Azura Amid

Bromelain: an overview of industrial application and purification strategies

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.

Identification of troponin I and actin, alpha cardiac muscle 1 as potential biomarkers for hearts of electrically stimulated chickens.

MethodsIn this study, proteomics methods have been used to study the effects of different currents and voltages used to stun chickens. Protein profiles of chicken hearts were constructed to detect differences in protein expression and modification. The different voltages studied were 10 V, 40 V and 70 V, while the currents examined were 0.25 A, 0.5 A, and 0.75 A. The profiles obtained from these stunning conditions were compared to the non-stunned (0 A, 0 V) sample.ResultsProteomics analyses using 2D Platinum ImageMaster 6.0 and Matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF) identified troponin I and alpha cardiac muscle actin 1 in the electrically stimulated heart samples. The overexpression of the proteins was further confirmed at the transcriptional level by Real Time PCR.ConclusionThe results from MALDI-TOF and Real Time PCR agreed; therefore, this method for identifying biomarkers of electrically stimulated chicken hearts provides a novel approach for differentiation the hearts of increased electrically stimulated chickens from those of non-stunned chickens.

Recombinant bromelain production in Escherichia coli: process optimization in shake flask culture by response surface methodology.

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.

Observation of antioxidant activity of leaves, callus and suspension culture of Justicia gendarusa

Antioxidant activity and phenolic compound was found in Justicia gendarussa via total phenolic content (TPC) and ,-diphenyl--pycrilhydrazil hydrate (DPPH) radical scavenging assays. The assays were applied on aqueous and methanolic extracts of leaves, callus culture and cell suspension culture of J. gendarussa . Callus was induced from green and matured leaves of J. gendarussa c ultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 2,4- dichlorophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) and benzylaminopurine (BAP). Callus induced on NAA showed higher phenolic content and antioxidant activity as compared to the one induced on 2,4-D. The highest total phenolic content (88 mg GAE/g) was observed in callus culture induced with NAA and BAP, while the highest antioxidant activity (lowest IC 50 ; 8.46 × 10 -3 mg/ml) was observed in the cell suspension. Key words: Anti-oxidant, callus, Justicia gendarusa , suspension culture.

Optimized preparation and characterization of CLEA-lipase from cocoa pod husk

Cross-linked enzyme aggregate (CLEA) is easily prepared from crude enzyme and has many advantages to the environment and it is considered as an economic method in the context of industrial biocatalysis compared to free enzyme. In this work, a highly active and stable CLEA-lipase from cocoa pod husk (CPH) which is a by-product after removal of cocoa beans, were assayed for their hydrolytic activity and characterized under the optimum condition successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of the three significant factors (concentration of ammonium sulfate, concentration of glutaraldehyde and concentration of additive) to achieve higher enzyme activity of CLEA. From 20 runs, the highest activity recorded was around 9.407U (83% recovered activity) under the condition of using 20% saturated ammonium sulfate, 60mM glutaraldehyde as cross-linker and 0.17mM bovine serum albumin as feeder. Moreover, the optimal reaction temperature and pH value in enzymatic reaction for both crude enzyme and immobilized were found to be 45°C at pH 8 and 60°C at pH 8.2, respectively. A systematic study of the stability of CLEA and crude enzyme was taken with regards to temperature (25-60°C) and pH (5-10) value and in both factors, CLEA-lipase showed more stability than free lipase. The Km value of CLEA was higher compared to free enzyme (0.55mM vs. 0.08mM). The CLEA retained more than 60% of the initial activity after six cycles of reuse compared to free enzyme. The high stability and recyclability of CLEA-lipase from CPH make it efficient for different industrial applications.

Enzymatic Hydrolysis of Plants and Algae for Extraction of Bioactive Compounds

Plants and algae contain novel biocompounds with therapeutic potentials. These compounds are either embedded within the cell wall matrix or bonded with polymers at cell cytoplasm, which hinders release of bioactive compounds during extraction. Enzymatic hydrolysis of cell wall and cytoplasm polymers enhances the release of biocompounds while preserving their biological potencies. Previous works have focused more on comparative studies of different enzymes, whereas other process parameters, such as agitation speed, substrate particle sizes, hydrolysis time, degree of hydrolysis, and multistage extraction, have received less attention. This review examines the extent of studies in this area and highlight current research gaps to be explored in future researches.

Statistical Analysis of Metal Chelating Activity of Centella asiatica and Erythroxylum cuneatum Using Response Surface Methodology

The purpose of the study is to evaluate the relationship between the extraction parameters and the metal chelating activity of Centella asiatica (CA) and Erythroxylum cuneatum (EC). The response surface methodology was used to optimize the extraction parameters of methanolic extract of CA and EC with respect to the metal chelating activity. For CA, Run 17 gave optimum chelating activity with IC50 = 0.93 mg/mL at an extraction temperature of 25°C, speed of agitation at 200 rpm, ratio of plant material to solvent at 1 g : 45 mL and extraction time at 1.5 hour. As for EC, Run 13 with 60°C, 200 rpm, 1 g : 35 mL and 1 hour had metal chelating activity at IC50 = 0.3817 mg/mL. Both optimized extracts were further partitioned using a solvent system to evaluate the fraction responsible for the chelating activity of the plants. The hexane fraction of CA showed potential activity with chelating activity at IC50 = 0.090 and the ethyl acetate fraction of EC had IC50 = 0.120 mg/mL. The study showed that the response surface methodology helped to reduce the extraction time, temperature and agitation and subsequently improve the chelating activity of the plants in comparison to the conventional method.

Cocoa pod husk: A new source of CLEA-lipase for preparation of low-cost biodiesel: An optimized process

Enzymatic reactions involving lipases as catalyst in transesterification can be an excellent alternative to produce environmental-friendly biodiesel. In this study, lipase extracted from Cocoa Pod Husk (CPH) and immobilized through cross linked enzyme aggregate (CLEA) technology catalysed the transesterification of Jatropha curcas oil successfully. Face centered central composite design (FCCCD) under response surface methodology (RSM) was used to get the optimal conditions of 3% (w/w) enzyme loading, 4h reaction time and 1:6 oil/ethanol ratio to achieve the highest conversion of free fatty acid and glycerides into biodiesel (93%). The reusability of CLEA-lipase was tested and after seven cycles, the conversion percentage reduced to 58%. The results revealed that CLEA lipase from CPH is a potential catalyst for biodiesel production.

Gene Expression Analysis in MCF-7 Breast Cancer Cells Treated with Recombinant Bromelain

The contributing molecular pathways underlying the pathogenesis of breast cancer need to be better characterized. The principle of our study was to better understand the genetic mechanism of oncogenesis for human breast cancer and to discover new possible tumor markers for use in clinical practice. We used complimentary DNA (cDNA) microarrays to compare gene expression profiles of treated Michigan Cancer Foundation-7 (MCF-7) with recombinant bromelain and untreated MCF-7. SpringGene analysis was carried out of differential expression followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We identified 1,102 known genes differentially expressed to a significant degree (p < 0.001) changed between the treatment. Within this gene set, 20 genes were significantly changed between treated cells and the control cells with cutoff fold change of more than 1.5. These genes are RNA-binding motif, single-stranded interacting protein 1 (RBMS1), ribosomal protein L29 (RPL29), glutathione S-transferase mu 2 (GSTM2), C15orf32, Akt3, B cell translocation gene 1 (BTG1), C6orf62, C7orf60, kinesin-associated protein 3 (KIFAP3), FBXO11, AT-rich interactive domain 4A (ARID4A), COPS2, TBPL1|SLC2A12, TMEM59, SNORD46, glioma tumor suppressor candidate region gene 2 (GLTSCR2), and LRRFIP. Our observation on gene expression indicated that recombinant bromelain produces a unique signature affecting different pathways, specific for each congener. The microarray results give a molecular mechanistic insight and functional effects, following recombinant bromelain treatment. The extent of changes in genes is related to and involved significantly in gap junction signaling, amyloid processing, cell cycle regulation by BTG family proteins, and breast cancer regulation by stathmin1 that play major roles.

Chinese hamster ovary (CHO-K1) cells expressed native insulin-like growth factor-1 (IGF-1) gene towards efficient mammalian cell culture host system

Insulin-like growth factor-1 (IGF-1) has been shown to promote cell proliferation and inhibit apoptosis of cells. These are two characteristics of mammalian cell culture which may lead to high density cell culture producing optimal desired yield of bioproducts. An inherent secretion of IGF-1 protein from host cells into the culture media is hypothesized to enable reduction or removable of serum from culture media, thus reducing cost. This study was set to investigate the IGF-1 gene expression in Chinese hamster ovary (CHO-K1) cells. The cells were first cultured in T-flask with three independent experiments. An 8-hourly sampling for responses (glucose, lactate, total protein and biomass) was done. PCR-based method was performed to study the expression of IGF-1 gene. To this end, it was confirmed that CHO-K1 cells used in this study expressed IGF-1 gene. The study also provides the baseline data on kinetics and biochemical responses of CHO-K1 cell growth. Together, the data would be particularly useful for further studies using CHO-K1 cells as efficient mammalian cell culture host system to produce biologics.