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Projects / Programmes source: ARIS

The development of enzyme formulation for disinfecting prion-contaminated areas.

Research activity

Code Science Field Subfield
4.06.06  Biotechnical sciences  Biotechnology  Finishing processes in biotechnology 

Code Science Field
B510  Biomedical sciences  Infections 

Code Science Field
2.09  Engineering and Technology  Industrial biotechnology 
Keywords
prions disinfection, thermostable proteases, penisin, heterologous expression system, Streptomyces sp.
Evaluation (rules)
source: COBISS
Researchers (18)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  28299  PhD Martina Avbelj  Biotechnology  Researcher  2017 - 2018  72 
2.  39095  PhD Miha Bahun  Biochemistry and molecular biology  Junior researcher  2017 - 2019  41 
3.  18332  PhD Neža Čadež  Biotechnology  Researcher  2017 - 2019  318 
4.  36374  PhD Jerneja Čremožnik Zupančič  Microbiology and immunology  Researcher  2019  67 
5.  04630  PhD Polona Juntes  Veterinarian medicine  Researcher  2017 - 2019  426 
6.  32503  PhD Katarina Karničar  Biotechnology  Researcher  2017 - 2019  23 
7.  50382  Vilma Lindner    Technical associate  2018 - 2019 
8.  50383  Irena Mubi    Technical associate  2018 - 2019 
9.  29336  PhD Ilja Gasan Osojnik Črnivec  Chemistry  Researcher  2018 - 2019  235 
10.  15618  PhD Maja Paš  Biotechnology  Researcher  2017 - 2019  103 
11.  13542  PhD Hrvoje Petković  Biotechnology  Researcher  2017 - 2019  299 
12.  10873  PhD Nataša Poklar Ulrih  Chemistry  Head  2017 - 2019  827 
13.  23417  PhD Iztok Prislan  Chemistry  Researcher  2017 - 2019  202 
14.  35360  PhD Sandra Ropret  Biochemistry and molecular biology  Researcher  2018  15 
15.  30763  PhD Marko Šnajder  Biotechnology  Researcher  2017 - 2019  39 
16.  29544  PhD Ajda Taler Verčič  Biochemistry and molecular biology  Researcher  2017 - 2019  78 
17.  04988  PhD Dušan Turk  Biochemistry and molecular biology  Researcher  2017 - 2019  621 
18.  08506  PhD Jelka Zabavnik Piano  Veterinarian medicine  Researcher  2017 - 2019  204 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0406  University of Ljubljana, Veterinary Faculty  Ljubljana  1627139  10,694 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  66,575 
3.  2990  Center of excellence for integrated approaches in chemistry and biology of proteins, Ljubljana  Ljubljana  3663388  725 
Abstract
Transmissible spongiform encephalopathy (TSE) forms a family of neurodegenerative diseases known for animal and humans. The emergence of a variant form of human Creutzfeldt-Jakob disease (vCJD), as a result of the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, has increased the profile of transmissible spongiform encephalopathies, as they have become a serious risk to public health policy worldwide. CJD is a fatal neurodegenerative disease that affects on average 1 person per million per year in the United States. Infective prions that cause CJD or other TSEs show high resistance to conventional chemical and physical decontamination methods. Prions are well-known to bind tightly to surfaces, including stainless still surfaces. The basic procedures for preventing iCJD transmission via surgical instruments are sterilisation or prion decontamination according to guidelines by autoclaving them at high temperatures (from 121°C to 132°C and using sodium hydroxide. These procedures usually take around 1 hour, but surgical equipment (eg. endoscopes) cannot resist these harsh conditions. Efficient removal of biofilms from medical devices is another big challenge in healthcare, to avoid hospital-acquired infections. Again, there can be problems with delicate devices like flexible endoscopes, which cannot be cleaned using harsh chemicals or high temperatures. There are a few alkaline and enzymatic detergents that can reduce the prion infectivity. Although that there are rare proved transmissions of CJD, as they so called iatrogenic CJD (iCJD), through medical equipment has been rare, the consequences can be far-reaching. With the evidence of tens of thousands infectious carriers just in the United Kingdom, there is increasing concern of potential transmission of infection. The long incubation periods for CJD, along with the need for diagnosis of CJD through brain tissue, might indeed are causes that can possibly lead to uncontrolled CJD transmission via contaminated surgical instruments. In 2011, we patented a procedure for enzymatic degradation of protein aggregates by pernisine, such as for infective prions (PrPSc) from different origins (i.e., mouse, bovine, deer, human), as well as aggregates related to Alzheimer’s disease. However, very low productions yield of pernisine, at ( 0.5 mg per 1 L of culture, has hampered its industrial applicability. Our breakthrough with an Escherichia coli expression system through codon optimization of the gene coding for pernisine can now provide ) 20-fold increases in yields, which has opened new opportunities for industrial purposes. However, the need for the activation of recombinant pernisine and the secretion of this protein extracellularly has raised new obstacles for industrial applications. Our proposed solution is to use a relatively new expression system with Streptomyces sp. and to make use of a shorter version of the pernisine gene, which is constructed without its proregion. The aim of the project is to develop this new and efficient formulation for cleaning/disinfection the prion's contaminants through a system based on recombinant pernisine, which will be carried out in collaboration with Borer Chemie AG, as one of a leading supplier of products for aqueous cleaning and disinfections.
Significance for science
Original scientific contribution: The applied research approach adopted within this project is itself novel and will significantly extend our understanding of the mechanism of action and physicochemical and biological properties of the archaeal protein pernisine expressed in Streptomyces. This will allow the development of a new formulation to disinfect and clean the prion contaminants in laboratories, and for surgical instruments (Poklar N. Vilfan T. 2012). The project is design so it contains the application note based on science. The scientific results obtained will be published in peer-reviewed journals. The impact of the original scientific project’s will be in-vitro validation of new formulation of pernisine at Borer Chemie AG. As well as development of the new formulations, the products also will also be improved in terms of their compositions. All of these properties are essential for high added value products. The main scientific contributions: (i)   Generating the basic knowledge of the mechanism of PrPSc and other TSE degradation processes by the thermostable protease pernisine (ii)   Knowledge of using the Streptomyces sp. as a heterologous expression system for archaeal proteins (iii)  Understanding the mechanism/correlation between thermal stability and activity of proteins found in hyperthermophilic archaea. (iv)  3D structure of recombinant thermostable serine protease (pernisine) should enable the further optimisation of the enzyme activity (v)  Development of different enzyme-based formulation(s) for the  decontamination of PrPSc or other protein aggregates
Significance for the country
With the presented project, we would like to use pernisine, which has shown potential for industrial applications as a prion decontaminating agent with a new expression system of Streptomyces sp. to use in a newly developed formulation. The results will be tested, along with the development and commercialisation, and these will have a rapid impact on the economic benefits for the collaborating partners. Potential enzyme-based new formula(e) can be used for cleaning of potential PrPSc infections in medical fields (hospitals, medical centre), to avoiding the fatal consequences of TSE. Our two already granted patents (one is European: Compositions and Methods for Degradation of Protein Deposits and Prions; another is Slovenian: Overproducing recombinant pernisine in heterologous expression systems) will have beneficial financial impacts. The development of technology for Streptomyces sp. as an expression system for producing pernisine can be further upgraded to a new patent. Successful collaboration can further result in a newly establish company for developing the new industrially important proteins or producing pernisine. Some preliminary results are already promising for new articles with relatively high impact factors. The biotechnology master students and PhD students will be involved in the research work. The partnership involved in the project will be strengthen on the basis of future collaboration and knowledge transfer between industrial and academic partners. Successful collaboration with industry can lead to further collaboration on similar application projects with other different promising enzymes from A. pernix.
Most important scientific results Interim report, final report
Most important socioeconomically and culturally relevant results Interim report, final report
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