An Analysis of the Proteolytic System Inaspergillus in Order to Improve

Total Page:16

File Type:pdf, Size:1020Kb

An Analysis of the Proteolytic System Inaspergillus in Order to Improve An analysis of theproteolyti c system inAspergillus in order to improve protein production Promotor: dr. ir. J.A.M. de Bont Hoogleraar in de Industriele Microbiologic Co-promotor: dr. ir. J. Visser Universitair hoofddocent sectie Moleculaire Genetica van Industriele Microorganismen /ucl)08?o 0^ Johannes Petrus Theodorus Wilhelmus van den Hombergh An analysis of theproteolyti c system in Aspergillus in order to improve protein production Proefschrift ter verkrijging van de graad van doctor in de Landbouw- en Milieuwetenschappen op gezag van de rector magnificus dr. CM. Karssen in het openbaar te verdedigen op dinsdag 11jun i 1996 des morgens om 11uu r in de Aula van de Landbouwuniversiteit te Wageningen r Sr\0[£>n~>C\\v The research described in this thesis was financially supported by Ciba Geigy AG (Basel, Switzerland) and by the European Community in the framework of BRIDGE (BIOTCT- 90169) and BIOTECH (BIO-CT9301974), and was performed in the labs of the section Molecular Genetics of Industrial Microorganisms at Wageningen Agricultural University BIBIJOTIU^K' lANDBOUWUNiVK.'s: V. An analysis of the proteolytic system in Aspergillus in order to improve protein producti­ on / Johannes P.T.W. van den Hombergh. - [S.I.: s.n.] Thesis Landbouwuniversiteit Wageningen. - With ref. - With Summary in Dutch. ISBN 90-5485-545-2 Subject headings: Aspergillus niger I proteolytic degradation / protease regulation Cover: Annemaria Bruinslot Gerrit Rietveld Akademie, Amsterdam , pi i0a2o'12 °^ Stellingen 1. Er wordt bij het optimaliseren van eiwitproduktie inAspergillus stammen ten onrechte te weinig aandacht besteed aan de mate waarin een dergelijke stam in staat is tot verzuring van het cultuurmedium in relatie tot proteolyse. 2. De door Caddick (1994) beschreven indeling van mutaties in het bij de ammonium metabolietrepressie betrokken areA gen in Aspergillus is arbitrair omdat een dergelijke indeling in sterke mate afhankelijk is van de keuze van de targetgenen die hiervoor worden gebruikt. Caddick (1994) Progress in Industrial Microbiol. 29:347-353 3. Bij het tijdens autoregulatie negatief moduleren van de functionaliteit van het PacC eiwit is deC-terminal e QED repeat niet betrokken. 4. De extracellulaire serinecarboxypeptidases in Aspergillus niger hebben mogelijk een functie bij de autokatalytisch gei'nduceerde maturatie van het extracellulaire pepstatine-rembare zure protease PEPA. Inouee tal . (1996)Eur .J .Biochem . 237:719-725 5. Bij het verklaren van de verschillen in opbrengsten tussen homologe en heterologe eiwitproduktie in filamenteuze schimmels wordt de vaak afwijkende aminozuursamenstelling van heterologe eiwitten en de invloed van de veranderde aminozuurbehoefte veelal over het hoofd gezien. 6. Omdat de door Tilburn et al. (1995) waargenomen pH regulatie van hetprtA gen in Aspergillus nidulans door Katz et al. (1996) niet gereproduceerd kan worden is het niet uitgsloten dat de xprE mutaties en xpr¥,G suppressoren in Aspergillus nidulans betrokken zijn bij pH regulatie. Tilburn et al. (1995)EMB O J. 14:779-790; Katze t al. (1996)Mol .Gen .Genet .250:715-72 4 7. Niet-functionerende gemeentesecretarissen, procureurs generaal, ABP functionarissen en besturen van toezichthoudende organen blijken bij niet functioneren alleen vergezeld van forse afkooppremies (de gouden handdruk) te kunnen vertrekken. Opmerkelijk hierbij is verder dat de hierover verontwaardigde Tweede-Kamerleden zelf bij vertrek gebruik maken van een zogenaamde 'aanvulling op hun nieuwe inkomen'. 8. Het voorgenomen teruggeven aan denatuu r van grote stukken cultuurgrond in deprovinci e Zeeland vereist een aanpassing van de Zeeuwse lijfspreuk in 'Luctor et Submergo'. 9. De vertaling van het Latijnse woord doctorandus suggereert ten onrechte dat een ingenieur niet zou kunnen promoveren. 10. Het beursale AlO systeem is een stap in de richting van het creeren van een wetenschapsproletariaat. 11. Bij het uit aerodynamisch oogpunt inklapbaar maken van de verlichting van bepaalde sportauto's hebben de constructeurs over het hoofd gezien dat de wetten van de aerodynamica ook 's nachts van toepassing zijn. 12. Het aanbieden van walkman-vrije coupes zal meer mensen overhalen tot het gebruik van de trein dan de uitgebreide 'We gaan ervoor' reclamecampagnes. Stellingen behorende bij het proefschrift An analysis of the proteolytic system in Aspergillus in order to improve protein production Wageningen, 11jun i 1996 J.P.T.W. van den Hombergh aan Caroline en mijn ouders Contents Chapter 1 General introduction; fungal proteases, structure, characteristics, 1 function and regulation Chapter 2 Aim and outline of the thesis 71 Chapter 3 Expression of an Erwinia pectate lyase in three species of Asper- Ti gillus Chapter 4 Cloning and characterization of the pepE gene of Aspergillus 89 niger encoding a new aspartic protease and regulation of the pepE and pepC genes Chapter 5 Cloning, characterization and expression ofpepF, a gene encoding 107 a serine carboxypeptidase from Aspergillus niger Chapter 6 Residual protease spectra in Aspergillus niger prt mutants contain 123 elevated metallo protease activities: identification, cloning, charac­ terization and expression ofpepH, a novel metallo protease Chapter 7 Disruption of three acid proteases in Aspergillus niger: effects on 137 residual protease spectra and degradation of target proteins Chapter 8 New protease mutants in Aspergillus niger result in strongly 157 reduced in vitro degradation of target proteins: genetic and bio­ chemical characterization of seven complementation groups Chapter 9 Production of the homologous pectin lyase B protein in six diffe- 175 rent genetically defined protease deficient Aspergillus niger mutant strains Chapter 10 Identification, cloning and analysis of the Aspergillus niger pacC 193 wide domain pH regulatory gene Chapter 11 Regulation of acid phosphatases in an Aspergillus niger paclQ" 209 disruption strain Chapter 12 Complex regulation of extracellular proteases in Aspergillus niger: 229 an analysis of wide domain regulatory mutants demonstrates CREA, AREA and PACC mediated control Chapter 13 Reduced degradation of Erwinia pectate lyase expressed in Asper- 251 gillus through modulation of pH regulation and cloning of pep A, an Aspergillopepsin from Aspergillus nidulans Chapter 14 Identification, cloning and analyses ofpepl and pep], two metallo 269 proteases from Aspergillus nidulans: indications for an unique acid metallo protease class in filamentous fungi Chapter 15 General discussion; towards further improving protein production 287 Summary 297 Samenvatting (Nederlands) 301 Curriculum vitae 305 List of publications 307 Nawoord 309 -| General Introduction; fungal proteases, structure, characteristics, function and regulation1 J.P.T.W. van den Hombergh and J. Visser 1.1 Introduction Traditionally the proteases have been regarded as degradative enzymes, capable of cleaving proteins into small peptides and/or amino acids, and whose role it ist o digest nutrient protein or to participate in the turnover of cellular proteins. However, although the best characterized proteases have this elementary function, it has also been shown that proteases play key roles in a wide range of cellular processes, via mechanisms of selectivemodificatio n by limited proteolysis,an d thusca nhav e essential regulatory functions (Holzer and Tschensche 1979; Holzer and Heinrich, 1980). There is a number of reasons why proteases of filamentous fungi are of particular interest. The basic process of hydrolytic cleavage of peptide bonds in proteins appears costly and potentially detrimental to an organism if not properly controlled. The desired limits to proteolytic action are achieved through the specificity of proteinases, by compartmentalization of proteases and substrates within the cell, through modification of the substrates allowing recognition by the respective proteases, by regulation via zymogen activation, and the presence or absence of specific inhibitors, and of course also by regulation of protease gene expression. Studying proteases in fungi will help to understand fundamental cellular functions involving proteolysis, including intracellular protein turnover, processing, translocation, sporulation, germination and differentiation. Filamentous fungi provide interesting possibilities to study proteases as it is relatively easy to generate mutants, and powerful genetical, biochemical and molecular biological techniquesar e availablefo r analysis. In fact, Aspergillus nidulans and Neurospora crassaar e used asmode l organisms for analyzing the molecular basis of a range of physiological and developmental processes. Their well-developed genetics enable direct access tobiochemica l and genetical studies,unde r defined nutritionalan d cultivationconditions . Furthermore, a large group of fungi pathogenic to humans, live-stock and crop, have been isolated and proteolysis could play a role in their pathogenicity (host penetration, countering host defense mechanisms and/or nutrition during infection). Proteases are also frequently used in laboratory and clinical practice and in industrial processes; proteases both microbial and non-microbial are in wide­ spread use in the food industry (baking, brewing, cheese manufacturing, meat tenderizing), in the ' Accepted for publication in Applied Molecular and Cellular Biology of Filamentous Fungi. S. Brown and R.M. Berka (Eds.) 2 Chapter 1 tanning industry and in the manufacture of biological detergents (Aunstrup, 1980). The commercial interest in exploiting certain filamentous
Recommended publications
  • Microbial Enzymes and Their Applications in Industries and Medicine
    BioMed Research International Microbial Enzymes and Their Applications in Industries and Medicine Guest Editors: Periasamy Anbu, Subash C. B. Gopinath, Arzu Coleri Cihan, and Bidur Prasad Chaulagain Microbial Enzymes and Their Applications in Industries and Medicine BioMed Research International Microbial Enzymes and Their Applications in Industries and Medicine Guest Editors: Periasamy Anbu, Subash C. B. Gopinath, Arzu Coleri Cihan, and Bidur Prasad Chaulagain Copyright © 2013 Hindawi Publishing Corporation. All rights reserved. This is a special issue published in “BioMed Research International.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contents Microbial Enzymes and Their Applications in Industries and Medicine,PeriasamyAnbu, Subash C. B. Gopinath, Arzu Coleri Cihan, and Bidur Prasad Chaulagain Volume 2013, Article ID 204014, 2 pages Effect of C/N Ratio and Media Optimization through Response Surface Methodology on Simultaneous Productions of Intra- and Extracellular Inulinase and Invertase from Aspergillus niger ATCC 20611, Mojdeh Dinarvand, Malahat Rezaee, Malihe Masomian, Seyed Davoud Jazayeri, Mohsen Zareian, Sahar Abbasi, and Arbakariya B. Ariff Volume 2013, Article ID 508968, 13 pages A Broader View: Microbial Enzymes and Their Relevance in Industries, Medicine, and Beyond, Neelam Gurung, Sumanta Ray, Sutapa Bose, and Vivek Rai Volume 2013, Article
    [Show full text]
  • Amyloid-Degrading Ability of Nattokinase from Bacillus Subtilis Natto
    J. Agric. Food Chem. 2009, 57, 503–508 503 Amyloid-Degrading Ability of Nattokinase from Bacillus subtilis Natto †,‡ § § † RUEI-LIN HSU, KUNG-TA LEE, JUNG-HAO WANG, LILY Y.-L. LEE, AND ,†,‡ RITA P.-Y. CHEN* Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, R. O. C., Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan, R. O. C., and Department of Biochemical Science and Technology, National Taiwan University, Taipei 106, Taiwan, R. O. C. More than 20 unrelated proteins can form amyloid fibrils in vivo which are related to various diseases, such as Alzheimer’s disease, prion disease, and systematic amyloidosis. Amyloid fibrils are an ordered protein aggregate with a lamellar cross- structure. Enhancing amyloid clearance is one of the targets of the therapy of these amyloid-related diseases. Although there is debate on whether the toxicity is due to amyloids or their precursors, research on the degradation of amyloids may help prevent or alleviate these diseases. In this study, we explored the amyloid-degrading ability of nattokinase, a fibrinolytic subtilisin-like serine protease, and determined the optimal conditions for amyloid hydrolysis. This ability is shared by proteinase K and subtilisin Carlsberg, but not by trypsin or plasmin. KEYWORDS: Nattokinase; amyloid; natto; subtilisin NAT; amyloid degradation; fibril; amyloidosis INTRODUCTION of cardiovascular disease. Dietary supplementation with natto Natto, a fermented food made from boiled soybeans, has suppresses the intimal thickening of arteries and leads to the been eaten for more than 1000 years in Asia. The fermenta- lysis of mural thrombi seen after endothelial injury (12). tion microbe isolated from natto is the Gram-positive Other clinically thrombolytic agents, such as urokinase and endospore-forming bacterium Bacillus subtilis natto (formerly streptokinase, are costly and unstable in the intestinal tract designated Bacillus natto)(1).
    [Show full text]
  • 1 Evidence for Gliadin Antibodies As Causative Agents in Schizophrenia
    1 Evidence for gliadin antibodies as causative agents in schizophrenia. C.J.Carter PolygenicPathways, 20 Upper Maze Hill, Saint-Leonard’s on Sea, East Sussex, TN37 0LG [email protected] Tel: 0044 (0)1424 422201 I have no fax Abstract Antibodies to gliadin, a component of gluten, have frequently been reported in schizophrenia patients, and in some cases remission has been noted following the instigation of a gluten free diet. Gliadin is a highly immunogenic protein, and B cell epitopes along its entire immunogenic length are homologous to the products of numerous proteins relevant to schizophrenia (p = 0.012 to 3e-25). These include members of the DISC1 interactome, of glutamate, dopamine and neuregulin signalling networks, and of pathways involved in plasticity, dendritic growth or myelination. Antibodies to gliadin are likely to cross react with these key proteins, as has already been observed with synapsin 1 and calreticulin. Gliadin may thus be a causative agent in schizophrenia, under certain genetic and immunological conditions, producing its effects via antibody mediated knockdown of multiple proteins relevant to the disease process. Because of such homology, an autoimmune response may be sustained by the human antigens that resemble gliadin itself, a scenario supported by many reports of immune activation both in the brain and in lymphocytes in schizophrenia. Gluten free diets and removal of such antibodies may be of therapeutic benefit in certain cases of schizophrenia. 2 Introduction A number of studies from China, Norway, and the USA have reported the presence of gliadin antibodies in schizophrenia 1-5. Gliadin is a component of gluten, intolerance to which is implicated in coeliac disease 6.
    [Show full text]
  • Identification and Phylogenetic Analysis of Keratinase Producing Bacteria SNP1 from Poultry Field
    International Journal of Biotechnology and Biochemistry ISSN 0973-2691 Volume 15, Number 1 (2019) pp. 39-51 © Research India Publications http://www.ripublication.com Identification and Phylogenetic Analysis of Keratinase Producing Bacteria SNP1 from Poultry Field Divya Balakrishnan and Nithadas Sathyadas Padmanabhan Department of Biotechnology, Sree Narayana College, Kollam-691001, Kerala, India. (*Corresponding author) Abstract The study was conduct to select the best promising keratinolytic bacterial strain. A keratinase positive bacterial strain was isolated from the soil samples of poultry field, Attingal, Thiruvananthapuram. Each sample was plated on skim milk agar and feather meal agar plates containing 5 g feather. The well grown isolates which produced the largest clear zone on skimmed milk plate were selected for keratinase assays. Out of 26 bacterial isolates, 7 isolates were selected. Among the selected strain, the best keratinase producing bacterium SNP1was selected for further analysis. The SNP1 potential strain was later confirmed as Bacillus haynessi based on molecular and phylogenetic analysis. The medium components and culture conditions were optimized to enhance keratinase production through optimization. Keratin and feather powder (10g/l) were identified as good substrates for the highest keratinase production. The strain SNP1 resulted maximum enzyme production at 96h of incubation at 37°C and pH 8 under 120 rpm. Therefore, Bacillus hayneisi might be used for large scale production of keratinase for industrial purposes. Keywords: Keratinase, Bacillus sp., Optimization, Enzyme activity, 16SrRNA Keratin is a hard-degrading fibrous and recalcitrant structural protein, which forms the third most abundant polymer in nature after cellulose and chitin (Lene et al ., 2016). In the native state, the feather keratin resists the degradation by proteolytic enzymes such as trypsin and pepsin due to tightpeptide-chains held together by disulfide bridges by means of cysteine residues (Weeranut, 2017; Korniłłowicz-Kowalska and Bohacz, 2011).
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002
    USOO6395889B1 (12) United States Patent (10) Patent No.: US 6,395,889 B1 Robison (45) Date of Patent: May 28, 2002 (54) NUCLEIC ACID MOLECULES ENCODING WO WO-98/56804 A1 * 12/1998 ........... CO7H/21/02 HUMAN PROTEASE HOMOLOGS WO WO-99/0785.0 A1 * 2/1999 ... C12N/15/12 WO WO-99/37660 A1 * 7/1999 ........... CO7H/21/04 (75) Inventor: fish E. Robison, Wilmington, MA OTHER PUBLICATIONS Vazquez, F., et al., 1999, “METH-1, a human ortholog of (73) Assignee: Millennium Pharmaceuticals, Inc., ADAMTS-1, and METH-2 are members of a new family of Cambridge, MA (US) proteins with angio-inhibitory activity', The Journal of c: - 0 Biological Chemistry, vol. 274, No. 33, pp. 23349–23357.* (*) Notice: Subject to any disclaimer, the term of this Descriptors of Protease Classes in Prosite and Pfam Data patent is extended or adjusted under 35 bases. U.S.C. 154(b) by 0 days. * cited by examiner (21) Appl. No.: 09/392, 184 Primary Examiner Ponnathapu Achutamurthy (22) Filed: Sep. 9, 1999 ASSistant Examiner William W. Moore (51) Int. Cl." C12N 15/57; C12N 15/12; (74) Attorney, Agent, or Firm-Alston & Bird LLP C12N 9/64; C12N 15/79 (57) ABSTRACT (52) U.S. Cl. .................... 536/23.2; 536/23.5; 435/69.1; 435/252.3; 435/320.1 The invention relates to polynucleotides encoding newly (58) Field of Search ............................... 536,232,235. identified protease homologs. The invention also relates to 435/6, 226, 69.1, 252.3 the proteases. The invention further relates to methods using s s s/ - - -us the protease polypeptides and polynucleotides as a target for (56) References Cited diagnosis and treatment in protease-mediated disorders.
    [Show full text]
  • In Escherichia Coli (Synthetic Oligonucleotide/Gene Expression/Industrial Enzyme) J
    Proc. Nati Acad. Sci. USA Vol. 80, pp. 3671-3675, June 1983 Biochemistry Synthesis of calf prochymosin (prorennin) in Escherichia coli (synthetic oligonucleotide/gene expression/industrial enzyme) J. S. EMTAGE*, S. ANGALt, M. T. DOELt, T. J. R. HARRISt, B. JENKINS*, G. LILLEYt, AND P. A. LOWEt Departments of *Molecular Genetics, tMolecular Biology, and tFermentation Development, Celltech Limited, 250 Bath Road, Slough SL1 4DY, Berkshire, United Kingdom Communicated by Sydney Brenner, March 23, 1983 ABSTRACT A gene for calf prochymosin (prorennin) has been maturation conditions and remained insoluble on neutraliza- reconstructed from chemically synthesized oligodeoxyribonucleo- tion, it was possible that purification as well as activation could tides and cloned DNA copies of preprochymosin mRNA. This gene be achieved. has been inserted into a bacterial expression plasmid containing We describe here the construction of E. coli plasmids de- the Escherichia coli tryptophan promoter and a bacterial ribo- signed to express the prochymosin gene from the trp promoter some binding site. Induction oftranscription from the tryptophan and the isolation and conversion of this prochymosin to en- promoter results in prochymosin synthesis at a level of up to 5% active of total protein. The enzyme has been purified from bacteria by zymatically chymosin. extraction with urea and chromatography on DEAE-celiulose and MATERIALS AND METHODS converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in Materials. DNase I, pepstatin A, and phenylmethylsulfonyl clotting milk as the natural enzyme isolated from calf stomach. fluoride were obtained from Sigma. Calf prochymosin (Mr 40,431) and chymosin (Mr 35,612) were purified from stomachs Chymosin (rennin) is an aspartyl proteinase found in the fourth from 1-day-old calves (1).
    [Show full text]
  • Molecular Markers of Serine Protease Evolution
    The EMBO Journal Vol. 20 No. 12 pp. 3036±3045, 2001 Molecular markers of serine protease evolution Maxwell M.Krem and Enrico Di Cera1 ment and specialization of the catalytic architecture should correspond to signi®cant evolutionary transitions in the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, St Louis, history of protease clans. Evolutionary markers encoun- MO 63110-1093, USA tered in the sequences contributing to the catalytic apparatus would thus give an account of the history of 1Corresponding author e-mail: [email protected] an enzyme family or clan and provide for comparative analysis with other families and clans. Therefore, the use The evolutionary history of serine proteases can be of sequence markers associated with active site structure accounted for by highly conserved amino acids that generates a model for protease evolution with broad form crucial structural and chemical elements of applicability and potential for extension to other classes of the catalytic apparatus. These residues display non- enzymes. random dichotomies in either amino acid choice or The ®rst report of a sequence marker associated with serine codon usage and serve as discrete markers for active site chemistry was the observation that both AGY tracking changes in the active site environment and and TCN codons were used to encode active site serines in supporting structures. These markers categorize a variety of enzyme families (Brenner, 1988). Since serine proteases of the chymotrypsin-like, subtilisin- AGY®TCN interconversion is an uncommon event, it like and a/b-hydrolase fold clans according to phylo- was reasoned that enzymes within the same family genetic lineages, and indicate the relative ages and utilizing different active site codons belonged to different order of appearance of those lineages.
    [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]
  • Cloning and in Vitro-Transcription of Chymosin Gene in E. Coli
    The Open Nutraceuticals Journal, 2010, 3, 63-68 63 Open Access Cloning and In Vitro-Transcription of Chymosin Gene in E. coli S.A. El-Sohaimy1 Elsayed. E, Hafez2,* and M.A. El-Saadani3 1Food Science and Technology Department, Arid land Research Institute, Alexandria, Egypt 2Plant Molecular Pathology Department, Arid land Research Institute, Alexandria, Egypt 3Mubarak City for Scientific Research and Technology Applications, Alexandria, Egypt Abstract: Chymosin, commonly known as rennin, is the main milk-coagulating enzyme available in rennet. RNA was extracted from the abomasum of a suckling calf water buffalo and was subjected to RT-PCR using degenerate primers to amplify 850bp of the chymosin gene. The sequence was aligned with 19 different mammals' chymosin genes. The sequence revealed that there is a similarity to them ranging from 64% to 98%. The purified recombinant proteins were obtained from the transformed E. coli and yeast. The clotting activity of both of the resulting proteins was examined compared to the commercial peers. It was noticed that the concentration of the purified protein ranged from 15,000 to 40,000 MCU. Therefore, the activity of the obtained proteins was the same and it was 105% when compared to the commercial peer. Having examined the cytotoxicity of the purified proteins, the results revealed no toxicity. We can conclude that the obtained recombinant protein is more active and safe even when expressed in bacteria rather than yeast. Keywords: Chymosin, Milk Clotting, Recombinant E. coli and Recombinant Protein. INTRODUCTION Many microorganisms are known to produce rennet-like proteinases which can replace the calf rennet.
    [Show full text]
  • Carboxypeptidase N (Kininase I) (Kdnins/Anaphylatoxins/Kallikrein/Proteases/Carboxypeptidase B) YEHUDA LEVIN*T, RANDAL A
    Proc. NatL Acad. Sci. USA Vol. 79, pp. 4618-4622, August 1982 Biochemistry Isolation and characterization of the subunits of human plasma carboxypeptidase N (kininase I) (kdnins/anaphylatoxins/kallikrein/proteases/carboxypeptidase B) YEHUDA LEVIN*t, RANDAL A. SKIDGEL*, AND ERVIN G. ERDOS*t§ Departments of *Pharmacology and UInternal Medicine, University of Texas Health Science Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235 Communicated by P. Kusch, May 13, 1982 ABSTRACT Carboxypeptidase N (kininase I, arginine car- MATERIALS AND METHODS boxypeptidase; EC 3.4.17.3) cleaves COOH-terminal basic amino the Parkland Me- acids of kinins, anaphylatoxins, and other peptides. The tetra- Outdated human plasma was obtained from meric enzyme of Mr 280,000 was purified from.human plasma by morial Hospital blood bank (Dallas, TX). Hippurylargininic acid ion-exchange and arginine-Sepharose affinity chromatography. (Vega Biochemicals, Tucson, AZ), bradykinin (Bachem Fine Treatment with 3 M guanidine dissociated the enzyme into sub- Chemicals, Torrance, CA), guanidine'HCI (Chemalog, South units of 83,000 and 48,000 molecular weight, which were sepa- Plainfield, NJ), trypsin (Worthington, Freehold, NJ), and hu- rated and purified by gel filtration or affinity chromatography. man plasmin [Committee on Thrombolytic Agents (CTA) Stan- When tested with hippurylarginine, hippurylargininic acid, ben- dard, 10 units/ml, the American National'Red Cross] were used zoylalanyllysine, or bradyldkini the Mr 48,000 subunit was as ac- as received. Human urinary kallikrein was donated by H. Fritz tive as the intact enzyme-and was easily distinguished from human (Munich, Federal Republic of Germany) and human plasma pancreatic carboxypeptidase B (EC 3.4.17.2).
    [Show full text]
  • Progress in the Field of Aspartic Proteinases in Cheese Manufacturing
    Progress in the field of aspartic proteinases in cheese manufacturing: structures, functions, catalytic mechanism, inhibition, and engineering Sirma Yegin, Peter Dekker To cite this version: Sirma Yegin, Peter Dekker. Progress in the field of aspartic proteinases in cheese manufacturing: structures, functions, catalytic mechanism, inhibition, and engineering. Dairy Science & Technology, EDP sciences/Springer, 2013, 93 (6), pp.565-594. 10.1007/s13594-013-0137-2. hal-01201447 HAL Id: hal-01201447 https://hal.archives-ouvertes.fr/hal-01201447 Submitted on 17 Sep 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Dairy Sci. & Technol. (2013) 93:565–594 DOI 10.1007/s13594-013-0137-2 REVIEW PAPER Progress in the field of aspartic proteinases in cheese manufacturing: structures, functions, catalytic mechanism, inhibition, and engineering Sirma Yegin & Peter Dekker Received: 25 February 2013 /Revised: 16 May 2013 /Accepted: 21 May 2013 / Published online: 27 June 2013 # INRA and Springer-Verlag France 2013 Abstract Aspartic proteinases are an important class of proteinases which are widely used as milk-coagulating agents in industrial cheese production. They are available from a wide range of sources including mammals, plants, and microorganisms.
    [Show full text]
  • A Label-Free Cellular Proteomics Approach to Decipher the Antifungal Action of Dimiq, a Potent Indolo[2,3- B]Quinoline Agent, Against Candida Albicans Biofilms
    A Label-Free Cellular Proteomics Approach to Decipher the Antifungal Action of DiMIQ, a Potent Indolo[2,3- b]Quinoline Agent, against Candida albicans Biofilms Robert Zarnowski 1,2*, Anna Jaromin 3*, Agnieszka Zagórska 4, Eddie G. Dominguez 1,2, Katarzyna Sidoryk 5, Jerzy Gubernator 3 and David R. Andes 1,2 1 Department of Medicine, School of Medicine & Public Health, University of Wisconsin-Madison, Madison, WI 53706, USA; [email protected] (E.G.D.); [email protected] (D.R.A.) 2 Department of Medical Microbiology, School of Medicine & Public Health, University of Wisconsin-Madison, Madison, WI 53706, USA 3 Department of Lipids and Liposomes, Faculty of Biotechnology, University of Wroclaw, 50-383 Wroclaw, Poland; [email protected] 4 Department of Medicinal Chemistry, Jagiellonian University Medical College, 30-688 Cracow, Poland; [email protected] 5 Department of Pharmacy, Cosmetic Chemicals and Biotechnology, Team of Chemistry, Łukasiewicz Research Network-Industrial Chemistry Institute, 01-793 Warsaw, Poland; [email protected] * Correspondence: [email protected] (R.Z.); [email protected] (A.J.); Tel.: +1-608-265-8578 (R.Z.); +48-71-3756203 (A.J.) Label-Free Cellular Proteomics of Candida albicans biofilms treated with DiMIQ Identified Proteins Accession # Alternate ID Gene names (ORF ) WT DIMIQ Z SCORE Proteins induced by DiMIQ Arginase (EC 3.5.3.1) A0A1D8PP00 CAR1 CAALFM_C504490CA 0.000 6.648 drug induced Glucan 1,3-beta-glucosidase BGL2 (EC 3.2.1.58) (Exo-1Q5AMT2 BGL2 CAALFM_C402250CA
    [Show full text]