An in silico analysis, purification and partial kinetic characterisation of a serine protease from Gelidium pristoides A dissertation submitted in fulfilment of the requirement for the degree of Master of Science (MSc) Biochemistry by Zolani Ntsata Supervisor: Prof. Graeme Bradley 2020 Department of Biochemistry and Microbiology Declaration I, Zolani Ntsata (201106067), declare that this dissertation, entitled ‘An in silico analysis and kinetic characterisation of proteases from red algae’ submitted to the University of Fort Hare for the Master’s degree (Biochemistry) award, is my original work and has NOT been submitted to any other university. Signature: __________________ I, Zolani Ntsata (201106067), declare that I am highly cognisant of the University of Fort Hare policy on plagiarism and I have been careful to comply with these regulations. Furthermore, all the sources of information have been cited as indicated in the bibliography. Signature: __________________ I, Zolani Ntsata (201106067), declare that I am fully aware of the University of Fort Hare’s policy on research ethics, and I have taken every precaution to comply with these regulations. There was no need for ethical clearance. Signature: _________________ i Dedication I dedicate this work to my grandmother, Nyameka Mabi. ii Acknowledgements Above all things, I would like to give thanks to God for the opportunity to do this project and for the extraordinary strength to persevere in spite of the challenges that came along. I am thankful to my family, especially my grandmother, for her endless support. I would also like to acknowledge Prof Graeme Bradley for his supervision and guidance. Thanks to my friends and colleagues, especially Yanga Gogela and Ntombekhaya Nqumla, and the plant stress group for their help and support. I would like to acknowledge the GMRDC, NRF, and SAIAB Phuhlisa programme for funding this project, and lastly, the University of Fort Hare for providing the facilities to do this project. iii Abstract The aim of this study was to characterize the protease enzyme (s) from red algae. An in silico analysis of red algae genomes was used to identify gene coding for protease. Protease sequences identified from these genomes were examined for conserved domains, active site and structures. The domain search revealed that the identified sequences were from the five classes of protease enzymes. For function inference, the red algae sequences were aligned to identify the catalytic sites, and the tertiary structures were predicted using homology modelling. An in silico analysis provides an indication of the class and potential functions of the enzymes. However, it cannot predict whether the gene is constitutively expressed in the red algae or under which conditions it may be induced, and it cannot determine the kinetic efficiency of an enzyme against various substrate, or the optimum conditions for the protein activity. Attempts to clone and recombinantly express selected red algae proteases, proved unsuccessful, as the available genomes where from red algae species found mainly in Asia, and the designed primers, therefore, did not amplify a corresponding PCR product from the red algae harvested in South Africa. Crude extracts of red algae collected from Kenton-on-Sea, along the East Coast of South Africa, were screened for protease activity using Benzoyl-Arginine-pNitroAnilide (BApNA) as substrate. The proteases detected in the crude extract were purified using ammonium sulphate precipitation and HiPrep DEAE FF 16/10; CM FF 16/10, and HiPrep Q FF 16/10 columns for ion-exchange chromatography. The HiPrep Q FF 16/10 column yielded active protein, which revealed two bands of 11kDa and 17kDa on SDS-PAGE. It was assumed that these bands represented two subunits of the purified protease. Kinetic characterisation of the purified protease revealed a pH optimum of 9, using BApNA as substrate, a temperature optimum at 60ºC, and sensitivity to temperature when stored above 4ºC. The protease activity was inhibited by Ferric chloride (32%), induced by calcium chloride (156%), no inhibition by magnesium chloride (97%) and slight inhibition by potassium chloride (77%) and manganese chloride (70%). Phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor, almost totally inhibited the protease activity, indicating that the protease from red algae was most likely a serine protease. The Km and kcat values were 1.96 µM, and 0.364 s-1, respectively using BApNA as the substrate. This study revealed that the red algae genome contains numerous genes that encode for proteases from almost all the classes of proteases. A serine protease from the red algae Gelidium pristoides was partially purified and kinetically characterised, confirming that red algae found along the Eastern Coast of South Africa contain genes that express active proteases that may be of medical or industrial interest. Further studies, however, are required to recombinantly express, purify and characterise the numerous proteases encoded by the genes identified in the in silico analysis of this study. iv Table of content 1. Introduction ............................................................................................ 2 1.1 Classification of proteases ................................................................................. 2 1.1.1 Serine protease ...................................................................................................... 3 1.1.2 Aspartic protease ................................................................................................... 4 1.1.3 Cysteine protease .................................................................................................. 4 1.1.4 Metalloprotease..................................................................................................... 4 1.1.5 Threonine protease ................................................................................................ 5 1.1.6 Glutamic protease .................................................................................................. 5 1.1.7 Asparagine peptide lyase (proteolytic enzyme but not protease) ........................ 5 1.2 Applications of protease .................................................................................... 5 1.2.1 Therapy .................................................................................................................. 6 1.2.2 Detergent ............................................................................................................... 6 1.2.3 Dairy industry ......................................................................................................... 7 1.2.4 Baking industry ...................................................................................................... 7 1.2.5 Meat tenderisation ................................................................................................ 7 1.2.6 Hydrolysis of protein .............................................................................................. 7 1.2.7 Silver recovery ........................................................................................................ 8 1.2.8 Dehairing industry .................................................................................................. 8 1.3 Sources of protease enzymes ............................................................................. 8 1.3.1 Plant proteases ...................................................................................................... 8 1.3.2 Animal protease ..................................................................................................... 9 1.3.3 Microbial protease ................................................................................................. 9 1.3.4 Seaweeds: a potential source of protease enzymes ........................................... 10 1.4 Production by recombinant technology ........................................................... 10 1.5 Problem statement .......................................................................................... 11 1.6 Research hypothesis ........................................................................................ 11 1.7 Aim and objectives .......................................................................................... 11 1.7.1 Aim ....................................................................................................................... 11 1.7.2 Objectives............................................................................................................. 11 1.7.2.1 To search available red algae genomes for genes encoding protease enzymes. 11 1.7.2.2 To clone and express a selected protease gene. ........................................ 11 1.7.2.3 To isolate and purify a selected protease enzyme(s) from red seaweed. 11 v 1.7.2.4 To characterise the biochemical and kinetic properties of the purified protease. 11 2. Introduction .......................................................................................... 13 2.1 Sequence-based approach ............................................................................... 14 2.2 Structure-based approach................................................................................ 15 2.2.1 Global structure-based method ........................................................................... 15 2.2.2 Local structure-based method ............................................................................. 16 2.2.3 Combined methods