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A Novel Microbial Transglutarninase Derived from Streptoverticillium Baldaccii

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A Novel Microbial Transglutarninase Derived from Streptoverticillium Baldaccii A Novel Microbial Transglutarninase Derived From Streptoverticillium baldaccii A Thesis submitted in fulfilment of the requirements of the degree of DOCTOR OF PHILOSOPHY of Griffith University by Suzanne Schleehauf Negus B.Sc, M.ScSt School of Biomolecular and Biomedical Science Faculty of Science Griffith University, Nathan Campus Queensland, Australia July 2001 STATEMENT OF ORIGINALITY This thesis contains original material that to the best of my knowledge has not been previously written or published by another person, except where due acknowledgment has been given in this thesis, nor has the material previously been submitted for a degree or diploma in any University. Suzanne Schleehauf Negus ACKNOWLEDGMENTS There are a number of people to whom I wish to acknowledge for their help, advice and friendship during the course of these studies. Firstly I would like to thank my supervisors, Dr. Peter Rogers, Dr. Kathryn Tonissen and Associate Professor Frank Clarke for the opportunity to undertake this research and for their support, advice and helpful guidance which has made my PhD a memorable experience. I would like to thank my colleagues in the laboratory, past and present, Michael Batzloff, Kelly Bloomfield, Simone Osborne, Ben Baldwin and Colm Cahill for their support, advice and friendship. I would also like to thank the Meat and Livestock Australia (MLA) for their generous contributions to this project and Ajinomoto for providing the partially purified microbial transglutaminase from Streptoverticillium S-8 112. Finally I would like to thank my husband Paul for his patience and support throughout my PhD. Transglutaminase (TGase; protein-glutamine y-glutamyltransferase, E.C. 2.3.2.13) is an enzyme that catalyses the acyl transfer reaction by introducing covalent cross-links between proteins, peptides and various primary amines. Until recently, commercial TGase has been derived from mammalian origin. Calcium-dependent TGase extracted from guinea-pig liver and blood plasma have been investigated for the purpose of their application in the food industry. However, supply, complicated separation and purification procedures as well as the requirement for calcium have made it almost impossible to apply mammalian TGase in food processing on an industrial scale. Microbial transglutaminase (MTGase) was first purified from the culture filtrate of Streptoverticillium S-8112, a variant of Stv. mobaraense and subsequently the extracellular enzyme has been purified from the culture filtrate of other Streptoverticillit~m and Streptomyces species. This enzyme is easily obtained by microbial fermentation and has been found to have the ability to induce cross-linking and gelation of food proteins. In addition, MTGase does not require calcium for activation which is of great advantage for the food industry as many food-proteins are easily precipitated in the presence of ca2+ thus rendering them less sensitive to the enzymatic reaction. A commercial source of MTGase derived from Stv. mobaraense is available, however the optimum temperature range of this enzyme is 50 to 55 OC. Important to the food industry is the requirement of catalytic activity at low temperatures so the need for a low temperature variant is desirable. This thesis explores the possibility of finding a bacterial source which retains MTGase activity at low temperatures and can be produced on an industrial scale. Thus provided the MTGase functions at the required temperature the reduced catalytic activity can be offset by using more enzyme. Psychrophilic, psychrotrophic and mesophilic bacteria were screened for the presence of a related TGase gene. A PCR strategy which amplified the region of the gene encoding the putative active site of MTGase was utilised for the selection and cloning of the gene. This successful screening strategy led to the cloning of the entire coding sequence of the mature form of MTGase from mesophilic actinomycetes including several Streptoverticillit~mspecies (Stv. mobnmerzse, Stv. griseocnmeum, Stv. cinnnmoneum ssp. cinrznmorzeum) and Streptomyces lnvendt~laeand also from a previously unreported mesophilic bacteria, Stv. bnldaccii. Structural relationships of the gene and protein were analysed by Southern and western blotting, respectively. MTGase derived from Stv. bnldaccii was examined to determine the optimal growth conditions for maximum enzyme activity and whether this enzyme could function at low temperatures. Sm. baldaccii TGase exhibits characteristics of cold-adapted enzymes found in psychrophilic bacteria. Stv. bnldnccii TGase has a lower temperature optimum, higher specific activity at low temperatures and thermal instability at moderately high temperatures. Industrial applications often require continuous large volumes of enzyme product. In this study a purification scheme was developed for the isolation of endogenous MTGase from the culture filtrate of Stv. baldnccii. However for commercial applications a recombinant source would overcome problems with supply, production time and complex and expensive growth requirements. MTGase gene encompassing the entire coding region for the protein from Stv. baldaccii was expressed in E. coli and produced an active enzyme. The recombinant MTGase shared similar immunological and enzymatic characteristics as the endogenous enzyme. The findings of this thesis are: (i) A PCR method was developed for selection and cloning of the gene based on the sequence encoding the mature active form and the putative active site encoding region of the TGase gene from Stv. mobarnense; (ii) The entire coding sequence of the mature form of MTGase from mesophilic acti~omycetes including several Streptoverticillium species (Stv. mobarnense, Stv. griseocnmeum, Stv. cinnamonetlm ssp. cinnanzo~zeum and Stv. balclnccii) and Streptomyces lavendulne were compared; (iii) Structural analysis of the protein by western blotting revealed that there is a related protein produced within the Streptoverticillium species with both the Pro-TGase and the IV active mature enzyme detected in the culture filtrate. Southern blot hybridisation revealed that MTGase produced within Streptoverticillizlm species is related by genomic organisation, with only one copy of the gene detected; (iv) Stv. baldaccii TGase production was optimised by a systematic analysis of growth conditions: TGase production was favoured by growth at low temperatures with maximum growth and enzyme activity occurring when cultured cells changed from exponential phase to stationary phase; (v) Stv. baldaccii TGase exhibits characteristics of cold-adapted enzymes found in psychrophilic bacteria with its low temperature optimum, higher specific activity at low temperatures and thermal instability at 55 OC; (vi) Comparison of the deduced amino acid sequences of the TGase gene cloned from Stv. rnobaraense and Stv. baldaccii showed approximately 80 % identity. This difference in the protein sequence of the two MTGases may be responsible for the lower activity optima and heat instability of Stv. baldaccii TGase; (vii) The MTGase gene encompassing the entire coding region for the protein from Stv. baldaccii was expressed in E. coli. The recombinant MTGase showed immunological and enzymatic characteristics similar to the endogenous form of the enzyme and therefore the same purification conditions were applied. Taken together I have shown that MTGase from Stv. baldaccii has high specific activity at low temperatures and the enzyme is produced at comparable levels to Stv. mobaraense under a variety of conditions. The enzyme can be over-expressed in E. coli thus providing a convenient production pathway since E. coli media has been optimised as a result of many studies to minimise the cost of production. A recombinant source wou,ld overcome supply, reduce production time and produce the enzyme cheaply and in abundant amounts. This thesis provides detailed proof of concept for the development of a commercial, high activity, temperature desensitised enzyme for use in biofilm production and protein manipulation with potential application in the food and beverage industry. TABLE OF CONTENTS STATEMENT OF ORIGINALITY I ACKNOWLEDGMENTS i1 ABSTRACT i11 TABLE OF CONTENTS VI LIST OF FIGURES XIV LIST OF TABLES XIX CHAPTER 1 General Introduction 1.1 Introduction 1.2 Enzymology of TGase 1.3 TGase Family 1.3.1 Mammalian TGase 1.3.2 Other varieties of TGase 1.4 Bacterial TGase 1.5 TGase from Streptoverticillium 5-8 112 1.6 Factors Affecting Activity of MTGase and Mammalian TGase 1.6.1 Influence of calcium and various metal ions 1.6.2 Secondary structural analysis and inhibition of catalytic activity 1.6.3 Thermal stability 1.7 Comparison of the Active Sites of TGases 1.8 Application of TGase in the Food Industry 1.8.1 Mammalian TGase 1.8.2 Microbial TGase 1.8.2.1 Benefits of using MTGase 1.8.2.2 Practical industrial uses of MTGase 1.8.2.3 Recombinant MTGase 1.9 Aims of this Study CHAPTER 2 Materials And Methods 2.1 Materials 2.1.1 List of Abbreviations 2.1.2 Culture Collections 2.1.3 Bacterial Strains and Plasmids 2.1.3.1 E. coli K12 strains 2.1.3.2 Streptoverticillium and Streptomyces species 2.1.3.3 Other bacteria 2.1.3.3a Psychrophilic and psychrotrophic bacteria (ACAM) 2.1.3.3b Actinomycete isolates (DRLMD) 2.1.3.4 Plasmids and constructs 2.1.4 Chemicals, Reagents and Kits 2.1.5 Enzymes 2.1.6 Growth Media 2.1.6.1 Streptoverticillium media 2.1.6. la Spore production 2.1.6. lb Production of TGase 2.1.6. lc Extraction of genomic DNA 2.1.6.2 E. coli media 2.1.6.3 Other bacteria media 2.1.6.3a Actinomycete
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