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Laura P Hale - One of the best experts on this subject based on the ideXlab platform.
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Bromelain treatment decreases neutrophil migration to sites of inflammation
Clinical Immunology, 2008Co-Authors: David J Fitzhugh, Siqing Shan, Mark W Dewhirst, Laura P HaleAbstract:Abstract Bromelain, a mixture of proteases derived from pineapple stem, has been reported to have therapeutic benefits in a variety of inflammatory diseases, including murine inflammatory bowel disease. The purpose of this work was to understand potential mechanisms for this anti-inflammatory activity. Exposure to Bromelain in vitro has been shown to remove a number of cell surface molecules that are vital to leukocyte trafficking, including CD128a/CXCR1 and CD128b/CXCR2 that serve as receptors for the neutrophil chemoattractant IL-8 and its murine homologues. We hypothesized that specific proteolytic removal of CD128 molecules by Bromelain would inhibit neutrophil migration to IL-8 and thus decrease acute responses to inflammatory stimuli. Using an in vitro chemotaxis assay, we demonstrated a 40% reduction in migration of Bromelain- vs. sham-treated human neutrophils in response to rhIL-8. Migration to the bacterial peptide analog fMLP was unaffected, indicating that Bromelain does not induce a global defect in leukocyte migration. In vivo Bromelain treatment generated a 50–85% reduction in neutrophil migration in 3 different murine models of leukocyte migration into the inflamed peritoneal cavity. Intravital microscopy demonstrated that although in vivo Bromelain treatment transiently decreased leukocyte rolling, its primary long-term effect was abrogation of firm adhesion of leukocytes to blood vessels at the site of inflammation. These changes in adhesion were correlated with rapid re-expression of the Bromelain-sensitive CD62L/L-selectin molecules that mediate rolling following in vivo Bromelain treatment and minimal re-expression of CD128 over the time period studied. Taken together, these studies demonstrate that Bromelain can effectively decrease neutrophil migration to sites of acute inflammation and support the specific removal of the CD128 chemokine receptor as a potential mechanism of action.
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Bromelain treatment decreases secretion of pro inflammatory cytokines and chemokines by colon biopsies in vitro
Clinical Immunology, 2008Co-Authors: Jane E Onken, Paula K Greer, Brian Calingaert, Laura P HaleAbstract:Abstract Oral Bromelain has been anecdotally reported to decrease inflammation in ulcerative colitis (UC). Proteolytically active Bromelain is known to decrease expression of mRNAs encoding pro-inflammatory cytokines by human leukocytes in vitro. To assess the effect of Bromelain on mucosal secretion of cytokines in inflammatory bowel disease (IBD), endoscopic colon biopsies from patients with UC, Crohn's disease (CD), and non-IBD controls were treated in vitro with Bromelain or media, then cultured. Secretion of pro-inflammatory cytokines and chemokines was measured. Significant increases in granulocyte colony-stimulating factor (G-CSF), interferon (IFN)-γ, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF) were detected in the media from actively inflamed areas in UC and CD as compared with non-inflamed IBD tissue and non-IBD controls. In vitro Bromelain treatment decreased secretion of G-CSF, granulocyte–macrophage colony-stimulating factor (GM-CSF), IFN-γ, CCL4/macrophage inhibitory protein (MIP)-1β, and TNF by inflamed tissue in IBD. Bromelain may be a novel therapy for IBD.
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Oral immunogenicity of the plant proteinase Bromelain.
International immunopharmacology, 2006Co-Authors: Laura P Hale, David J Fitzhugh, Herman F StaatsAbstract:Bromelain is a natural mixture of proteolytic enzymes derived from pineapple stem that has been shown to have anti-inflammatory activity when administered orally. Although most proteins given orally without adjuvant (e.g., food) result in tolerance, we previously reported that long-term oral exposure to Bromelain stimulated the development of high serum anti-Bromelain antibody titers. The purpose of these studies was to further investigate the mechanisms responsible for the immunogenicity of oral Bromelain. Results showed that repeated exposure was required for development of anti-Bromelain antibodies, with strong antibody responses in all mice that received at least 12 doses of Bromelain either orally or intragastrically over 3-6 weeks. Proteolytic activity was required for strong oral immunogenicity in the absence of conventional adjuvant, with strong serum antibody responses generated against proteolytically active Bromelain and trypsin, but not against ovalbumin, lysozyme, or inactivated Bromelain. Significantly higher anti-Bromelain antibody titers were seen in IL-10-deficient versus wild-type mice, suggesting that simultaneous treatments that decrease IL-10 activity may further enhance systemic antibody responses following oral exposure. The antibodies generated did not affect the proteolytic activity of Bromelain. The data demonstrate that proteolytically active antigens such as Bromelain can stimulate both systemic and mucosal immune responses following repeated oral exposure. Further studies of the mechanisms involved in generation of immune responses following oral exposure to proteolytically active antigens can lead to a better understanding of mechanisms of oral tolerance and to the development of novel adjuvants for oral vaccines.
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treatment with oral Bromelain decreases colonic inflammation in the il 10 deficient murine model of inflammatory bowel disease
Clinical Immunology, 2005Co-Authors: Laura P Hale, Paula K Greer, Chau T Trinh, Marcia R GottfriedAbstract:Abstract Bromelain is a mixture of proteinases derived from pineapple stem that is marketed in health food stores as a “digestive aid”. Orally administered Bromelain was anecdotally reported to induce clinical and endoscopic remission of ulcerative colitis in two patients whose disease was refractory to multi-agent conventional medical therapy. However, the potential efficacy of Bromelain in colitis has not yet been tested rigorously in either animals or humans. In this study, the clinical and histologic severity of inflammatory bowel disease (IBD) was determined in IL-10−/− mice treated orally with Bromelain in vivo. Daily treatment with oral Bromelain beginning at age 5 weeks decreased the incidence and severity of spontaneous colitis in C57BL/6 IL-10−/− mice. Bromelain also significantly decreased the clinical and histologic severity of colonic inflammation when administered to piroxicam-exposed IL-10−/− mice with established colitis. Proteolytically active Bromelain was required for anti-inflammatory effects in vivo. Adverse effects of dermatitis, hair loss, and weight loss due to mucositis were rare, dose related, and were not seen in wild-type mice treated orally with up to 1000 mg Bromelain/kg/day for 18 weeks. Although the exact mechanisms by which exogenous proteinases affect bowel inflammation have not yet been determined, the results justify additional studies of this complementary biologically based approach to treatment of IBD.
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proteinase activity and stability of natural Bromelain preparations
International Immunopharmacology, 2005Co-Authors: Laura P Hale, Paula K Greer, Chau T Trinh, Cindy L JamesAbstract:Abstract Bromelain is a complex mixture of proteinases typically derived from pineapple stem. Similar proteinases are also present in pineapple fruit. Beneficial therapeutic effects of Bromelain have been suggested or proven in several human inflammatory diseases and animal models of inflammation, including arthritis and inflammatory bowel disease. However, it is not clear how each of the proteinases within Bromelain contributes to its anti-inflammatory effects in vivo. Previous in vivo studies using Bromelain have been limited by the lack of assays to control for potential differences in the composition and proteolytic activity of this naturally derived proteinase mixture. In this study, we present model substrate assays and assays for cleavage of Bromelain-sensitive cell surface molecules can be used to assess the activity of constituent proteinases within Bromelain without the need for biochemical separation of individual components. Commercially available chemical and nutraceutical preparations of Bromelain contain predominately stem Bromelain. In contrast, the proteinase activity of pineapple fruit reflects its composition of fruit Bromelain>ananain∼stem Bromelain. Concentrated Bromelain solutions (>50 mg/ml) are more resistant to spontaneous inactivation of their proteolytic activity than are dilute solutions, with the proteinase stability in the order of stem Bromelain>fruit Bromelain∼ananain. The proteolytic activity of concentrated Bromelain solutions remains relatively stable for at least 1 week at room temperature, with minimal inactivation by multiple freeze–thaw cycles or exposure to the digestive enzyme trypsin. The relative stability of concentrated versus dilute Bromelain solutions to inactivation under physiologically relevant conditions suggests that delivery of Bromelain as a concentrated bolus would be the preferred method to maximize its proteolytic activity in vivo.
Yogeshwer Shukla - One of the best experts on this subject based on the ideXlab platform.
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Bromelain inhibits nuclear factor kappa b translocation driving human epidermoid carcinoma a431 and melanoma a375 cells through g2 m arrest to apoptosis
Molecular Carcinogenesis, 2012Co-Authors: Kulpreet Bhui, Shilpa Tyagi, Amit Srivastava, Madhulika Singh, Richa Singh, Yogeshwer ShuklaAbstract:Bromelain, obtained from pineapple, is already in use clinically as adjunct in chemotherapy. Our objective was to test its ability to act as a sole anti-cancer agent. Therefore, we describe its anti-proliferative, anti-inflammatory and subsequent anti-cancer effects in vitro, against human epidermoid carcinoma-A431 and melanoma-A375 cells. Bromelain exhibited reduction in proliferation of both these cell-lines and suppressed their potential for anchorage-independent growth. Further, suppression of inflammatory signaling by Bromelain was evident by inhibition of Akt regulated-nuclear factor-kappaB activation via suppression of inhibitory-kappaBα phosphorylation and concomitant reduction in cyclooxygenase-2. Since, the inflammatory cascade is well-known to be closely allied to cancer; we studied the effect of Bromelain on events/molecules central to it. Bromelain caused depletion of intracellular glutathione and generation of reactive oxygen-species followed by mitochondrial membrane depolarization. This led to Bromelain-induced cell-cycle arrest at G2/M phase which was mediated by modulation of cyclin B1, phospho-cdc25C, Plk1, phospho-cdc2, and myt1. This was subsequently followed by induction of apoptosis, indicated by membrane-blebbing, modulation of Bax-Bcl-2 ratio, Apaf-1, caspase-9, and caspase-3; chromatin-condensation, increase in caspase-activity and DNA-fragmentation. Bromelain afforded substantial anti-cancer potential in these settings; hence we suggest it as a potential prospect for anti-cancer agent besides only an additive in chemotherapy. © 2011 Wiley Periodicals, Inc.
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pineapple Bromelain induces autophagy facilitating apoptotic response in mammary carcinoma cells
Biofactors, 2010Co-Authors: Kulpreet Bhui, Shilpa Tyagi, Bharti Prakash, Yogeshwer ShuklaAbstract:Bromelain, from pineapple, possesses potent anticancer effects. We investigated autophagic phenomenon in mammary carcinoma cells (estrogen receptor positive and negative) under Bromelain treatment and also illustrated the relationship between autophagy and apoptosis in MCF- 7 cells. MCF- 7 cells exposed to Bromelain showed delayed growth inhibitory response and induction of autophagy, identified by monodansylcadaverine localization. It was succeeded by apoptotic cell death, evident by sub-G1 cell fraction and apoptotic features like chromatin condensation and nuclear cleavage. 3 -Methyladenine (MA, autophagy inhibitor) pretreatment reduced the Bromelain-induced autophagic level, also leading to decline in apoptotic population, indicating that here autophagy facilitates apoptosis. However, addition of caspase- 9 inhibitor Z-LEHD-FMK augmented the autophagy levels, inhibited morphological apoptosis but did not prevent cell death. Next, we found that Bromelain downregulated the phosphorylation of extracellular signal-regulated kinase ½ (ERK½), whereas that of c-jun N-terminal kinase (JNK) and p38 kinase were upregulated. Also, MA had no influence on Bromelain-suppressed ERK½ activation, yet, it downregulated JNK and p38 activation. Also, addition of mitogen-activated protein kinase (MAPK) inhibitors enhanced the autophagic ratios, which suggested the role of MAP kinases in Bromelain-induced autophagy. All three MAPKs were seen to be constantly activated over the time. Bromelain was seen to induce the expressions of autophagy-related proteins, light chain 3 protein B II (LC 3 BII), and beclin-1. Using ERK½ inhibitor, expressions of LC 3 BII and beclin-1 increased, whereas p38 and JNK inhibitors decreased this protein expression, indicating that Bromelain-induced autophagy was positively regulated by p38 and JNK but negatively regulated by ERK½. Autophagy-inducing property of Bromelain can be further exploited in breast cancer therapy.
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Bromelain inhibits cox 2 expression by blocking the activation of mapk regulated nf kappa b against skin tumor initiation triggering mitochondrial death pathway
Cancer Letters, 2009Co-Authors: Kulpreet Bhui, Sahdeo Prasad, Jasmine George, Yogeshwer ShuklaAbstract:Abstract Chemoprevention impels the pursuit for either single targeted or cocktail of multi-targeted agents. Bromelain, potential agent in this regard, is a pharmacologically active compound, present in stems and fruits of pineapple ( Ananas cosmosus ), endowed with anti-inflammatory, anti-invasive and anti-metastatic properties. Herein, we report the anti tumor-initiating effects of Bromelain in 2-stage mouse skin tumorigenesis model. Pre-treatment of Bromelain resulted in reduction in cumulative number of tumors (CNT) and average number of tumors per mouse. Preventive effect was also comprehended in terms of reduction in tumor volume up to a tune of ∼65%. Components of the cell signaling pathways, connecting proteins involved in cell death were targeted. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in Bcl-2. A marked inhibition in cyclooxygenase-2 (Cox-2) expression and inactivation of nuclear factor-kappa B (NF-κB) was recorded, as phosphorylation and consequent degradation of Iκ Bα was blocked by Bromelain. Also, Bromelain treatment curtailed extracellular signal regulated protein kinase (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and Akt activity. The basis of anti tumor-initiating activity of Bromelain was revealed by its time dependent reduction in DNA nick formation and increase in percentage prevention. Thus, modulation of inappropriate cell signaling cascades driven by Bromelain is a coherent approach in achieving chemoprevention.
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regulation of p53 nuclear factor κb and cyclooxygenase 2 expression by Bromelain through targeting mitogen activated protein kinase pathway in mouse skin
Toxicology and Applied Pharmacology, 2008Co-Authors: Neetu Kalra, Kulpreet Bhui, Sahdeo Prasad, Jasmine George, Smita Srivastava, Yogeshwer ShuklaAbstract:Abstract Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of Bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that Bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-κB), we also investigated the effect of Bromelain on Cox-2 and NF-κB expression. Results showed that Bromelain application significantly inhibited Cox-2 and inactivated NF-κB by blocking phosphorylation and subsequent degradation of IκBα. In addition, Bromelain treatment attenuated DMBA–TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that Bromelain induces apoptosis-related proteins along with inhibition of NF-κB-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA–TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects.
Azura Amid - One of the best experts on this subject based on the ideXlab platform.
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Functional characterization of recombinant Bromelain
2015Co-Authors: Azura Amid, Muntari Bala, Hamzah Mohd. SallehAbstract:Stem Bromelain is a plant protease with a number of industrial and therapeutic applications. This study investigated the functional properties of commercial Bromelain and recombinant Bromelain (expressed in E. coli BL 21-AI). In vitro cytotoxicity assays were conducted to evaluate the effect of both Bromelains on tumour cell lines (murine melanoma and breast cancer MCF-7) and normal cells (Vero and Chinese hamster ovary) using Sulforhodamine B and MTT tests. Anti-inflammatory activities of the enzymes were also assessed on murine macrophage cell lines using Griess reagents and ELISA test. The beef tenderizing effects of both Bromelains and in-situ localization of the recombinant Bromelain were analysed using Transmission Electron Microscopy. The results obtained revealed that both Bromelains were more cytotoxic to tumour cell lines (IC50 values ≈ 0.16-0.23mg/mL) than normal cell lines (IC50 values ≈ 0.27-0.51mg/mL). Moreover, the two enzymes had effectively inhibited the pro-inflammatory mediators’ production and thus, had good anti-inflammatory activity. Both enzymes were found to have comparative anti-cancer and anti-inflammatory properties. The expression of recombinant enzyme was discovered to occur in the cytoplasm of E. coli BL 21-AI and the purified enzyme efficiently degraded both actin and myosin of fresh beef. Hence, it served as a good meat tenderizer.
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Case Study: Recombinant Bromelain Downstream Processing
Recombinant Enzymes - From Basic Science to Commercialization, 2015Co-Authors: Azura Amid, Zatul Iffaf Mohd Arshad, Muhd. Ezza Faiez OthmanAbstract:This chapter presents details on the purification, formulation and drying of recombinant Bromelain. The wide range of applications of recombinant Bromelain has increased interest in finding viable purification techniques for large-scale production. An affinity chromatography technique was developed by Amid and co-workers (Expression, purification, and characterization of a recombinant stem Bromelain from Ananas comosus, Process Biochem 46:2232–2239, 2011) to purify recombinant Bromelain. However, this technique presented low recovery and small sample loading capacity and thus is not suitable as a purification tool in the large-scale production of recombinant Bromelain. An aqueous two-phase system is one alternative method that we use to purify recombinant Bromelain, as it reliable and easy to scale up and has a low cost. As part of avoiding cysteine degradation, spray drying the purified recombinant protein with maltodextrin as an excipient provides the possibility of preserving its activity and creating fine particles that are suitable for end-product application. The processes of the purification, formulation and spray drying of recombinant Bromelain are explained briefly.
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Bromelain: an overview of industrial application and purification strategies
Applied Microbiology and Biotechnology, 2014Co-Authors: Zatul Iffah Mohd Arshad, Azura Amid, Faridah Yusof, Kausar Ahmad, Irwandi Jaswir, Show Pau LokeAbstract:This review highlights the use of Bromelain in various applications with up-to-date literature on the purification of Bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially in many applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of Bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant Bromelain for novel applications in the future.
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recombinant Bromelain production in escherichia coli process optimization in shake flask culture by response surface methodology
AMB Express, 2012Co-Authors: Azura Amid, Mohammed Saedi Jami, Bala Muntari, Hamzah Mohd. SallehAbstract:Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced Bromelain activity of 9.2 U/mg while validation experiments gave Bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant Bromelain in shake flask culture.
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Bromelain production current trends and perspective
2012Co-Authors: Muntari Bala, Hamzah Mohd. Salleh, Nurul Azira Ismail, Mohammed Saedi Jami, Azura AmidAbstract:Bromelain is a mixture of proteolytic enzymes derived from the stems and juice of pineapples. It has been extensively used in food industry; for meat tenderization, baking processes, and in prevention of browning of apple juice. Stem Bromelain is a highly accepted phytotherapeutic agent. It has anti-tumor and antiinflammatory effects, use in wound healing, as digestive aids, etc. This review aims at presenting a detailed account on the level of achievement on Bromelain production from the conventional methods. Specifically, the isolation, purification and the biochemical properties of Bromelain are discussed. The study also focused on the current trends and the progress made in the production of recombinant stem Bromelain in Escherichia coli. This involved cloning of stem Bromelain gene and its expression, optimization of labscale fermentation conditions for E. coli harboring recombinant Bromelain as well as the enzyme thermal and storage stability measured using differential scanning calorimetry. It is hoped that this review would provide relevant information to contemporary researchers who are active in the field of Bromelain and other related proteases.
Hamzah Mohd. Salleh - One of the best experts on this subject based on the ideXlab platform.
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Functional characterization of recombinant Bromelain
2015Co-Authors: Azura Amid, Muntari Bala, Hamzah Mohd. SallehAbstract:Stem Bromelain is a plant protease with a number of industrial and therapeutic applications. This study investigated the functional properties of commercial Bromelain and recombinant Bromelain (expressed in E. coli BL 21-AI). In vitro cytotoxicity assays were conducted to evaluate the effect of both Bromelains on tumour cell lines (murine melanoma and breast cancer MCF-7) and normal cells (Vero and Chinese hamster ovary) using Sulforhodamine B and MTT tests. Anti-inflammatory activities of the enzymes were also assessed on murine macrophage cell lines using Griess reagents and ELISA test. The beef tenderizing effects of both Bromelains and in-situ localization of the recombinant Bromelain were analysed using Transmission Electron Microscopy. The results obtained revealed that both Bromelains were more cytotoxic to tumour cell lines (IC50 values ≈ 0.16-0.23mg/mL) than normal cell lines (IC50 values ≈ 0.27-0.51mg/mL). Moreover, the two enzymes had effectively inhibited the pro-inflammatory mediators’ production and thus, had good anti-inflammatory activity. Both enzymes were found to have comparative anti-cancer and anti-inflammatory properties. The expression of recombinant enzyme was discovered to occur in the cytoplasm of E. coli BL 21-AI and the purified enzyme efficiently degraded both actin and myosin of fresh beef. Hence, it served as a good meat tenderizer.
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recombinant Bromelain production in escherichia coli process optimization in shake flask culture by response surface methodology
AMB Express, 2012Co-Authors: Azura Amid, Mohammed Saedi Jami, Bala Muntari, Hamzah Mohd. SallehAbstract:Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced Bromelain activity of 9.2 U/mg while validation experiments gave Bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant Bromelain in shake flask culture.
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Bromelain production current trends and perspective
2012Co-Authors: Muntari Bala, Hamzah Mohd. Salleh, Nurul Azira Ismail, Mohammed Saedi Jami, Azura AmidAbstract:Bromelain is a mixture of proteolytic enzymes derived from the stems and juice of pineapples. It has been extensively used in food industry; for meat tenderization, baking processes, and in prevention of browning of apple juice. Stem Bromelain is a highly accepted phytotherapeutic agent. It has anti-tumor and antiinflammatory effects, use in wound healing, as digestive aids, etc. This review aims at presenting a detailed account on the level of achievement on Bromelain production from the conventional methods. Specifically, the isolation, purification and the biochemical properties of Bromelain are discussed. The study also focused on the current trends and the progress made in the production of recombinant stem Bromelain in Escherichia coli. This involved cloning of stem Bromelain gene and its expression, optimization of labscale fermentation conditions for E. coli harboring recombinant Bromelain as well as the enzyme thermal and storage stability measured using differential scanning calorimetry. It is hoped that this review would provide relevant information to contemporary researchers who are active in the field of Bromelain and other related proteases.
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expression purification and characterization of a recombinant stem Bromelain from ananas comosus
Process Biochemistry, 2011Co-Authors: Azura Amid, Faridah Yusof, Nurul Azira Ismail, Hamzah Mohd. SallehAbstract:Abstract Commercially available Bromelain is prepared by performing a tedious and costly purification method that yields Bromelain at different degrees of purity. In the current study, a gene encoding stem Bromelain from Ananas comosus was amplified using polymerase chain reaction. This Bromelain gene was initially cloned into the pENTR/TEV/D-TOPO vector before being sub-cloned into the pDEST17 expression vector. DNA sequencing of the amplified products exhibited a high level of homology to the corresponding gene from the NCBI public database. Protein expression was conducted in the BL21-AI Escherichia coli strain. The recombinant Bromelain was then purified in a single step using immobilized metal affinity chromatography, specifically a Ni-NTA spin column. The purified recombinant Bromelain was detected by Western blotting. In addition, the purified enzyme exhibited hydrolytic activity towards gelatin and a synthetic substrate, LNPE. The purified recombinant Bromelain exhibited optimum activity at pH 4.6 and 45 °C.
Umesh H Hebbar - One of the best experts on this subject based on the ideXlab platform.
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extraction of Bromelain from pineapple core and purification by rme and precipitation methods
Separation and Purification Technology, 2013Co-Authors: Ram Saran Chaurasiya, Umesh H HebbarAbstract:Demand for Bromelain is increasing because of its application in various fields like medical, food and cosmetics. While Bromelain extracted from pineapple stem is commercially known, presence of Bromelain is reported also in core, peel and crown of pineapple fruit. Core, which is slightly harder in texture, is one of the major wastes generated (15% of the fruit) by the pineapple processing industry. The present work mainly focuses on optimization of conditions for extraction of Bromelain from pineapple (Ananas comosus L.) core and purification of Bromelain by reverse micellar extraction (RME) and precipitation techniques. Extraction conditions like type of buffer, pH, temperature and strength of buffer on Bromelain activity and protein content were standardized. Storage stability study of the extract under refrigerated condition (5–8 °C) showed decrease in Bromelain activity with duration. Mineral analysis of extract showed the presence of calcium, sodium, potassium, and magnesium in the extract. RME, one of the promising liquid–liquid extraction techniques was employed for the primary purification of Bromelain from pineapple core. Selective separation of Bromelain (excluding polyphenol oxidase (PPO) and peroxidase (POD)) with 3.96-fold purification and activity recovery of 78.90% was achieved with this method. Acetone and ammonium salt precipitation methods resulted in 4.90 and 3.07-fold purification under optimized conditions. The studies indicate that extraction followed by RME or precipitation method could be employed for separation and purification of Bromelain from extract obtained from pineapple core.