Dissertations / Theses: 'Proteolytic enzymes'

1

Ekici, Özlem Doğan. "Design, synthesis, and evaluation of novel irreversible inhibitors for caspases." Available online, Georgia Institute of Technology, 2004:, 2003. http://etd.gatech.edu/theses/available/etd-04062004-164633/unrestricted/ekici%5Fozlem%5Fd%5F200312%5Fphd.pdf.

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2

Ahmed, Naveed. "The synthesis of inhibitors of proteolytic enzymes." Thesis, University of Huddersfield, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.416765.

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3

Ekici, Ozlem Dogan. "Design, synthesis, and evaluation of novel irreversible inhibitors for caspases." Diss., Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/5333.

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4

Verity, Christiana Kelsick. "Cathepsin D-like aspartic protease from Schistosoma japonicum : developmental, enzymological and immunological studies /." [St. Lucia, Qld.], 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16289.pdf.

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5

Chaveesuk, Ravipim. "Accleration of fish sauce fermentation using proteolytic enzymes." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60539.

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First grade and second grade Nampla, commercially produced Thai fish sauces, were analyzed for their chemical and microbiological composition. First grade commercially produced Nampla contained higher amounts of total nitrogen, formol nitrogen, free and total amino acids compared to second grade sauce. Most of the essential amino acids were present in both grades of sauces. Low microbial counts of halotolerant microorganisms were observed in both sauces. The use of trypsin and chymotrypsin to accelerate the rate of fish sauce fermentation produced from herring, one of the underutilized fish species in Quebec, was investigated. Results showed that supplementation with trypsin and chymotrypsin increased significantly the rate of proteolysis, the amounts of total nitrogen, formol nitrogen and free amino acids in the final fish sauces (p 0.05). (Abstract shortened by UMI).

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6

陳臻 and Chun Jade Chan. "Proteolytic enzyme in soy sauce fermentation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31223977.

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7

Chan, Chun Jade. "Proteolytic enzyme in soy sauce fermentation." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk:8888/cgi-bin/hkuto%5Ftoc%5Fpdf?B22713384.

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8

Deane, Shelly May. "Molecular biology studies on the extracellular serine proteases of Vibrio alginolyticus." Doctoral thesis, University of Cape Town, 1989. http://hdl.handle.net/11427/21895.

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Bibliography: pages 161-176.
Vibrio alginolyticus is a gram-negative aerobic bacterium that produces several extracellular serine proteases and a collagenase during the stationary growth phase. The aim of this study was to investigate alkaline serine protease production by this organism, and to attempt the cloning and expression of a V.alginolyticus protease gene in Escherichia coli.

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9

賴玉耀 and Yuk-yeu William Lai. "Identification and characterization of proteolytic enzymes in Trichinella spp." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1996. http://hub.hku.hk/bib/B31214228.

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10

Hinchliffe, Paul Stuart. "The synthesis of #beta#-sultam inhibitors of proteolytic enzymes." Thesis, University of Huddersfield, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368328.

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11

Lai, Yuk-yeu William. "Identification and characterization of proteolytic enzymes in Trichinella spp /." Hong Kong : University of Hong Kong, 1996. http://sunzi.lib.hku.hk/hkuto/record.jsp?B19739898.

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12

Faro, Andrew. "Recombinant expression and full backbone assignment of the human DWNN using heteronuclear NMR." Thesis, University of the Western Cape, 2005. http://etd.uwc.ac.za/index.php?module=etd&amp.

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The cellular levels of a number of proteins have been found to be regulated by the ubiquitin-proteasome pathway. In this pathway, proteins are covalently tagged (&ldquo
ubiquitinated&rdquo
) by ubiquitin, which acts as a signal for degradation by the proteasome. A number of key cellular processes, including cell-cycle progression, transcription and DNA repair, are regulated in this way. In recent years a number of cellular proteins resembling ubiquitin in structure or function, the so-called ubiquitin-like proteins, have been identified. Ubiquitin-like proteins can be divided into two classes-the so-called &ldquo
ubiquitin-like modifiers&rdquo
, which consist of a single domain that structurally resembles ubiquitin, and &ldquo
ubiquitin-domain&rdquo
proteins, which are multi-domain proteins, which include domains that resemble ubiquitin.

This thesis describes the recombinant expression, purification and full backbone assignment of the human DWNN domain, a novel ubiquitin-like domain. The DWNN domain occurs at the N-terminus of RBBP6, a protein that has been shown to interact with p53 and Rb as well as to be involved in mRNA processing and apoptosis. A bacterial expression system was used to overexpress the DWNN domain as a GST fusion protein. The domain was labelled with 15N and 13C to perform triple-resonance heteronuclear NMR experiments, from which full backbone assignments were obtained.

Although full structure determination of the DWNN domain falls outside the scope of this thesis, the backbone assignments formed the basis for the subsequent structure determination, which confirmed that the DWNN domain is indeed a novel ubiquitin-like domain. The RBBP6 protein may therefore represent a novel E3 ubiquitin ligase that plays a role in regulating the cellular levels of p53 and Rb.

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13

Gordon, Nathaniel Charles. "Protease engineering for therapeutic applications." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648185.

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14

Duman, Ramona Elena. "Structural studies of bacterial Lon ATP-dependent proteases." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609086.

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15

Seo, Sang-Beom. "Purification and characterization of protease C2 : the protease which initiates the degradation of the B-subunit of B-conglycinin in soybean (Glycine max) seedlings /." Online version via UMI:, 1999.

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16

Dotse, Anthony Kwabla. "Design, synthesis and evaluation of novel inhibitors of cysteine proteases, metalloproteases and the proteasome, a unique high molecular weight proteolytic enzyme." Diss., Georgia Institute of Technology, 1997. http://hdl.handle.net/1853/29979.

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17

Cameron, Angus. "Proteolytic enzymes from the hepatopancreas of the Kamchatkan King crab." Thesis, London South Bank University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264822.

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18

Vaiyapuri, Sakthivel. "Sequence, structure and functional studies of viper venom proteolytic enzymes." Thesis, University of Reading, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501321.

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Snake venom proteins are potential sources for novel drug design both for treatment of snake bites and for human genetic disorders. To achieve these, the basic sequence, structure and functional relationships of venom proteins should be understood. Proteolytic enzymes such as metallo and serine proteases are the major components of venom in vipers and are responsible for local and systematic envenomation effects. We have purified, partially sequenced and functionally characterised a serine protease, rhinocerase from the venom of Bitis gabonica rhinoceros. Rhinocerase is a multifunctional enzyme with kininogenase, clotting and defibrase activities.

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19

Deng, Meihong. "Proteolytic cleavage of FOXM1 by caspases /." View the Table of Contents & Abstract, 2006. http://sunzi.lib.hku.hk/hkuto/record/B36396503.

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20

Deng, Meihong, and 邓美虹. "Proteolytic cleavage of FOXM1 by caspases." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B45010675.

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21

Saldanha, Rohit Gregory Medical Sciences Faculty of Medicine UNSW. "Proteolytic enzymes in grass pollen and their relationship to allergenic proteins." Awarded by:University of New South Wales. School of Medical Sciences, 2005. http://handle.unsw.edu.au/1959.4/20824.

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Pollen grains are ubiquitous triggers of allergic asthma and seasonal rhinitis. Proteolytic enzymes in pollen as well as other sources are capable of disrupting airway epithelial integrity in vivo and in vitro. This provides a plausible mechanism for the initiation of sensitisation of the respiratory immune system to inhaled pollen allergens, comparable to that suggested for Group 1 allergens from house dust mites and cat dander, which are known to possess intrinsic proteolytic activity. This thesis explores the relationship between pollen allergens and proteolytic enzymes. It describes the different strategies used for the characterisation, purification and identification of immunogenic and proteolytic proteins in the complex mixtures of pollen diffusates. The peptidases in the diffusates of Kentucky blue grass, ryegrass and Bermuda grass pollens were characterised by a sensitive fluorescence assay and gelatin zymography. Among these, Bermuda grass pollen demonstrated the presence of a serine peptidase at Mr ~30,000 Da, which corresponded to an intense band by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen, Phl p 1. Purification of this enzyme from Bermuda grass was complicated by the low levels of the enzyme present in the diffusate, as well as by its autohydrolysis. Partial purification of the serine peptidase activity by affinity chromatography using Concanavalin A Sepharose demonstrated that the diffusate contained a trypsin-like peptidase, detected by the fluorescent assay, in addition to the ~30,000 Da serine endopeptidase, detected on gelatin zymography. Proteomic analysis of the ~30,000 Da protein using one- and two-dimensional electrophoresis and mass spectrometry identified it as the major pollen allergen of Bermuda grass, Cyn d 1. The studies reported here provide, for the first time, evidence that a pollen allergen may possess intrinsic proteolytic activity. This activity may play a role in the initiation of airway inflammation and allergic sensitisation.

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22

Heidlebaugh, Nancy Marie. "Analysis of nitrogen reallocation from senescing barley leaves characterization of the influence of a high-grain protein content locus on chromosome six, and molecular cloning and heterologous expression of a serine carboxypeptidase /." Thesis, Montana State University, 2008. http://etd.lib.montana.edu/etd/2008/heidlebaugh/HeidlebaughN0508.pdf.

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23

Lacroix-Desmazes, Sebastien. "Influence of flow environment on the production and secretion of metalloproteinases and urokinase-type plasminogen activator by cultured bovine aortic endothelial cells." Thesis, Georgia Institute of Technology, 1993. http://hdl.handle.net/1853/15826.

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24

Ye, Yu. "Targeting ubiquitin chains with deubiquitinases." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610783.

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25

Gibb, Gregory Donald. "Physiological regulation of Streptomycete Proteases /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487327695620921.

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26

Gençkal, Hande Tarı Canan. "Studies On Alkaline Protease Production From Bacillus Sp./." [s.l.]: [s.n.], 2004. http://library.iyte.edu.tr/tezler/master/biyoteknoloji/T000505.pdf.

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27

Beaumont, Marie Louise. "Rapid screening of proteolytic enzymes and their inhibitors using fluorescence methods." Thesis, Loughborough University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.394876.

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28

Haycock, John W. "Degradation of human muscle proteins by free radicals and proteolytic enzymes." Thesis, University of Newcastle upon Tyne, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260052.

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29

Poliukh, К. "Modern state in the research and application of proteolytic enzymes of fungi." Thesis, National Aviation University, 2021. https://er.nau.edu.ua/handle/NAU/50640.

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1. Braga-Silva L.A., Santos A.L. Aspartic protease inhibitors as potential anti-Candida albicans drugs: impacts on fungal biology, virulence and pathogenesis. Curr Med Chem. 2011. Vol.18 (16). P. 2401-19. 2. Ghosh A.K., Osswald H.L., Prato G. Recent Progress in the developent of HIV-1 protease inhibitors for the treatment of HIV/AIDS. Journal of medicinal chemistry. 2016. Vol. 59 (11). P. 5172–5208. 3. Kim S.Y., Gunasekaran S., Olson N.F. Combined use of chymosin and protease from Cryphonectria parasitica for control of meltability and firmness of cheddar cheese. J Dairy Sci. 2004. Vol. 87(2). P. 274-83. URL: doi: 10.3168/jds.S0022-0302(04)73166-5. 4. Suwannarach N., Kumla J., Sujarit K. et al. Natural bioactive compounds from fungi as potential candidates for protease inhibitors and immunomodulators to apply for coronaviruses. Molecules. 2020. Vol. (25 (8). P.1800. 5. Варбанець Л.Д., Мацелюх О.В. Протеолітичні ферменти мікроорганізмів та методи їх дослідження: монографія. Київ. 2008. 108 с.
Proteolytic enzymes are studied as tools for understanding protein structure and the mechanisms of enzymatic catalysis, or as well as bioactive substances applied in medicine, agriculture and industry. The growing needs of biotechnology, changes in environmental and radiation backgrounds, widespread usage of drugs encourages the search for a new, effective, safe species and strains of fungi which could be the base for new probiotics production and serve in biotransformations of food products.
Протеолітичні ферменти вивчаються як інструменти для розуміння білкової структури та механізмів ферментативного каталізу, або, а також біологічно активних речовин, застосовуваних у медицині, сільському господарстві та промисловості. Зростаючі потреби біотехнології, зміни в екологічних та умовах випромінювання, широке використання наркотиків сприяє пошуку нових, ефективних, безпечних видів та штамів грибів, які можуть бути базою для виробництва нових пробіотиків та служать у біотрансформаціях харчових продуктів.

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30

Boräng, Jennifer, and Adam Boucher. "Green tea inhibits proteolytic enzymes in GCF from patients with chronic periodontitis." Thesis, Malmö högskola, Odontologiska fakulteten (OD), 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-19944.

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Kronisk parodontit orsakar vävnadsdestruktion till följd av matrixmetalloproteinasaktivitet. Dessa enzym härrör från värdcellerna och är en del av det immunologiska svaret på bakteriella virulensfaktorer. Grönt te har studerats för sina hälsofrämjande egenskaper, som omfattar bland annat anti-inflammatoriska effekter. Effekten beror delvis på enzyminhibering av tepolyfenoler. Syftet med denna studie var att ytterligare undersöka den inhiberande effekten av grönt te, med fokus på enzymatisk aktivitet i gingivalvätska från patienter med parodontal sjukdom. Patienter med kronisk parodontit valdes ut för att delta i studien. Gingivalvätska extraherades med mikropipetter från patienternas gingivala sulci. Proverna behandlades med grönt te och jämfördes med obehandlade prover från samma försöksperson. Fluorescens proteasanalys med kasein som substrat utfördes på fjorton prover för att detektera skillnader i kaseinolytisk aktivitet. Zymogramanalys med användning av gelatin som substrat utfördes på fyra prover, för att undersöka skillnader i gelatinolytisk aktivitet och analysera molekylvikter för de olika enzymerna. Den fluorometriska analysen visade en signifikant lägre enzymaktivitet i prover med tillsatt grönt te jämfört med obehandlade prover (p<0.001). Zymogramanalysen visade en skillnad i enzymaktivitet som var mest uttalad i banden för molekylär vikt runt 255 kDa, analogt med komplex av matrixmetalloproteinas-9. Sammanfattningsvis har det i denna studie påvisats att grönt te har en hämmande effekt på kaseinolytisk aktivitet och en mindre, mer specifik, hämmande effekt på gelatinasaktivitet.
Chronic periodontitis involves tissue destruction by matrix metalloproteinase, derived from the host cells, as part of the immunological response to bacterial virulence factors. Green tea has been studied for its health promoting properties, which includes anti-inflammatory effects. The effect is in part due to enzyme inhibition by tea polyphenols. The aim of this study was to further investigate the inhibitory effect of green tea, focusing on enzymatic activity in gingival crevicular fluid from patients with periodontal disease. Patients with chronic periodontitis were selected for participation in the study. Gingival crevicular fluid was extracted with micropipettes from the gingival sulci of the patients. Samples were treated with green tea and compared with untreated samples from the same subject. Fluorescence protease assay with casein as substrate was made using fourteen samples for detecting differences in caseinolytic activity. Zymogram assay using gelatin as substrate was done using four samples to test gelatinolytic activity and analyse molecular weights of the different enzymes. The fluorometric assay showed a significantly lower enzyme activity in samples mixed with green tea than untreated samples (p<0.001). The zymogram assay showed a difference in band strength which was most pronounced in the bands of molecular weight around 255 kDA, analogous to complexes of matrix metalloproteinase-9. In conclusion, green tea has been shown in this study to have a strong inhibitory effect on caseinolytic activity and a lesser, more specific, inhibitory effect on gelatinase activity.

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31

Shakery, Javad. "The isolation and characterisation of antibiotics and exo-enzymes from strains of Trichoderma harzianum." Thesis, University of Salford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366021.

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32

Baska, Kathleen M. "Ubiquitin-proteaseome : pathway in bovine epididymal sperm maturation /." free to MU campus, to others for purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?p1426044.

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33

Krause, Jason. "Purification and partial characterisation of cathepsin D from ostrich skeletal muscle, and its activity during meat maturation." Thesis, Nelson Mandela Metropolitan University, 2009. http://hdl.handle.net/10948/1461.

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Cathepsin D, a muscle proteinase, participates in lysosomally mediated protein degradation in vivo. This enzyme has been proposed to play a significant role in the postmortem proteolysis process apparently associated with tenderisation. The lack of data on the postmortem characteristics of ostrich meat, especially on the ageing process and its influence on meat tenderness, called for an investigation into this process. There is no data available for purified ostrich cathepsin D, and the aim of this study was, therefore, to isolate, purify and characterise cathepsin D from ostrich skeletal muscle and subsequently investigate the possible role that it may have in the tenderisation process of meat. Cathepsin D was successfully isolated and purified from ostrich skeletal muscle using pepstatin A-agarose chromatography. The purified enzyme was composed of two subunits (14 and 29kDa). The amino acid composition as well as the N-terminal amino acid sequence of both subunits were determined. Kinetic parameters (Km and Vm), thermodynamic parameters (Ea, ∆H, ∆S and ∆G) and functional characteristics (effect of pH, temperature and various inhibitors on cathepsin D activity) were determined and are reported in this study. Ostrich muscle cathepsin D showed a pH optimum of 4 and a temperature optimum of 45°C. The activity of cathepsin D was strongly inhibited by pepstatin A and DTT. Purified ostrich cathepsin D displayed kinetic and functional properties similar to previously reported values from various species. The effect of storage on the activity of cathepsin D was investigated over a 30 day period. It was established that substantial postmortem cathepsin D activity remained throughout the storage period, to implicate cathepsin D, fulfilling a possible role in meat maturation.

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34

Tshidino, Shonisani Cathphonia. "Purification and partial characterization of a Myofibril-Bound Serine Protease and its endogenous inhibitor from skeletal muscle of the ostrich." Thesis, Nelson Mandela Metropolitan University, 2008. http://hdl.handle.net/10948/703.

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The ostrich is becoming an important source of meat for humans in developed and developing countries. This study was conducted to purify and characterize myofibrilbound serine protease (MBSP) and its endogenous inhibitor (MBSPI) from skeletal muscle of the ostrich. It is well documented that MBSP is tightly bound to myofibrils and its endogenous inhibitor has been purified from the same tissue of other studied mammalian species. Literature supports an association of MBSP and its endogenous inhibitor with the degradation of myofribrillar proteins, resulting in the softening of muscle that lead to the conversion of muscle into meat with the control of the inhibitor. MBSP was successfully dissociated from washed myofibrils by 40 percent ethylene glycol at pH 8.5. Following centrifugation, MBSP was partially purified in two chromatographic steps, namely Toyopearl Super Q 650S and p-aminobenzamidine-Agarose. On the other hand, MBSPI was fractionated from the sarcoplasmic fraction using 75 percent ammonium sulfate saturation, followed by centrifugation and partially purified by three chromatographic steps, namely Toyopearl Super Q 650S, Superdex 200 and HiTrap SP HR. Ostrich MBSP was physicochemically and kinetically characterized, while MBSPI was only physicochemically characterized. Ostrich MBSP revealed an Mr of 21 kDa, cleaving synthetic fluorogenic substrates specifically at the carboxyl side of arginine residues. Optimum pH and temperature of ostrich MBSP were 8.0 and 40˚C, respectively. Kinetic parameters (Km and Vmax values) were calculated from Lineweaver-Burk plots. The characteristics of ostrich MBSP were compared to the values obtained for commercial bovine trypsin in this study, as well as that obtained for MBSP from various fish species and mouse. The results suggest that ostrich MBSP is a trypsin-like serine protease, thereby confirming the existence of MBSP in ostrich skeletal muscle. Partially purified ostrich MBSPI (Mr 17 kDa) (one form) shares 100 percent identity to myoglobin from the same species, while 2 other forms of MBSPI (Mr values of 35 and 36 kDa) exhibited high sequence identity to glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (76 percent) from human and rat.

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35

Thomas, Adele René. "Purification and characterisation of 20S proteasome from ostrich skeletal muscle and its role in meat tenderisation." Thesis, University of Port Elizabeth, 2004. http://hdl.handle.net/10948/320.

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The proteasome is renowned for its high molecular weight, multisubunit and mulicatalytic nature. One of its many suggested roles is the degradation of myofibrillar proteins, and therefore it has been proposed to play a role in the meat tenderisation process. The aim of this study was therefore to isolate, purify and characterise the 20S proteasome from ostrich skeletal muscle, with a view to ultimately investigating its role in the tenderisation process of ostrich meat. The 20S proteasome was successfully isolated and purified from ostrich skeletal muscle using Toyopearl Super Q-650S, Sephacryl S-300, hydroxylapatite and Mono Q chromatographies. The intact molecule showed a molecular weight of 725 K and a pI of 6.67. The subunits showed a molecular weight range of 22.2-33.5 K and a pI range of 3-9. 2D-PAGE revealed at least 14 polypeptides. The amino acid composition of the intact enzyme and of each of the eight subunits separating on SDSPAGE, as well as the N-terminal sequences of five of the eight subunits, were determined. The trypsinlike (Tr-L), chymotrypsin-like (ChT-L), peptidylglutamyl peptide hydrolase (PGPH) and caseinolytic activities showed pH optima of 11, 9, 7-8 and 10.3, and temperature optima of 40, 60, 70 and 60oC, respectively. The pH stability range for all four activities was 5-12. The ChT-L and PGPH activities showed thermostabilities up to 60oC, whereas the Tr-L and caseinolytic activities were stable up to 40o C. The enzyme showed complex kinetics. It was inhibited by the peptide aldehyde Z-LLL-CHO and cysteine protease inhibitors. Cations had negligible effects on the enzyme, excepting for Ca2+ and Mg2+. Of the detergents tested, SDS had the most potent stimulatory effect, particularly on the PGPH and caseinolytic activities. The fatty acid studies showed that unsaturation enhanced the ChT-L and the caseinolytic activities, while it completely suppressed the Tr-L activity. Heating at 60oC for 1-2 min stimulated the caseinolytic and PGPH activities. The studies on the role of ostrich skeletal muscle 20S proteasome in ostrich meat tenderisation suggested a definite but minor role of this enzyme, based on the fact that it remained active throughout the 12 days of storage of ostrich M. iliofibularis meat at 4oC and that it participated in myofibril degradation of post-mortem muscle, but to a small degree. These results support the proposal that the proteasome comes into play after the calpains have initiated degradation. However, there was a lack of improvement in tenderness values and minimal myofibrillar degradation over the 12-day storage period of the ostrich M. iliofibularis meat, leading to the conclusion that the tenderisation of this meat was incomplete after 12 days.

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36

Abuelyaman, Ahmed Salih. "Synthesis and evaluation of fluorescent and biotinylated derivatives of diphenyl peptidylphosphonate esters and biotinylated isocoumarins as inhibitors of serine proteases." Diss., Georgia Institute of Technology, 1994. http://hdl.handle.net/1853/27556.

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37

Hewin, David Fitzgerald. "An investigation of the plasminogen activator system in human oesophageal and gastric carcinoma." Thesis, University of Bristol, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326095.

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38

Lu, Stephen Hsueh-Jeng. "The molecular interactions and mechanisms of intramembrane-cleaving aspartyl proteases." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610144.

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39

Hassanein, Mohamed. "Biochemical and functional characterization of a novel placental protease, cathepsin P, in rat trophoblasts." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 148 p, 2007. http://proquest.umi.com/pqdweb?did=1654487491&sid=6&Fmt=2&clientId=8331&RQT=309&VName=PQD.

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40

Vrhovski, Bernadette. "Recombinant human tropoelastin : production, properties and interactions." Thesis, The University of Sydney, 1997. https://hdl.handle.net/2123/27631.

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41

Molawa, Letshego Gloria. "SphereZyme (TM) technology for enhanced enzyme immobilisation application in biosensors." Thesis, Rhodes University, 2011. http://hdl.handle.net/10962/d1004048.

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Self-immobilisation enzyme technologies, such as SphereZyme™, suffer from the lack of applicability to hydrolyse large substrates. Solid support immobilisation is usually a method of choice, to produce a stable biocatalyst for large substrates hydrolysis in the industry. In order to investigate this limitation, a commercial protease called Alcalase® was chosen as a model enzyme due to its natural activity (hydrolysis of large substrates-proteins). Prior to immobilising through the SphereZyme™ technology, Alcalase® was partially purified through dialysis followed by CM Sepharose™ FF cation exchanger. Sample contaminants, such as salts and stabilisers can inhibit protein crosslinking by reacting with glutaraldehyde. Alcalase® was successfully separated into 3 proteases with the major peak correlating to a positive control run on native PAGE, indicating that it was likely subtilisin Carlsberg. A 16% alkaline protease activity for azo-casein hydrolysis was retained when 5% v/v PEI: 25% v/v glutaraldehyde solution was used as a crosslinking agent in Alcalase® SphereZyme™ production. An increase in activity was also observed for monomeric substrates (PNPA) where the highest was 55%. The highest % activities maintained when 0.33 M EDA: 25% v/v glutaraldehyde solution was initially used as crosslinking agent were 4.5% and 1.6% for monomeric and polymeric substrates, respectively. PEI is a hydrophilic branched polymer with an abundance of amine groups compared to EDA. A comparison study of immobilisation efficiencies of SphereZyme™, Eupergit® and Dendrispheres was also performed for large substrate biocatalysis. The two latter technologies are solid-support immobilisation methods. Dendrispheres reached its maximum loading capacity in the first 5 minute of the one hour binding time. Twenty minutes was chosen as a maximum binding time since there was constant protein maintained on the solid support and no enzyme loss was observed during the 1 hour binding time. PEI at pH 11.5, its native pH, gave the highest immobilisation yield and specific activity over the PEI pH range of 11.5 to 7. SphereZyme™ had the highest ratio for azocasein hydrolysis followed by Dendrispheres and Eupergit®. The SphereZyme™ was also shown to be applicable to biosensors for phenol detection. Different modifications of glassy carbon electrode (GCE) were evaluated as a benchmark for the fabrication of SphereZyme™ modified phenol biosensor. GCE modified with laccase SphereZyme™ entrapped in cellulose membrane was the best modification due to the broad catechol range (<0.950 mM), high correlation coefficient (R2, 0.995) and relative high sensitivity factor (0.305 μA.mM-1). This type of biosensor was also shown to be electroactive at pH 7.0 for which its control, free laccase, lacked electroactivity. From the catalytic constants calculated, GCE modified with laccase SphereZyme™ entrapped in cellulose membrane also gave the highest effectiveness factor (Imax/Km app) of 1.84 μA.mM-1. The modified GCE with Alcalase® SphereZyme™ was relatively more sensitive than GCE modified with free Alcalase®.

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42

Bokrová, Jitka. "Příprava enkapsulovaných enzymů pro využití v kosmetice." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2014. http://www.nusl.cz/ntk/nusl-217068.

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Presented diploma thesis is focused on testing of an appropriate form of encapsulated enzymes intended for application in cosmetic and pharmaceutical industry. For encapsulation, proteolytic enzymes bromelain, papain and collagenase were used. These enzymes were encapsulated into alginate and chitosan microparticles prepared by an encapsulator and packed into liposomes. Encapsulation effectiveness was evaluated by analysis of total proteins. Particles stability was evaluated in model and real conditions by photometrical analysis of released proteins. Proteolytic activity of released enzymes in model and real conditions were observed too. Alginate and chitosan microparticles prepared by the encapsulator were found as an appropriate form of encapsulated enzymes designed to wound healing. Encapsulation effectiveness of these particles and stability in model conditions were good in comparison with liposomes. Hydrogel and water-oil emulsion were used for analysis of particles stability at real conditions. Hydrogel was found as a good option for preservation of particles as well as proteolytic enzyme activity. Emulsion made particles less stable and proteolytic activity of enzymes decreased rapidly. Encapsulation enables long-term stabilization of biologically active compounds as well as possibility of targeted transport and controlled releasing. Presented diploma thesis suggests possibilities of application encapsulated enzymes in designing more effective formulations for wound healing.

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43

Hultmann, Lisbeth. "Endogenous proteolytic enzymes - Studies of their impact on fish muscle proteins and texture." Doctoral thesis, Norwegian University of Science and Technology, Department of Biotechnology, 2004. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-178.

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This thesis covers studies on endogenous proteolytic enzymes and their impact on fish muscle proteins and texture. The studies have been performed using Atlantic salmon (Salmo salar) and cod (Gadus morhua) subjected to different treatments and storage conditions.

The textural properties were very different in the two species. Salmon fillets were significantly softer and less resilient than cod fillets, and the properties changed somewhat differently during storage experiments. Different proteolytic enzymes have been reported to participate in muscle softening. Some of these enzymes were investigated, and specific proteolytic activities were detected throughout the storage periods. Collagenase-like enzymes seem to be the most important for cod muscle texture. Microorganisms and/or microbial enzymes seem not to be important for changes in salmon muscle texture. Results suggest that the cathepsin B-like enzymes are important for salmon texture. The activities of the proteolytic enzymes may be greatly affected by the muscle pH, and by the treatment(s) the fish are subjected to. In any case, changes caused by differences in proteolytic activities may need some time to be detectable or have significant impact on fish quality.

When cod fillets are stored in ice, it is highly recommended to keep the temperature low. Even a relatively mild temperature abuse was sufficient to result in less favorable textural characteristics, and make the fillets seem older than their days of storage.

Salmon fillets are often subjected to cold-smoking. The smoking temperature was important for the solubility properties of the muscle proteins, and for their composition, but did not affect the proteolytic activity. The effects of the processing parameters were most important early in the product’s shelf life, as the differences caused by the different smoking temperatures were reduced by further storage of the smoked samples.

Paper II and III are reprinted with kind permission of Elsevier, sciencedirect.com

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44

Anderson, Paulette S. (Paulette Sue) 1952. "Exoprotease Production by Aeromonas hydrophila in a Chemically Defined Medium." Thesis, North Texas State University, 1985. https://digital.library.unt.edu/ark:/67531/metadc504602/.

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Wretlind, Heden, and Wadstrom found ammonium sulfate to be inhibitory for the formation of extracellular protease in Aeromonas hydrophila grown in Brain Heart Infusion medium. They demonstrated by manipulating the iron and zinc content within their medium that it is possible to differentially affect the accumulation of hemolysin and protease by A. hydrophila grown in batch culture. Further manipulation of the composition of this medium was done in the present study to determine the effect of other components on the production of protease. The purpose of this study was to determine the factors affecting the level of A. hydrophila protease produced in a chemically defined medium.

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45

Tong, Raymond Cheuk Wa. "Topological analysis of the transhydrogenase in Escherichia coli membranes using proteolytic probes." Thesis, University of British Columbia, 1991. http://hdl.handle.net/2429/30401.

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Using proteolytic probes, the pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was analyzed for its native topography in the cytoplasmic membrane. Before analyses could be performed, the isolation of transhydrogenase-enriched ISO (inside-out) cytoplasmic membrane vesicles was accomplished by modification of the procedure followed by Clarke (Clarke, D. M. and Bragg, P. D. (1985) Eur. J. Biochem. 149, 517-523) in purifying the enzyme from overexpressing E.coli JM83pDC21 cells. Two major changes were made. One was that the solubilization of the bacterial membrane and subsequent purification steps were omitted. The other was the separation of outer membranes from the cytoplasmic membrane preparation by sucrose gradient density centrifugation. This was essential owing to the contaminating presence of a 30 kD protein in the outer membrane of the original preparation. Transhydrogenase-enriched RSO (right-side-out) membrane vesicles were isolated by a different procedure using lysozyme-mediated breakage of E.coli spheroplasts and subsequent vesicular reformation. To identify possible transhydrogenase fragments arising from proteolytic cleavage, anti-E.coli transhydrogenase polyclonal antibodies were generated in rabbits. Two sets of polyclonal antibodies were produced. One set cross-reacted with both the α (52 kD) and β (48 kD) subunits of the transhydrogenase. The other reacted with the α subunit only. Trypsin and proteinase K were the main proteolytic probes used against both ISO and RSO cytoplasmic membrane vesicles, although chymotrypsin was also used in preliminary experiments with ISO membrane vesicles. Identification of fragments resulting from proteolytic cleavage of the enzyme was obtained using anti-transhydrogenase antibodies and by N-terminal sequencing and/or C-terminal sequencing. In some of these experiments, isolation of the proteolytic fragments was necessary prior to analysis. This was done using a number of different methods. The particular methods applied, which included column chromatography strategies and elution procedures from SDS-Polyacrylamide gels, depended on the type of analysis carried out. The analyses indicated that the α subunit has at least a 41 kD sequence extending from its N-terminus which is exposed to the cytoplasmic side of the membrane. This sequence may contain an active site of the enzyme. This is suggested by the binding of this fragment to a NAD-affinity column. The membrane-imbedded region of the α subunit anchoring the 41 kD region predicted by hydropathy plotting (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) could not be detected by our methods. Susceptible tryptic cleavage sites along the 41 kD region were identified by partial proteolysis and may reflect areas in the subunit's tertiary or quaternary structure that are exposed to the surrounding medium. Major cleavage sites were at arg₁₅, Iys₂₂₇, Iys₂₆₄, arg₂₆₈, Iys₂₇₅, arg₃₅₅, and arg₃₆₁. There do not appear to be significant portions of the subunit protruding into the periplasm as neither trypsin nor proteinase K had any effect on the subunit in RSO-oriented membrane vesicles. Proteinase K experiments with ISO and RSO membrane vesicles suggest that a 20 kD portion of the β subunit is protected from cleavage and is imbedded in the membrane. The identity of this fragment could not be confirmed. Hydropathy analysis of the transhydrogenase gene-derived amino acid sequence (Clarke, D. M., Loo, Tip W., Gilliam, S. and Bragg, P. D. (1986), Eur. J. Biochem. 158, 647-653) suggests that this could be a sequence extending from the N-terminus of the β subunit. This is a hydrophobic sequence containing 7 possible transmembranous helices and having a theoretical molecular weight in the range of 20 kD. The proteinase K results also indicate that the rest of the β subunit is exposed to the cytoplasmic side of themembrane rather than the periplasmic side. The results obtained here are consistent with hydropathy predictions made with regard to this subunit. In addition, two different experiments indicate that an α-α subunit interaction may be present in the oligomeric structure of the membrane-bound enzyme (Hou, C, Potier, M. and Bragg, P. D. (1990), Biochim. Biophys. Acta 1018, 61-66). Substrates of the enzyme did not appear to affect the transhydrogenase's general conformation upon binding as detected by experiments using partial tryptic proteolysis. Partial trypsinolysis also revealed that selective detergent extraction of transhydrogenase-enriched ISO vesicles with Triton X-100 and sodium cholate did not affect the overall conformation of the membrane-bound enzyme despite greatly reducing the enzymatic activity.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

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46

Al-Omirah, Husam F. "Proteolytic degradation products as indicators of quality in meat and fish." Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=27268.

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Assessment of freshness and quality of meat and fish is a major activity of both food regulatory agencies and the food industry. Various methods are used for measuring fish and meat quality, each with its particular advantages and limitations. However, methods based on monitoring the products of proteolysis have received relatively little attention. The objective of the present study was to identify specific protein and peptide products of proteolysis as indicators of freshness and quality during chilled storage of fresh fish and meat.
Samples of meat and fish were subjected to chilled storage; at intervals of 0, 2, 4, 8, 12 and 16 days, samples were subjected to protein and peptide extraction, and separation of individual sarcoplasmic and myofibrillar proteins by SDS and native electrophoresis. These extracted proteins along with acid soluble nitrogen (ASN) were separated by RP-HPLC, fractions were collected and identified by electrospray ionization mass spectrometry (ESI-MS).
RP-HPLC separated at least thirty fractions from the ASN extract of fresh fish. ESI-MS revealed the presence of at least twenty-five polypeptides with molecular weights (MW) ranging from 2 to 32 kDa. The relative area % of the polypeptides with MW 32.8 kDa and 42.8 kDa decreased during the storage while polypeptides of MW of 10.9 kDa and 16.7 kDa increased during storage. Changes in polypeptides of MW 12, 34.2 and 42.8 kDa was also observed. The sarcoplasmic protein extracted from ground and whole meat contained at least 12 polypeptides with MW ranging from 11 to 42 kDa. The relative area % of polypeptide of MW of 35.7 kDa decreased during storage. The results suggest that changes in proteins and polypeptides of MW 10.9, 12, 16.7, 32.8, 34.2 and 42.88 kDa in fish and 35.7 kDa in meat could serve as indicators of spoilage.

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47

Wang, Hsin Jayaswal Radheshyam K. Wilkinson Brian J. "Molecular analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity." Normal, Ill. Illinois State University, 1991. http://wwwlib.umi.com/cr/ilstu/fullcit?p9219089.

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Thesis (Ph. D.)--Illinois State University, 1991.
Title from title page screen, viewed January 5, 2006. Dissertation Committee: Radheshyam K. Jayaswal, Brian J. Wilkinson (co-chairs), Herman E. Brockman, Anthony J. Otsuka, Hou Tak Cheung. Includes bibliographical references (leaves 117-129) and abstract. Also available in print.

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48

Arteaga, Mac Kinney Guillermo Eleazar. "Protection of the proteolytic activity of crude papain and chemical modification of papain by tetrathionate." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/27792.

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In the first chapter, sodium tetrathionate (TT), a sulfhydryl blocking agent, is assessed for its ability to protect the proteolytic activity (PA) of papaya latex during air, sun or vacuum drying, and of crude papain during storage. By means of Taguchi's L₂₇ (3¹³) fractional factorial design, it was found that the addition of 1% TT significantly increased the retention of PA of papaya latex when it was air dried at a temperature of 55°C. This protection of PA was found to be 23% higher than the one given by the addition of 1% sodium metabisulfite, the compound commonly used in the commercial processing of papaya latex. When drying was carried out either under 27 inches vacuum at 50°C or in the sun, the protective effect of TT on the PA was not significantly different from that of metabisulfite. The PA of crude papain during storage at room temperature was also protected by TT. A loss of 20% of the original PA occurred over a period of 13 wk when crude papain contained 1% TT, compared to a loss of 45% when the crude enzyme preparation contained 1% metabisulfite. In the same chapter five different oxidants for synthesis of TT from thiosulfate are compared, namely: iodine, hydrogen peroxide, ferric chloride, cupric sulfate and sodium vanadate. The results indicated that hydrogen peroxide or sodium vanadate were not only effective in the oxidation but also much less expensive than iodine, which is the most popular oxidant for the synthesis of TT. The results obtained in this chapter warrant the use of TT in the commercial production of commercial papain to prevent the destruction of the enzymes during harvesting, storage, transportation and processing. In the second chapter, chemical modification of pure papain by TT is discussed. Optimization techniques were applied for improving the precision of two methods used in this study: circular dichroism (CD) and proteolytic activity determination. Simplex optimization significantly improved repeatability and signal to noise ratio of the CD scan of papain. A new optimization approach, which was a combination of a central composite rotatable design and simplex optimization, was successfully applied to achieve maximum precision for the proteolytic activity assay of papain using casein as a substrate. This approach may also be applied to other analytical methods to improve the reliability of the experimental data. Influential factors in the inactivation of PA of papain by using TT and reactivation of the inactivated papain by cysteine were carried out using two Taguchi's L₁₆ (2¹⁵) fractional factorial designs. The results indicated that when inactivation was carried out at pH 6.8, with a reaction time of 5 min at 22°C, and a molar ratio of TT to papain of 10, the inactivation reaction was highly reversible upon addition of 20 mM cysteine. Although some interactions of the factors were significant, 70% reactivation was achieved in most cases. Analysis of UV absorbance, near-UV and far-UV CD spectra indicated that there were no major changes in the spectra in papain upon the chemical modification of the enzyme with TT. Secondary structure computed from far-UV CD spectra also demonstrated no significant changes upon this modification. Sulfhydryl data and pH-fluorescence profiles of the modified papain support the hypothesis that reversible blocking by TT results from binding with the single reactive cysteine residue present in papain. Quenching of the intrinsic fluorescence of papain when the modification was carried out using high molar ratios of TT to papain was suggestive of modification of tryptophan residues in the enzyme during the oxidation reaction with TT. Precipitation or insolubilization of pure papain, and of the proteins of papaya latex and commercial papain was observed upon the chemical modification with TT under certain conditions. Addition of β-mercaptoethanol and TT at levels of 100 mM and 50 mM, respectively, precipitated 90% of pure papain. Solubility studies together with electrophoretic analysis of the precipitated papain suggested formation of insoluble aggregates due to the insoluble aggregation as a result of inter-molecular disulfide bonds formation. TT was found to be a competitive inhibitor of both reversible and irreversible inhibition of the enzyme action, when carbobenzoxyglycine p-nitrophenyl ester was used as a substrate. The second order inactivation constant in the absence of substrate was computed to be 16,919 M⁻¹sec⁻¹, indicating that the reaction had a high rate.
Land and Food Systems, Faculty of
Graduate

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49

Boehringer, Jonas. "Substrate recognition by the proteasome." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.669968.

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The ubiquitin proteasome system targets proteins to the proteasome where they are degraded. Substrate recognition and processing prior to degradation take place at the 19S regulatory particle of the proteasome. A polyubiquitin chain, linked through isopeptide bonds formed between the C-terminal G76 and K48, is the signal responsible for delivery to the proteasome. Because chains linked via any of the seven lysine residues of ubiquitin exist in vivo and encode signals unrelated to protein degradation it is crucial for cells to avoid crosstalk between these different pathways. Several ubiquitin receptors related to proteasomal degradation have been identified but the selectivity between the different ubiquitin chains has not been assessed quantitatively while avoiding artefacts attributed to GST-dimerisation. By employing isothermal titration calorimetry, analytical ultracentrifugation and nuclear magnetic resonance, discrimination between K48- and K63-linked diubiquitin was established for the S. pombe proteasomal receptor Rpn10 and the shuttle protein Rhp23. The same methods allowed us to propose a discriminatory model for Rpn10. The crystal structures of the 19S regulatory particle subunits Rpn101-193 and Rpn121-224 have been determined and possible protein-protein interaction sites were identified by surface conservation and electrostatics analysis. Rpn12 surface residues were identified that had a negative effect on Rpn10-binding. This interaction was studied by surface plasmon resonance, fluorescence anisotropy and nuclear magnetic resonance. These experiments revealed a binding site on Rpn10 that is exclusively occupied by either ubiquitin or Rpn12 and for the first time demonstrated the interaction of a ubiquitin interacting motif with a protein other than ubiquitin.

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50

Dennison, Kelly J. "Development of a structural model of human T-cell leukemia virus type-I protease." Thesis, Georgia Institute of Technology, 2002. http://hdl.handle.net/1853/30060.

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Dissertations / Theses on the topic 'Proteolytic enzymes'

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