Ananas Comosus (also known as pineapple) is a part of Bromeliaceae family and it is consumed as food as well as folk medicine for the treatment of various diseases. It is reported that pineapple is a rich source of bromelain, a cysteine protease and it is considered as an important enzyme in different industries due to its significant therapeutic and industrial applications such as anticancer, anti-inflammatory and meat tenderizing. Bromelain is mostly present in fruit and stem of pineapple, but it is reported that crown, core, and peels, which constitute the waste of the pineapple plant, also contain bromelain but limited data is available. Therefore, the proposed study aimed at utilizing pineapple waste for the extraction and characterization of bromelain. Firstly, crude bromelain was extracted with phosphate buffer (pH 7), then it was subjected to partial purification using different fractions of ammonium sulphate (NH4)2SO4 such as 30, 40, 50 and 60% followed by desalting and concentration. Enzyme activity was calculated by using casein digesting unit (CDU) method. The results demonstrated that the crown bromelain showed highest purification of 4.34-fold at 30% (NH4)2SO4 saturation, whereas core and peel bromelain showed highest purification of 2.75 and 2.59-fold at 40% (NH4)2SO4 saturation. The molecular weight of crude and partially purified bromelain was determined by SDS-PAGE analysis and found to be 26 KDa. The pH and thermal stability of all the parts of pineapple showed maximum stability at pH 7 and at 35oC temperature.

• Background of study
Bromelain is defined as a natural complex mixture of various proteolytic enzymes which has a cysteine amino acid side chain. It is also stated that bromelain is a combination of various thiol endopeptidases and other constituents like peroxidase, phosphatase, cellulase, glucosidase and several protease inhibitors [1]. Bromelain is mainly derived from pineapple (Ananas comosus), which is a well-known plant of Bromeliaceae family that is widely cultivated in tropical and subtropical areas [2]. Thailand is one of the major cultivators and exporter of pineapple around the world [3]. It is used for edible purposes and the fruit has been popularly used in different cuisine. Moreover, pineapple is considered as a nutrient rich plant and has been traditionally known for having a wide array of potential biological compounds among which bromelain is of great importance [2]. Bromelain has been used in folk medicine for so many years to address various health problems which causes persistent interest in this protease [4]. It possesses a wide range of therapeutic applications like fibrinolytic, anti-thrombotic, anti-cancer, anti-inflammatory and anti-bacterial activity. In addition to this, bromelain has various industrial applications also that include pharmaceutical, food, textile, and cosmetic industry [5].

Bromelain has captured attention in vast range of industrial applications due to its higher commercial values and potential properties. The proteases including bromelain holds approximately 60% share of the global enzyme market. This growth is associated with the increasing general awareness about the protection of environment from the harmful impacts of chemical industrialization [6]. The robust protein degrading activity of bromelain has formed a widespread interest in a number of applications, mainly in the tenderization of meat in food industry. The pharmaceutical and the food and beverages industries have the largest applications of bromelain for a variety of purposes, both of these sections throughout the forecast period of 2017-2025, have mutually expected to hold more than 85% of the global bromelain market share [7]. The boundless interest in a wide variety of bromelain applications has encouraged many researchers to isolate and characterize the protease enzyme from pineapple waste or juice. It is reported that the stem of ripped pineapple has the highest concentration of bromelain. However, pineapple waste parts such as peel, core, and crown which serve as a byproduct in the pineapple processing industry are also rich in bromelain and are still not utilized properly. The commercial bromelain, most of the time is extracted from pineapple stem by means of centrifugation, ultrafiltration, lyophilization and two-step Fast Protein Liquid Chromatography (FPLC) [8].

Moreover, with the increase in pineapple production worldwide, pineapple waste (which includes peel, leaves, stem, core, and crown) are also proportionally increasing [3]. These waste parts have low commercial importance, and, in most cases, they are discarded, and this waste is occasionally utilized as compost for soil fertilization [9]. Therefore, proper waste disposal is the biggest concern around the world as throwing them as garbage causes serious environmental problems as well as it is usually prone to microbial spoilage which later can cause biohazards [3]. Considering the wide range of bromelain application in various industries, and the potential of pineapple waste for the extraction of bromelain, the aim of the present study was to extract and partially purify bromelain using ammonium sulphate precipitation method using pineapple waste (crown, core and peel) as an alternate and cheap source for this enzyme and to perform biochemical characterization studies of the extracted bromelain enzyme which will serve as the basis for further studies.

Modern technological advancements have raised the protease production industrially worldwide. Today, proteases dominate with approximately 60 % market share of the total enzyme market worldwide where the major producers are Novo Industries, Gist-Brocades, Genencor International, and Miles Laboratories (Feijoo-Siota and Villa 2011). This growth is entrenched with the rising general awareness of the need to protect the environment from the impact of chemical indus- trialization. Recently, bromelain has drawn attention in di- verse industrial applications owing to its properties and higher commercial values (Heinicke and Gortner 1957). Bromelain is a protease derived from the stem and fruit of pineapples (Ananas comosus). Stem bromelain (EC, ananain (EC, and comosain are extracted from pineapple stems, while fruit bromelain (EC is mainly from fruit juice (Rowan et al. 1990). Similar proteases are also present in pineapple peel, core, crown, and leaves, with the highest proteolytic activity and protein contents detected in the extract of pineapple crown (Ketnawa et al. 2012).

Stem bromelain can be isolated from stem juices by means of precipitation and centrifugation (Devakate et al. 2009; Heinicke and Gortner 1957). Moreover, using chromato- graphic methods, other basic proteolytic (ananain, comosain, F4, F5, and F9) and acidic components of stem bromelain fraction A (SBA/a) and fraction B (SBA/b) have been partially and fully purified (Feijoo-Siota and Villa 2011; Harrach et al. 1995, 1998; Murachi et al. 1964; Napper et al. 1994; Rowan et al. 1990; Wharton 1974). The molecular weight of purified stem bromelain is 23.40–35.73 kDa, fruit bromelain31.00 kDa, ananain 23.43–23.42 kDa, and comosain 23.56– 24.51 kDa. The isoelectric point of stem and fruit bromelain is at pH 9.55 and 4.6, respectively (Murachi et al. 1964; Yamada et al. 1976).

1.2 Statement of problem
Stem bromelain contains 285 amino acids where the most abundant amino acids are alanine and glycine, while histidine and methionine are present in the lowest amounts (Murachi 1964; Ota et al. 1964). It appeared to differ significantly in the number of lysine, arginine, and isoleucine compared to ananain and comosain (Napper et al. 1994). Ananain possesses more hydrophobicity in the region near histidine-157 and sequence insert between 170 and 174 residues which cannot be found in stem bromelain (Lee et al. 1997). Stem bromelain also contains four hexosamines, and 2.1 % carbohydrate, meaning it is a glycoprotein (Murachi 1964; Murachi et al. 1964). This observation was corroborated by the findings of Ota et al. (1964), who detected 1.5 % carbohydrate, including six glucosamines, in purified stem bromelain. Later, Murachi et al. (1967) isolated 30 mg of glycopeptides from 1 g of stem bromelain using gel filtration in an attempt to confirm the presence of a glycoprotein. Murachi et al. (1967) and Yasuda et al. (1970) reported that the carbohydrate composition in stem bromelain consists of mannose, fucose, xylose, and glucosamine in the ratio of 3:1:1:4 using gas chromatography analysis, whereas Scocca and Lee (1969) obtained the same oligosaccharide group with a different ratio of 2:1:1:2 using automated borate chromatography.

Stem bromelain is an endoprotease that breaks peptide bonds within the protein molecule. Stem bromelain is under the papain family which resembles papain unequivocally in the alignment of amino acid sequence. A prominent similarity is observed in their mechanism of action. Initially, noncovalent bonding involves the free enzyme (structure a) and substrate to form the complex (structure b).

1.3 Objectives of study
1. Develop and optimize efficient extraction techniques for bromelain from pineapple sources.

2. Purify bromelain to achieve high enzyme purity and activity.

3. Characterize the biochemical properties and evaluate the therapeutic potential of purified bromelain.

1.4 Significance of the Study:
Advancement in Enzyme Technology:

Contribute to the field of biotechnology by improving extraction and purification techniques for plant-derived enzymes.

Provide a reliable source of high-purity bromelain for research and industrial applications.

Medical and Health Benefits:
Enhance the availability of bromelain for therapeutic purposes, including its use in treating inflammation, thrombosis, and digestive disorders.

Support the development of bromelain-based pharmaceuticals and health supplements.

Economic and Environmental Impact:
Promote the utilization of pineapple waste (stems and cores) for bromelain extraction, adding value to agricultural by-products.

Encourage sustainable practices by reducing waste and promoting green technologies.

Innovation in Food Industry:
Facilitate the production of natural meat tenderizers, enhancing food quality and consumer acceptance.

Enable the creation of functional foods with added health benefits through bromelain fortification.

Foundation for Further Research:
Provide a basis for future studies on the genetic modification of pineapples to enhance bromelain yield.

Stimulate research on the synergistic effects of bromelain with other bioactive compounds.

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