alkaline protease in detergent

The effectiveness of alkaline protease in detergent additive was studied for this experiment white cotton cloth pieces (5×5 cm) were stained with blood. The following set experiments were performed and tested with a) blood-stained cloth dipped in flask with distilled water (100 ml); b) blood-stained cloth dipped in flask with distilled water (100 ml) + 1ml detergent (Ghadi, Nirma; 7 mgml^-1); c) blood-stained cloth dipped in flask with distilled water (100ml) + 2ml enzyme solution; d) blood-stained cloth dipped in flask with distilled water (100ml) + 1ml detergent (7mgml^-1) + 2ml enzyme solution; e) blood-stained cloth dipped in flask with glycine-NaOH buffer, pH 10.0. All the five flasks were incubated at 60°C for 15 min, after incubation blood-stained cloth pieces were taken out, rinsed with water and air dried for visual examination of cloth pieces exhibited the effect of enzymes in removal of blood strains (Mahakhan et al., 2023).

RESULTS AND DISCUSSION

Isolation and qualitative screening of alkali-stable protease producing bacteria

The pH values of all the collected soil samples were in alkaline region. As a result of initial screening of the soil samples, 24 bacterial isolates were found having proteolytic activity on CPYA medium (pH 10.0) contained casein as substrate (Figure 1). Most of the bacterial isolates are found to be Bacillus, filamentous and rest are of cocci in their morphology. Extracellular proteolytic activity of bacterial isolates resulted in partial hydrolysis of casein present in the CPYA agar medium resulting in the formation of clear zones of different sizes (Figure 2). Alkaline protease producing Bacillus species were isolated from dairy sludge and used for the extracellular enzyme production (Sedaghat et al., 2022).  Isolates forming larger zones of hydrolysis were further screened quantitatively for the production of alkaline protease in the shake flask containing 50 mL of CPYA medium. Isolate number DF-10 (1.9 cm), DF-6 (1.2 cm) PF-4 (1.9 cm) and CM-6 (1.6 cm), CM-3 (1.2 cm) showed the maximum zone of hydrolysis activity on casein containing medium (Table 1).

Protease production and assay

Colonies appeared on CPYA medium plates have capability to utilize the proteinaceous substrate casein and are supposed to possess proteolytic activity under given condition. To determine the cell structure and arrangement, we performed Grams staining and observed under microscope in immersion oil.  Pure culture was maintained on slants (CPYA medium). Qualitative estimation of protease activity performed on solid medium have been categorized in 4 categories on basis of their zone of clearing i.e. excellent, good, fair and poor. A culture of selected isolate having Excellent, Good zone of clearing is transferred to broth (CPYA medium) for enzyme production. In stationary condition the growth gave the medium turbid appearance.  For detection of extracellular protease in broth, first cell free culture filtrate was obtained via centrifugation and well diffusion assay was performed at pH 10.0. Activity was compared by means of REA in solid medium (Table 2; Figure 3).  A comparative study of the previous literature of the characteristics of alkali stable Bacillus strains produces alkaline protease revealed that most of the Bacillus strains grow on temperature optima of 35-40°C (Gupta et al., 2002).

Effect of Alkaline protease for wash performance test with detergent

The increased usage of alkaline proteases as detergent additives is primarily due to the cleaning efficiency of these enzymes and approach is environmentally friendly. In addition, the use of enzymes confers lower wash temperatures and less agitation (Kumara and Takagi, 1999). The compatibility of alkaline protease wash studied in with Ghadi and Nirma commercial detergent for a period of 15 min at 60^°C were studied. The protease retained about 60% relative activity after 2 hours. As the alkaline protease produced by test isolate DF-10, CM-6 and PF-4 was highly stable at pH 10.0.  The supplementation of the enzyme preparation of isolate DF-10 in Ghadi detergent could significantly improve the cleaning of the blood strains. The ability of alkaline protease to remove blood stain was measured by wash performance test (Figure 4). Mumecha et al. (2021) reported the role of alkaline protease produced by Bacillus sp. for the removal of blood stain. Detergent compatible alkaline protease from Geobacillus thermoglucosidasius showed comparable stability when mixed with commercial detergents. Wash performance test confirmed that blood stained removed when protease enzyme and 1% (v/v) detergent used for 45 min at 80°C (Allison et al., 2023).

CONCLUSION

Three bacterial strains were selected on the basis of zone of clearing on CPYA medium for extracellular alkaline protease production. Isolate number DF-10, CM-6 and PF-4 are prominent producer of alkaline protease at alkaline pH 10.0. The enzyme of DF-10 was highly effective for blood stain removal and suitable as detergents additive. Furthermore, a characterization and purification of this enzyme would be performed in future. Considering the biodiversity of environment, it is very important to find out novel microbial source of enzymes for industrial purpose.

ACKNOWLEDGMENTS

Authors are also thankful for Prof. Naveen Kango, Head, Department of Microbiology, Dr. Harisingh Gour Central University, Sagar, MP, for providing research facility.

Conflicts of interest

The authors have declared no conflicts of interest.

REFERENCES

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13. Zhou C, Qin H, Chen X, Zhang Y, Xue Y, Ma Y. A novel alkaline protease from alkaliphilic Idiomarina sp. C9-1 with potential application for eco-friendly enzymatic dehairing in the leather industry. Sci Rep. 2018;7;8(1):16467. doi: 10.1038/s41598-018-34416-on regulatory standards for stability studies.

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                            Figure 1-Colonial characteristics of alkalophilic bacterial isolate on CPYA medium.

           
               
           
        Figure 2-Zone of hydrolysis by alkalophilic bacterial isolates on CPYA medium

           
               
           
        Figure 3 Caseinase activity produced by culture filtrate of selected bacterial alkaline protease on solid agar medium (pH 10.0)

           
               
           
    Figure 4 Analysis of Bacillus alkaline protease (DF-10) for wash performance test with detergent

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    Table 1 Source of samples and zone of hydrolysis of clearing of isolated bacteria

    SN	Samples source	pH	No of isolates	Isolate No	Zone of hydrolysis (cm)	Type of clearing
    1	Detergent Factory           (DF)	10.0	12	DF -1	0.6	Good
    				DF-2	0.9	Good
    				DF-3	0.8	Good
    				DF-4	0.4	Fair
    				DF-5	0.7	Good
    				DF-6	1.2	Excellent
    				DF-7	0.4	Fair
    				DF-8	0.7	Good
    				DF-9	1.1	Good
    				DF-10	1.9	Excellent
    				DF-11	0.8	Good
    				DF-12	0.7	Good
    2	Paper Factory       (PF)	10.0	4	PF-1	0.9	Good
    				PF-2	0.1	Poor
    				PF-3	1.0	Good
    				PF-4	1.8	Excellent
    				PF-5	0.2	Fair
    				PF-6	0.2	Fair
    3	Chicken Market         (CM)	10.0	8	CM-1	0.4	Fair
    				CM-2	0.6	Fair
    				CM-3	1.2	Excellent
    				CM-4	0.9	Good
    				CM-5	0.8	Good
    				CM-6	1.6	Excellent
    				CM-7	0.7	Good
    				CM-8	1.0	Good

     

     

     

     

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    Table 2 Diameter of zone of hydrolysis of the culture filtrates of selected bacterial isolates on solid agar medium containing casein (1% w/v) as substrate after optimized condition

    SN	Isolate No	Diameter of zone of hydrolysis (mm, pH 10)	Relative enzyme activity (mm)
    1	DF-10	23	5.6
    2	DF-6	19	3.7
    3	PF-4	21	4.9
    4	CM-6	17	3.1
    5	CM-3	22	5.2

     

     

     

     

     

     

     

     

     

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    Table 3 Alkaline protease activity estimated in culture filtrate of alkalophilic isolates using      
                  haemoglobin (1% w/v) as substrate

1. SN	Isolate No	Tyrosine released (mg/ml)	Alkaline protease activity (U/ml)
   	DF-10	59.13	18.10
   	DF-6	34.57	9.19
   	PF-4	41.24	10.91
   	CM-6	36.96	12.80
   	CM-3	31.71	10.43