I. Introduction

Proteases are biocatalyst that perform a multitude of chemical reaction and are commercially used in detergents, food, pharma, diagnostic and fine chemical industries [1, 2]. They are group of enzymes which catalyse the cleavage of peptide bonds, generally referred to as proteolytic enzymes or proteinases. Proteases are ubiquitous in nature widely distributed in plants, animals and microorganisms.

The significant role of proteases has made them industrially important. They account for 65% of total worldwide enzyme sale. Several proteases have been isolated from latex, fruits and seeds. They have major applications in industrial process such as laundry, silk, pharmaceutical, food, degradation of gelatin on X-ray films [3] and bioremediation process. They are widely used in detergents, leather, waste management and silver recovery [4].

A lot of work has been reported on proteases of microbial origin holding industrial potential due to their biochemical diversity. A variety of microorganisms such as bacteria, fungi, yeast and actinomycetes are known to produce these enzymes [5]. It would be advantageous to utilize a fungal protease as fungal expression systems are capable of producing larger quantities of enzymes than bacterial expression system [6]. Proteases are produced by many species of fungi such as Aspergillus [7], Mucor [8], Fusarium [9], Cephalosporium [10] and Rhizopus [11]. Filamentous fungi, such as Aspergillus have been the organism of choice for large scale production of bulk industrial enzymes [12]. Actinomycetes being a protease producing bacteria have the potential for use industrial purpose, pharmaceutical and cytotoxic agent [13]. The sources of protease are enormous. Bacillus sp. was found to be predominant and rich source of alkaline proteases. Many of the fungi have also been reported to produce extracellular alkaline protease [14]. B. substilis yield maximum protease production and be used efficiently for industrial purposes [15]. Among various proteases, bacterial proteases are more significant compared to with animal and fungal protease [16]. They are present in Bacillus subtilis, B. amyloliquefaciens, Pseudomonas sp., Lysobacter enzymogene, E. coli etc. [17]. B. subtilis produce alkaline protease that can be used in textiles, leather and food industries [18]. Plants are also the rich source of protease enzyme [19] but not been worked upon much. In this study, spices such as Carum copticum, Syzygium aromaticum, Cuminum cyminum, Nigella sativa, Cinnamon verum, Foeniculum vulgare, Zingiber officinale, Cinnamomum tamala and Curcuma longa have been explored as source of protease enzyme.

II. Materials and methods

Spices were procured from Green Earth products Pvt. Ltd., New Delhi. All chemicals were of reagent grade and obtained from standard commercial firms.

A. Screening of various spices for Protease enzyme

Screening of various spices viz; Ajwain (Carum copticum), Cloves (Syzygium aromaticum), Cumin (Cuminum cyminum), Black cumin (Nigella sativa), Dal chini (Cinnamon verum), Fennel (Foeniculum vulgare), Tej patta (Cinnamomum tamala), Ginger (Zingiber officinale) and Turmeric (Curcuma longa) was done for protease activity by using standard protocols.

B. Extraction of Protease enzyme

The pre-weighed samples were crushed in sodium acetate buffer (pH 5.0, 0.05 M), filtered through Whatman filter paper and centrifuged for 20 minutes at 10,000 rpm at 4°C. The pellet was discarded, supernatant collected and subjected to further purification.

C. Determination of specific activity

Specific activity was determined by using the following relationship:

Specific activity= Total enzyme units /Total protein (mg)

  • Protein determination: Protein content of the enzyme extract was determined by Lowry method [20] using BSA as standard.

  • Protease Assay: Protease activity was assayed using Folin-Ciocalteau method [21] The reaction mixture containing casein (1%), enzyme, 0.05 M sodium acetate buffer (pH 5.0) was incubated for 30 min at 30°C followed by addition of 0.5 M NaOH and folins reagent. 1 International Unit of protease enzyme is defined as 1μg of tyrosine released per minute per ml under standard assay conditions. The amount of protease produced was measured with the help of a tyrosine standard graph [22]. The activity was reported as mean of three determinations.

D. Partial purification of protease enzyme

  • Ammonium sulphate fractionation: The crude extract of the screened spices were subjected to precipitation using salting out process. Ammonium sulphate fractionation (0-30%) was done by adding salt in the extract according to the required saturation level, slowly while keeping on ice [23]. The ice-cold saturated solution of the protein was stirred continuously and kept at 0-4°C for at least one hour followed by centrifugation at 10,000 rpm for 10-15 min. Pellets were collected and dissolved in minimal amount of sodium acetate buffer (0.05 M, pH 5.0) and used as 0-30% fraction, the supernatant being subjected to next fractionation steps and further two fractions (30-60% and 60-90%) were obtained in the similar manner.

E. Biochemical characterization of Protease enzyme

  • Time course: To determine the time course, the reaction mixture was incubated at different time intervals ranging from 10 min to 90 min and the protease activity was determined using standard conditions.

  • Temperature Optima: The reaction mixture containing enzyme and substrate was incubated at 30°C for time period ranging between 10-90 minutes and the product released estimated by Folin’s method.

  • pH optima: To determine the pH optima, suitable buffers of different pH values ranging from 3.0 to 9.0 were used. The reaction mixture was incubated for optimum time period and activity determined using standard assay.

  • pH stability: The enzyme alone was incubated for 2 hours with suitable buffers of pH ranging from 3.0 to 9.0 and further assayed using optimum assay conditions and residual protease activity, determined.

  • Heat Resistance: The enzyme was incubated alone at different temperature ranging from 10°C to 90°C for 2h followed by standard assay.

III. Results and discussion

Protease plays an essential role in various pathological processes. Arthritis, tumor invasion and metastasis, infections and number of degenerative disease have been linked with proteolytic enzymes which have widespread applications in bioremediation processes [24]. This research work was undertaken to study the various biochemical aspects of high specific activity protease enzyme isolated from spices. Out of the nine spices studied, the extract of Nigella sativa (204 units/mg of protein) and Curcuma longa (124 units/mg) was found to contain protease with maximum specific as shown in Table I, and was further characterized. A better understanding of the functions of enzymes could be determined by purification of enzyme [25]. Figure 1 shows the specific activity of fractions of protease isolated from Nigella sativa and Curcuma longa at three different saturation levels of (NH4)2SO4. It can be seen from Table II that 0-30% fraction has highest specific activity with 2- fold and 4.8- fold purification level in N. sativa (409 U/mg) and C. longa (590 U/mg), respectively. Therefore, 0-30% enzyme fraction from both the sources was used for further characterization studies. The time course of enzyme catalyzed reaction for protease isolated from N. sativa and C. longa (figure 2). It shows that the enzyme substrate reaction reached to maximum value in 20 min and 30 min for N. sativa and C. longa, respectively, later no change in activity was observed. Figure 3 illustrates temperature optima of protease isolated from N. sativa and C. longa, showing maximum activity at 40°C, in both the cases. Increase in temperature above optimum level affects important factors like protein denaturation, protein ionization state and solubility of species in solution reducing enzyme activity [26]. Enzymes, being proteinaceous in nature, have properties that are quite pH sensitive. pH can affect activity by changing the charges on an amino acid residue which is functional in substrate binding or catalysis [27]. The pH optima of protease from N. sativa and C. longa were found to be (Figure 4) 5.0, in both the cases. Similar observation was reported in metalloprotease production in P. fluorescens [28] and Adhatoda vasica [29] with an optimum activity at pH 5.0. The proteases produced by isolates with enzymatic activity optima at pH 5.0 could be used to coagulate milk proteins for the dairy industry, as debittering agents in cheese and in peptide synthesis [30]. Figure 5 shows the pH stability curve of protease from N. sativa and C. longa. The protease from N. sativa was found to retain its activity in the pH range of 5.0 to 9.0 whereas respective values were from 4.0 to 8.0 in C. longa. The most significant result is the heat resistant property of the enzymes obtained from the two species, the temperature values being 60°C and 50°C for Nigella sativa and Curcuma longa (figure 6), respectively. Proteases from various sources with temperature stability upto 50°C have been reported by various workers [31-36]. Thus, it is imperative to state that spices may serve as good source of thermostable proteases which further have great importance in therapeutics and many other industries.

TABLE I. Specific activity of protease isolated from various spices.
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Figure 1.
figure 1

Specific activity of various fractions of protease enzyme isolated from N. sativa and C. longa after (NH4)2SO4 fractionation.

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TABLE II. Specific activity and purification-fold of various fractions of protease isolated from Nigella sativa and Curcuma longa after ammonium sulphate fractionation. 0
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Figure 2.
figure 2

Time course of enzyme catalyzed reaction for proteases isolated from N. sativa and C. longa. The reaction mixture was incubated at different time intervals ranging from 10 min to 90 min and the activity was determined using standard conditions.

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Figure 3.
figure 3

Temperature optima of proteases isolated from N. sativa and C. longa. The reaction mixture containing enzyme and substrate was incubated at different temperature ranging from 10°C to 90° C for time period of 20 minutes and further assayed under standard conditions.

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Figure 4.
figure 4

pH optima of proteases isolated from N. sativa and C. longa using suitable buffers of different pH values ranging from 3.0 to 9.0 at 40°C for 20 minutes.

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Figure 5.
figure 5

pH stability curve of proteases isolated from N. sativa and C. longa by incubating enzyme alone for 2 hours with buffers of pH values ranging from 3.0 to 9.0 and further assayed using optimum conditions.

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Figure 6.
figure 6

Profile showing heat resistance pattern of proteases isolated from Nigella sativa and Curcuma longa by incubating the enzyme alone at different temperature ranging from 10°C to 90° C for 2h followed by standard assay.

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IV. Conclusion

In the present work out of the nine spices studied, Nigella sativa and Curcuma longa were found to be good source of protease enzyme. Very limited work has been done on enzymes from these spices which call for a more detailed research in future. Proteases obtained from spices may serve as potential candidate for targeting many diseases. From the results obtained, it may be concluded that spices which are of great medicinal and culinary importance can be explored for heat resistant protease; an enzyme of immense value in therapeutics, chemical, leather, food and other bio-industries.

Acknowledgment

We are grateful to Director, Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, Uttar Pradesh, for his constant support and encouragement during this study.