DOCSLIB.ORG

Kinetic Studies on Recombinant Stem Bromelain

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Kinetic Studies on Recombinant Stem Bromelain Vol.1, No.3, 52-60 (2013) Advances in Enzyme Research http://dx.doi.org/10.4236/aer.2013.13006 Kinetic studies on recombinant stem bromelain Muntari Bala1,2, Maizirwan Mel1,3, Mohamed Saedi Jami1, Azura Amid1,3, Hamzah Mohd Salleh1,3* 1Bioprocess and Molecular Engineering Research Unit, Department of Biotechnology Engineering, International Islamic University Malaysia, Kuala Lumpur, Malaysia; *Corresponding Author: [email protected] 2Department of Biochemistry, Bayero University, Kano, Nigeria 3International Institute for Halal Research & Training (INHART) International Islamic University Malaysia, Kuala Lumpur, Malaysia Received 22 May 2013; revised 30 June 2013; accepted 8 July 2013 Copyright © 2013 Muntari Bala et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT cause they represent about 60% of all commercial en- zymes worldwide. They are widely used in food, phar- Stem bromelain is a plant thiol protease with maceutical and detergent industries [1]. Plant proteases several industrial and therapeutic applications. have been gaining unique attention in the field of bio- This current work presents kinetic studies of technology and medicine due to their exploitable proper- recombinant bromelain (recBM) expressed in ties. The most recognized plant proteases with greater Escherichia coli BL21-AI on four synthetic sub- commercial values are papain from Carica papaya, ficin strates, N-α-carbobenzoxy-L-alanyl-p-nitrophenyl from Ficus spp. and bromelain from Ananas comosus ester (ZANPE), N-α-carbobenzoxy-L-arginyl-L-ar- [2]. ginine-p-nitroanilide (ZAANA), N-α-carbobenzo- Bromelain is a crude, aqueous extract from the stems xy-L-phenylalanyl-L-valyl-L-arginine-p-nitroanili- and fruits of pineapples (Ananas comosus) derived from de (ZPVANA) and L-pyroglutamyl-L-phenylala- Bromeliaceae family. It contains a mixture of different nyl-L-leucine-p-nitroanilide (PFLNA). Hydrolytic proteases as well as phosphatase, glucosidases, peroxi- activities of recBM at various pH and tempera- dases, cellulases and glycoproteins [3]. Stem bromelain ture conditions were compared to that of com- mercial bromelain (cBM). Both enzymes dem- (EC 3. 4. 22. 32) is the major protease present in extracts onstrated high activities at 45˚C and pH 5 - 8 for of pineapple stem while fruit bromelain (EC 3. 4. 22. 33) recBM and pH 6 - 8 for cBM. recBM showed is the major enzyme present in pineapple fruit juice [3]. Some other minor thiol endopeptidases—ananain and marginally lower Km and slightly higher kcat/Km for ZAANA, ZANPE and ZPVANA in comparison comosain—are also present in the pineapple stem bro- to cBM. trans-Epoxysuccinyl-L-leucylamido {4- melain. All commercially available bromelain are de- guanidino} butane (E-64) severely affected recBM rived from the stem. Stem bromelain is activated by cys- and cBM hydrolysis of the synthetic substrates teine while hydrogen sulphide and sodium cyanide are less effective [4]. The enzyme is inhibited by heavy met- by competitive inhibition with Ki values of 3.6 - 5.1 µM and 5.5 - 6.9 µM for recBM and cBM, re- als such as mercury and silver as well as trans-epoxysu- spectively. The evaluated properties of recBM ccinyl-L-leucylamido {4-guanidino} butane, commonly including temperature and pH optima, substrate known as E-64. specificity and sensitivity to inhibitors or acti- Bromelain has numerous therapeutic, industrial and vators, satisfy the requisites required for food other applications. It has been widely used in food indus- industries. try for baking processes, meat tenderization, clarification of beer, as food supplement and in prevention of brown- Keywords: Protease; Recombinant Bromelain; ing of apple juice [5]. Additionally, it is used as active Substrates; Inhibitors; Activators ingredient to provide mild peeling effects in cosmetic industries [6]. Furthermore, it has also been used in lea- 1. INTRODUCTION ther industries for skin pre-tanning, softening and bating [7]. In textile industries, bromelain is used for improving Proteases are very significant industrial enzymes be- the dyeing qualities of protein fibers, decomposing or Copyright © 2013 SciRes. OPEN ACCESS M. Bala et al. / Advances in Enzyme Research 1 (2013) 52-60 53 partially solubilizing protein fiber from silk and wool [8]. 2.2. Microbial Strain Similarly, bromelain is used as hydrolyzing agent for the The Escherichia coli BL21-AI strain (Invitrogen, USA) release of antimicrobial peptides of leatherjacket’s in- harboring stem bromelain gene used in this study was as soluble proteins [9]. described in our earlier study [12]. Specifically, the gene Stem bromelain is a highly accepted phytotherapeutic encoding stem bromelain was initially amplified from agent. It has anti-tumor and anti-inflammatory effects [4]. pineapple’s stem cloned into pENTR/TEV/D-TOPO be- In addition, it exerts several inhibitory effects on platelet fore being sub-cloned into the expression vector aggregation, bronchitis, angina pectoris, surgical traumas, pDEST17 (Invitrogen, USA). The expression vector sinusitis, thrombophlebitis and pyelonephritis. Moreover, containing stem bromelain gene was then transformed it enhances absorption of drugs, especially antibiotics into E. coli BL21-AI competent cells. [10]. The advances in molecular genetics and genetic engineering in the last decades have made it possible to 2.3. Enzyme Expression clone and express virtually any gene into a suitable mi- crobial host, so that new enzymes from other microor- Recombinant bromelain expression was conducted as ganisms and also from higher organisms can be produced described in our earlier studies [12-14]. E. coli BL21-AI in convenient microbial hosts like bacteria, yeasts and cells harboring recombinant bromelain gene were grown fungi [11]. in shake flasks overnight in LB media containing 100 Considering the numerous applications of stem bro- μg/ml ampicillin. The overnight culture was then diluted melain, it is imperative to produce the enzyme in recom- 50 folds in a fresh LB media and was grown in a 2 liter binant form so as to achieve improved purification, bio- bioreactor at 27˚C, air flow rate of 1.0 vvm with 250 rpm chemical characterization and formulation. In that regard, agitation until a cell density of OD600 nm = 0.6 was we have reported the cloning and expression of recom- reached. This was followed by addition of L-arabinose binant stem bromelain (recBM) [12], recovery from solu- (0.15% w/v, final concentration) and the induction was ble and insoluble enzyme forms [13], lab-scale optimiza- allowed to continue for 8 hours. Cells were harvested tion of some of its culture cultivation conditions [14] and from the spent media by centrifugation (6000 × g) at 4˚C the enzyme thermal and storage stability [15]. The pur- for 20 min and stored at −20˚C for further use. All the pose of the current research is to further characterize the experiments were performed in triplicates. enzyme by focusing on the kinetic studies of recBM us- ing four synthetic substrates; N-α-carbobenzoxy-L-phe- 2.4. Enzyme Purification nylalanyl-L-valyl-L-arginine-p-nitroanilide (ZPVANA), The harvested cells were subjected to sonication (so- N-α-carbobenzoxy-L-alanyl-p-nitrophenyl ester (ZANPE), nicator, 150 v/t model, Biologics, Inc. USA) on ice using N-α-carbobenzoxy-L-arginyl-L-arginine-p-nitroanilide 6 - 10 sec burst, with 10 sec interval at high amplitude. (ZAANA) and L-pyroglutamyl-L-phenylalanyl-L-leuci- This was followed by centrifugation (8000 × g) at 4˚C, ne-p-nitroanilide (PFLNA). The results are then com- for 30 min and the supernatant was collected and purified pared to that of commercial bromelain (cBM) as control. by AKTA purifier FPLC system (GE Healthcare Bio- Sciences, USA) attached with a glass column (0.7 × 10 2. MATERIALS AND METHODS cm, Bio-Rad, USA) that is packed with 4 ml Ni-NTA 2.1. Reagents His•bind resin (Novagen, Germany). Purification of recBM was conducted in accordance to the manufac- Synthetic amide and peptide substrates, N-α-carbo- turer’s instructions. The supernatant (10 ml) was loaded benzoxy-L-alanyl-p-nitrophenyl ester (ZANPE), N-α-car- onto a pre-equilibrated Ni-NTA His•bind column in ac- bobenzoxy-L-arginyl-L-arginine-p-nitroanilide (ZAANA), cordance to the manufacturer’s protocol. The FPLC sys- N-α-carbobenzoxy-L-phenylalanyl-L-valyl-L-arginine-p- tem was set at a flow rate of 1 ml/min throughout the nitroanilide (ZPVANA) and L-pyroglutamyl-L-phenyla- operation. Washing step was achieved by using a wash lanyl-L-leucine-p-nitroanilide (PFLNA) were purchased buffer (50 mM NaH2PO4 pH 8, 300 mM NaCl, 20 mM from Bachem, Germany. Luria Bertani (LB) was a prod- imidazole) while purified recBM was recovered using an uct of Merck, Germany. A single lot (Ref. No. 61391) of elution buffer (50 mM NaH2PO4 pH 8, 300 mM NaCl stem bromelain (EC3. 4. 22. 32) was purchased from MP and 500 mM imidazole). Biomedicals, LLC (France). Coomassie brilliant blue R-250, acrylamide, bisacrylamide and low-range mo- 2.4.1. SDS-PAGE and Western Blot lecular weight markers were obtained from Bio-Rad After each step of enzyme recovery and purification, (Hercules, CA). All other reagents were purchased from the protein fractions were tested by SDS-PAGE in 12.5% Sigma (St. Louis, MO). polyacrylamide gels as described earlier [12]. Visualiza- Copyright © 2013 SciRes. OPEN ACCESS 54 M. Bala et al. / Advances in Enzyme Research 1 (2013) 52-60 tion was conducted by Coomassie staining [16]. On the on the hand, for the remaining three substrates The protein bands on the gel were transferred to poly- (ZAANA, ZPVANA and PFLNA) changes in the ab- vinylidene fluoride (PVDF) membrane (Bio-Rad Labo- sorbance were measured at 410 nm for the p-nitroaniline ratory, Inc., USA) using Mini-Trans-Blot system (Bio- generated [19,20].
Load more

Recommended publications
  • Enzymes Handling/Processing
    Enzymes Handling/Processing 1 Identification of Petitioned Substance 2 3 This Technical Report addresses enzymes used in used in food processing (handling), which are 4 traditionally derived from various biological sources that include microorganisms (i.e., fungi and 5 bacteria), plants, and animals. Approximately 19 enzyme types are used in organic food processing, from 6 at least 72 different sources (e.g., strains of bacteria) (ETA, 2004). In this Technical Report, information is 7 provided about animal, microbial, and plant-derived enzymes generally, and more detailed information 8 is presented for at least one model enzyme in each group. 9 10 Enzymes Derived from Animal Sources: 11 Commonly used animal-derived enzymes include animal lipase, bovine liver catalase, egg white 12 lysozyme, pancreatin, pepsin, rennet, and trypsin. The model enzyme is rennet. Additional details are 13 also provided for egg white lysozyme. 14 15 Chemical Name: Trade Name: 16 Rennet (animal-derived) Rennet 17 18 Other Names: CAS Number: 19 Bovine rennet 9001-98-3 20 Rennin 25 21 Chymosin 26 Other Codes: 22 Prorennin 27 Enzyme Commission number: 3.4.23.4 23 Rennase 28 24 29 30 31 Chemical Name: CAS Number: 32 Peptidoglycan N-acetylmuramoylhydrolase 9001-63-2 33 34 Other Name: Other Codes: 35 Muramidase Enzyme Commission number: 3.2.1.17 36 37 Trade Name: 38 Egg white lysozyme 39 40 Enzymes Derived from Plant Sources: 41 Commonly used plant-derived enzymes include bromelain, papain, chinitase, plant-derived phytases, and 42 ficin. The model enzyme is bromelain.
    [Show full text]
  • Biochemistry and the Genomic Revolution 1.1
    Dedication About the authors Preface Tools and Techniques Clinical Applications Molecular Evolution Supplements Supporting Biochemistry, Fifth Edition Acknowledgments I. The Molecular Design of Life 1. Prelude: Biochemistry and the Genomic Revolution 1.1. DNA Illustrates the Relation between Form and Function 1.2. Biochemical Unity Underlies Biological Diversity 1.3. Chemical Bonds in Biochemistry 1.4. Biochemistry and Human Biology Appendix: Depicting Molecular Structures 2. Biochemical Evolution 2.1. Key Organic Molecules Are Used by Living Systems 2.2. Evolution Requires Reproduction, Variation, and Selective Pressure 2.3. Energy Transformations Are Necessary to Sustain Living Systems 2.4. Cells Can Respond to Changes in Their Environments Summary Problems Selected Readings 3. Protein Structure and Function 3.1. Proteins Are Built from a Repertoire of 20 Amino Acids 3.2. Primary Structure: Amino Acids Are Linked by Peptide Bonds to Form Polypeptide Chains 3.3. Secondary Structure: Polypeptide Chains Can Fold Into Regular Structures Such as the Alpha Helix, the Beta Sheet, and Turns and Loops 3.4. Tertiary Structure: Water-Soluble Proteins Fold Into Compact Structures with Nonpolar Cores 3.5. Quaternary Structure: Polypeptide Chains Can Assemble Into Multisubunit Structures 3.6. The Amino Acid Sequence of a Protein Determines Its Three-Dimensional Structure Summary Appendix: Acid-Base Concepts Problems Selected Readings 4. Exploring Proteins 4.1. The Purification of Proteins Is an Essential First Step in Understanding Their Function 4.2. Amino Acid Sequences Can Be Determined by Automated Edman Degradation 4.3. Immunology Provides Important Techniques with Which to Investigate Proteins 4.4. Peptides Can Be Synthesized by Automated Solid-Phase Methods 4.5.
    [Show full text]
  • Cellulases: Characteristics, Sources, Production, and Applications
    8 CELLULASES: CHARACTERISTICS, SOURCES, PRODUCTION, AND APPLICATIONS Xiao-Zhou Zhang and Yi-Heng Percival Zhang 8.1 INTRODUCTION lulases: (1) endoglucanases (EC 3.2.1.4), (2) exogluca- nases, including cellobiohydrolases (CBHs) (EC Cellulose is the most abundant renewable biological 3.2.1.91), and (3) β -glucosidase (BG) (EC 3.2.1.21). To resource and a low-cost energy source based on energy hydrolyze and metabolize insoluble cellulose, the micro- content ($3–4/GJ) ( Lynd et al., 2008 ; Zhang, 2009 ). The organisms must secrete the cellulases (possibly except production of bio-based products and bioenergy from BG) that are either free or cell-surface-bound. Cellu- less costly renewable lignocellulosic materials would lases are increasingly being used for a large variety of bring benefi ts to the local economy, environment, and industrial purposes—in the textile industry, pulp and national energy security ( Zhang, 2008 ). paper industry, and food industry, as well as an additive High costs of cellulases are one of the largest obsta- in detergents and improving digestibility of animal cles for commercialization of biomass biorefi neries feeds. Now cellulases account for a signifi cant share of because a large amount of cellulase is consumed for the world ’ s industrial enzyme market. The growing con- biomass saccharifi cation, for example, ∼ 100 g enzymes cerns about depletion of crude oil and the emissions of per gallon of cellulosic ethanol produced ( Zhang et al., greenhouse gases have motivated the production of bio- 2006b ; Zhu et al., 2009 ). In order to decrease cellulase ethanol from lignocellulose, especially through enzy- use, increase volumetric productivity, and reduce capital matic hydrolysis of lignocelluloses materials—sugar investment, consolidated bioprocessing ( CBP ) has been platform ( Bayer et al., 2007 ; Himmel et al., 1999 ; Zaldi- proposed by integrating cellulase production, cellulose var et al., 2001 ).
    [Show full text]
  • Application of Plant Proteolytic Enzymes for Tenderization of Rabbit Meat
    Biotechnology in Animal Husbandry 34 (2), p 229-238 , 2018 ISSN 1450-9156 Publisher: Institute for Animal Husbandry, Belgrade-Zemun UDC 637.5.039'637.55'712 https://doi.org/10.2298/BAH1802229D APPLICATION OF PLANT PROTEOLYTIC ENZYMES FOR TENDERIZATION OF RABBIT MEAT Maria Doneva, Iliana Nacheva, Svetla Dyankova, Petya Metodieva, Daniela Miteva Institute of Cryobiology and Food Technology, Cherni Vrah 53, 1407, Sofia, Bulgaria Corresponding author: Maria Doneva, e-mail: [email protected] Original scientific paper Abstract: The purpose of this study is to assess the tenderizing effect of plant proteolytic enzymes upon raw rabbit meat. Tests are performed on rabbit meat samples treated with papain and two vegetal sources of natural proteases (extracts of kiwifruit and ginger root). Two variants of marinade solutions are prepared from each vegetable raw materials– 50% (w/w) and 100 % (w/w), with a duration of processing 2h, 24h, and 48h. Changes in the following physico- chemical characteristics of meat have been observed: pH, water-holding capacity, cooking losses and quantity of free amino acids. Differences in values of these characteristics have been observed, both between control and test samples, as well as depending of treatment duration. For meat samples marinated with papain and ginger extracts, the water-holding capacity reached to 6.74 ± 0.04 % (papain), 5.58 ± 0.09 % (variant 1) and 6.80 ± 0.11 % (variant 2) after 48 hours treatment. In rabbit meat marinated with kiwifruit extracts, a significant increase in WHC was observed at 48 hours, 3.37 ± 0.07 (variant 3) and 6.84 ± 0.11 (variant 4).
    [Show full text]
  • Evidence for an Active-Center Cysteine in the SH-Proteinase Cu-Clostripain Through Use of IV-Tosyl-L-Lysine Chloromethyl Ketone
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 173, number 1 FEBS 1649 July 1984 Evidence for an active-center cysteine in the SH-proteinase cu-clostripain through use of IV-tosyl-L-lysine chloromethyl ketone A.-M. Gilles and B. Keil Unitt! de Chimie des Protknes, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris CPdex 15, France Received 30 May 1984 The rapid reaction of a-clostripain with tosyl-L-lysine chloromethyl ketone results in a complete loss of activity and in the disappearance of one titratable SH group whereas the number of histidine residues is not affected. Tosyl-L-phenylalanine chloromethyl ketone and phenylmethylsulfonyl fluoride have no effect on the catalytic activity. From the molar ratio and under the assumption of 1: 1 molar interaction, the fully active enzyme has a specific activity of 650-700 units/mg [twice the value proposed by Porter et al. (J. Biol. Chem. 246 (1971) 76757682)]. Partial oxidation makes it experimentally impossible to attain this maximal value. ff-Clostripain Cysteine proteinase Active site 1. INTRODUCTION was due to the modification of a thiol group in an analogous way with other cysteine proteinases such Clostripain (EC 3.4.4.20) is a sulfhydryl protein- as papain [6] and ficin [7]. Recently [8], we eluci- ase isolated from the culture filtrate of Clostridium dated the amino acid sequence around this acces- histolyticum with a highly limited specificity sible thiol group after labelling with radioactive directed at the carboxyl bond of arginyl residues in iodoacetic acid.
    [Show full text]
  • Serine Proteases with Altered Sensitivity to Activity-Modulating
    (19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants.
    [Show full text]
  • Current IUBMB Recommendations on Enzyme Nomenclature and Kinetics$
    Perspectives in Science (2014) 1,74–87 Available online at www.sciencedirect.com www.elsevier.com/locate/pisc REVIEW Current IUBMB recommendations on enzyme nomenclature and kinetics$ Athel Cornish-Bowden CNRS-BIP, 31 chemin Joseph-Aiguier, B.P. 71, 13402 Marseille Cedex 20, France Received 9 July 2013; accepted 6 November 2013; Available online 27 March 2014 KEYWORDS Abstract Enzyme kinetics; The International Union of Biochemistry (IUB, now IUBMB) prepared recommendations for Rate of reaction; describing the kinetic behaviour of enzymes in 1981. Despite the more than 30 years that have Enzyme passed since these have not subsequently been revised, though in various respects they do not nomenclature; adequately cover current needs. The IUBMB is also responsible for recommendations on the Enzyme classification naming and classification of enzymes. In contrast to the case of kinetics, these recommenda- tions are kept continuously up to date. & 2014 The Author. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). Contents Introduction...................................................................75 Kinetics introduction...........................................................75 Introduction to enzyme nomenclature ................................................76 Basic definitions ................................................................76 Rates of consumption and formation .................................................76 Rate of reaction .............................................................76
    [Show full text]
  • A Process for Combined Biopolishing
    (19) TZZ _¥_T (11) EP 2 164 943 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C11D 3/386 (2006.01) D06M 16/00 (2006.01) 12.03.2014 Bulletin 2014/11 C12N 9/08 (2006.01) C11D 3/00 (2006.01) C12N 9/42 (2006.01) (21) Application number: 08760676.0 (86) International application number: (22) Date of filing: 06.06.2008 PCT/EP2008/057106 (87) International publication number: WO 2008/151999 (18.12.2008 Gazette 2008/51) (54) A PROCESS FOR COMBINED BIOPOLISHING AND BLEACH CLEAN-UP VERFAHREN FÜR KOMBINIERTE BIOPOLIERUNG UND BLEICHREINIGUNG PROCEDE POUR BIOPOLISSAGE ET NETTOYAGE PAR UN AGENT DE BLANCHIMENT COMBINES (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR WO-A-00/71808 WO-A-99/32708 HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT WO-A-03/002810 WO-A1-02/38717 RO SE SI SK TR WO-A1-91/17243 WO-A1-96/29397 WO-A1-2004/059075 WO-A2-2006/101584 (30) Priority: 11.06.2007 EP 07109969 WO-A2-2007/019442 CN-A- 1 584 192 GB-A- 2 216 149 PT-A- 101 152 (43) Date of publication of application: US-A- 5 700 769 US-A- 6 140 109 24.03.2010 Bulletin 2010/12 • ROY S K, DEY S K, RAHA S K, CHAKRABARTY S (60) Divisional application: L: "Purification and properties of an extracellular 13196997.4 endoglucanase from Myceliophthora thermophila", JOURNAL OF GENERAL (73) Proprietor: Novozymes A/S MICROBIOLOGY, vol.
    [Show full text]
  • The Proteolysis of Apolipoprotein E in Alzheimer's Disease
    THE PROTEOLYSIS OF APOLIPOPROTEIN E IN ALZHEIMER’S DISEASE by Julia Love A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology Boise State University August 2016 © 2016 Julia Love ALL RIGHTS RESERVED BOISE STATE UNIVERSITY GRADUATE COLLEGE DEFENSE COMMITTEE AND FINAL READING APPROVALS of the thesis submitted by Julia Love Thesis Title: The Proteolysis of Apolipoprotein E in Alzheimer’s Disease Date of Final Oral Examination: 26 April 2016 The following individuals read and discussed the thesis submitted by student Julia Love, and they evaluated her presentation and response to questions during the final oral examination. They found that the student passed the final oral examination. Troy Rohn, Ph.D. Chair, Supervisory Committee Kenneth A. Cornell, Ph.D. Member, Supervisory Committee Juliette Tinker, Ph.D. Member, Supervisory Committee The final reading approval of the thesis was granted by Troy Rohn, Ph.D., Chair of the Supervisory Committee. The thesis was approved for the Graduate College by Jodi Chilson, M.F.A., Coordinator of Theses and Dissertations. DEDICATION This thesis is dedicated to my parents Paul and Cynthia Love, my brother Philip Love, and all of my friends who have supported and encouraged me along the way. iv ACKNOWLEDGEMENTS There have been many people who have contributed to this work and my academic growth over the course of pursuing my Master’s degree. These individual contributions have not gone unnoticed and are an important part of my thesis work. First and foremost, I would like to thank Dr. Troy Rohn for being available with a willing attitude whenever I needed assistance, for his steadfast support and care, and for providing me with every opportunity to exceed what I thought were my limitations.
    [Show full text]
  • Molecular Insights Into Bromelain Application in Industry and Health Care
    Biosc.Biotech.Res.Comm. Special Issue Vol 13 No 15 (2020) Pp-36-46 Molecular Insights into Bromelain Application in Industry and Health Care Sushma S. Murthy and T. Bala Narsaiah 1Research Scholar, Department of Chemical Engineering, JNTUA College of Engineering, Ananthapuram-515002, Andhra Pradesh, India 2Department of Chemical Engineering, JNTUA College of Engineering, Ananthapuram-515002, Andhra Pradesh, India ABSTRACT Bromelain is a cysteine protease derived from the stem and fruit of the pineapple. It has a significant role in pharmacological and clinical applications. Studies have shown Bromelain to be a potent photoactive compound that has a wide application in industry. It has also been shown to be effective in treatment of cancer, inflammation, and allergies. It has a distinct immunomodulatory activity which forms an important strategy in its utilization as a therapeutic agent. Bromelain plays a significant role at molecular level by regulating the expression of proteins that are potential therapeutic targets. Bromelain is used extensively worldwide as an herbal medicine as it promises good efficacy and has no side effects. This paper reviews the general characteristics of Bromelain, its separation process, and its use in industries and healthcare as a therapeutic agent. The present review identifies that there is lack of knowledge pertaining to the mode of action of Bromelain in inhibiting transcriptional factors and in controlling cancer. An in-detail analysis in this area might help in expanding the therapeutic scope of Bromelain. KEY WORDS: BROMELAIN, CANCER, PHARMacOLOGICAL actiVITY, SEpaRatiON, TRANSCRIPTION FactORS. INTRODUCTION because of its wide benefits to the human system. The stem part of the plant, which is inexpensive and is usually The Bromelain protease is isolated from the stem and discarded, has a high concentration of bromelain and fruit of Pineapple (Ananas comosus,).
    [Show full text]
  • Collection of Information on Enzymes a Great Deal of Additional Information on the European Union Is Available on the Internet
    European Commission Collection of information on enzymes A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server (http://europa.eu.int). Luxembourg: Office for Official Publications of the European Communities, 2002 ISBN 92-894-4218-2 © European Communities, 2002 Reproduction is authorised provided the source is acknowledged. Final Report „Collection of Information on Enzymes“ Contract No B4-3040/2000/278245/MAR/E2 in co-operation between the Federal Environment Agency Austria Spittelauer Lände 5, A-1090 Vienna, http://www.ubavie.gv.at and the Inter-University Research Center for Technology, Work and Culture (IFF/IFZ) Schlögelgasse 2, A-8010 Graz, http://www.ifz.tu-graz.ac.at PROJECT TEAM (VIENNA / GRAZ) Werner Aberer c Maria Hahn a Manfred Klade b Uli Seebacher b Armin Spök (Co-ordinator Graz) b Karoline Wallner a Helmut Witzani (Co-ordinator Vienna) a a Austrian Federal Environmental Agency (UBA), Vienna b Inter-University Research Center for Technology, Work, and Culture - IFF/IFZ, Graz c University of Graz, Department of Dermatology, Division of Environmental Dermatology, Graz Executive Summary 5 EXECUTIVE SUMMARY Technical Aspects of Enzymes (Chapter 3) Application of enzymes (Section 3.2) Enzymes are applied in various areas of application, the most important ones are technical use, manufacturing of food and feedstuff, cosmetics, medicinal products and as tools for re- search and development. Enzymatic processes - usually carried out under mild conditions - are often replacing steps in traditional chemical processes which were carried out under harsh industrial environments (temperature, pressures, pH, chemicals). Technical enzymes are applied in detergents, for pulp and paper applications, in textile manufacturing, leather industry, for fuel production and for the production of pharmaceuticals and chiral substances in the chemical industry.
    [Show full text]
  • Biocatalysis, Enzyme Engineering and Biotechnology G
    P1: SFK/UKS P2: SFK BLBS102-c07 BLBS102-Simpson March 21, 2012 11:12 Trim: 276mm X 219mm Printer Name: Yet to Come 7 Biocatalysis, Enzyme Engineering and Biotechnology G. A. Kotzia, D. Platis, I. A. Axarli, E. G. Chronopoulou, C. Karamitros, and N. E. Labrou Preface Enzyme Utilisation in Industry Enzyme Structure and Mechanism Enzymes Involved in Xenobiotic Metabolism and Nomenclature and Classification of Enzymes Biochemical Individuality Basic Elements of Enzyme Structure Phase I The Primary Structure of Enzyme Phase II The Three-Dimensional Structure of Enzymes Phase III Theory of Enzyme Catalysis and Mechanism Acknowledgements Coenzymes, Prosthetic Groups and Metal Ion Cofactors References Kinetics of Enzyme-Catalysed Reactions Thermodynamic Analysis Abstract: Enzymes are biocatalysts evolved in nature to achieve Enzyme Dynamics During Catalysis the speed and coordination of nearly all the chemical reactions that Enzyme Production define cellular metabolism necessary to develop and maintain life. Enzyme Heterologous Expression The application of biocatalysis is growing rapidly, since enzymes The Choice of Expression System offer potential for many exciting applications in industry. The ad- Bacterial Cells vent of whole genome sequencing projects enabled new approaches Mammalian Cells for biocatalyst development, based on specialised methods for en- Yeast zyme heterologous expression and engineering. The engineering of Filamentous Fungi enzymes with altered activity, specificity and stability, using site- Insect Cells directed mutagenesis and directed evolution techniques are now Dictyostelium discoideum Trypanosomatid Protozoa well established. Over the last decade, enzyme immobilisation has Transgenic Plants become important in industry. New methods and techniques for en- Transgenic Animals zyme immobilisation allow for the reuse of the catalysts and the Enzyme Purification development of efficient biotechnological processes.
    [Show full text]