Browsing by Subject "biotechnology"

Microbial cellulases, e.g. cellobiohydrolases, are able to degrade cellulose and lignocellulosic biomass to smaller glucose-containing monomers and oligomers. Cellulases are often multi-domain enzymes comprised of different protein domains (i.e. modules), which have different functions. The main two components, which often appear in cellulases, are the cellulose-binding module (CBM) and the catalytic domain. The CBMs bind to cellulose, bringing the catalytic domains close to their substrate and increasing the amount of enzymes on the substrate surface. The catalytic domain performs the cleavage of the substrate, e.g. in the case of cellobiohydrolases hydrolyses or “cuts” the crystalline cellulose chain into smaller soluble saccharides, mainly cellobiose. Unlike aerobic fungi, which utilize free extracellular enzymes to break down cellulose, anaerobic microbes often use a different kind of strategy. Their cellulases are organized and bound to the cell surface in a macromolecular protein complex, the cellulosome. The core of the cellulosome is formed of a scaffolding protein (the scaffoldin) consisting mainly of multiple consecutive cohesin domains, into which the catalytic subunits of enzymes attach via a dockerin domain. This creates a protein complex with multiple different catalytic domains and activities arranged in close proximity to each other. Dockerins and cohesins are known to bind each other with one of the strongest receptor-ligand -pair forces known to nature. Dockerin containing fusion proteins have also been successfully combined in vitro with proteins containing their natural counterparts, cohesins, to create functional multiprotein complexes. In this Master’s thesis work the goal was to 1) produce fusion proteins in which different CBMs were connected to dockerin domains, 2) combine these fusions with cohesin-catalytic domain fusion proteins to create stable CBM and catalytic domain containing enzyme complexes, 3) to characterize these enzyme complexes in respect of their thermostability and cellulose hydrolysis capacity and 4) to ultimately create a robust and fast domain shuffling method for multi-domain cellobiohydrolases (CBH) to facilitate their faster screening. The hypothesis of the experiments was that different CBMs fused with a dockerin domain and the cellobiohydrolase catalytic domain fused with a cohesin domain could be produced separately and then be combined to produce a functional two-domain enzyme with a dockerin-cohesin “linker” in between. In this way time and work could be saved because not every different CBM- catalytic domain -pair would have to be cloned and produced separately. Several CBM-dockerin fusion proteins (in which the CBM were of fungal or bacterial origin) were tested for expression in heterologous hosts, either in Saccharomyces cerevisiae or Escherichia coli. The purified proteins were combined with a fungal glycoside hydrolase family 7 (GH7) cellobiohydrolase-cohesin fusion protein produced in S. cerevisiae. The characterization of the catalytic domain-CBM -complexes formed through cohesin-dockerin interaction included thermostability measurements using circular dichroism and activity assays using soluble and insoluble cellulosic substrate. The results were compared to enzyme controls comprising of the same CBM and catalytic domain connected by a simple peptide linker. The results showed that the cohesin-dockerin –linked cellobiohydrolase complex performed in the cellulose hydrolysis studies in a similar manner as the directly linked enzyme controls at temperature of 50˚C and 60 ˚C. At temperatures of 70 ˚C the complex did not perform as well as the control enzymes, apparently due to the instability of the dockerin-cohesin interaction. The thermostability measurements of the enzymes, together with the previously published data supported the hydrolysis results and this hypothesis. The future work should be aimed at enhancing the thermostability of the cohesin-dockerin interaction as well as on verifying the results on different cellulase fusion complexes.

Patents may be refused in Europe on the grounds that the commercial exploitation of otherwise patentable inventions would be considered contrary to ordre public and morality. Whereas patent offices have applied these exclusions only infrequently in the past, recent developments in the research of arising gene editing technologies such as CRISPR/Cas9 and prime editing have given rise to varying opinions as to the approach that should be adopted in relation to patents. While genetics have been described as promising to offer an unprecedented contribution to improve health care, from the perspective of social sustainability, these advancements should be shared with society as a whole and with the international community. Despite the visions of a promising future, not much progress has been shown in advancing these goals – instead, considerable resources have been allocated for managing the perceived risks of gene editing rather than providing incentives to research new opportunities for its use. This is embodied in the restrictive policy that the European Union has adopted towards inventions involving germline gene therapy with the Directive 98/44/EC of the European Parliament and of the Council of 6 July 1998 on the legal protection of biotechnological inventions which unambiguously prevents the patentability of processes for modifying the germline genetic identity of human beings. Patents act as a negative force that simply allow the patent holder to prohibit others from commercially exploiting the invention conferred by the patent; they do not, however, form any absolute positive right to use an invention. A strong patent position is nevertheless understood to be a prerequisite for investment, and although patent law is not considered the primary control mechanism for regulating innovation, exclusions on patenting de facto indirectly regulate the types of therapies for which capital may be attracted in order to fund the significant costs associated with bringing new therapies or medicinal products to the market. Given the justification of the patent system providing economic incentives for the technologies the development of which we want to promote in our society, as European Union Member States have played an important role in pioneering new technologies which have later transferred to developing countries, this approach may have effects on a global scale, preventing individuals suffering from severe hereditary illnesses from achieving the right to the highest attainable standard of health which along with the strengthened view on human rights is understood to extend to preventive medicine. Reports from more recent sources such as the Nuffield Council on Bioethics have opened the discussion on how the concept of human dignity is not helpful in the context of germline gene therapy, proposing alternative principles such as the welfare of the future person as well as social justice and solidarity to provide help with the assessment on the acceptability of human applications. While the preamble in the Directive 98/44 declares there to be a “consensus within the Community” that interventions in the human germline offend against ordre public and morality, through balancing these divergent legal-ethical values, this thesis argues how these unequivocal prohibitions to patenting in fact fail to respect the social and cultural context of each Member State. Influenced through time and historical events, the basic European bioethical principles are differently reflected in the legal culture of each Member State – and as ordre public and morality should correspond in particular to ethical or moral principles recognized in each Member State, considering all human applications of these technologies unequivocally contrary to ordre public and morality does not adequately reflect the values and ethical positions of all Member States. From the perspective of social sustainability, as ordre public and morality patenting exclusions discourage investing in the research and development of these technologies, they also work as to hinder the right to benefit from scientific and technological progress. The policy decisions that the European Union adopts around the economic incentives on investing in further research has consequences on the global level, as these technologies would eventually spread to developing nations that are disadvantaged under current conditions of scientific research and innovation. In terms of benefit sharing and solidarity, it is just as important to ensure that effective policies exist to secure that patents do not end up acting as limiters of welfare and that the growing developments in knowledge and technology will not widen the existing social inequities but rather act as to reduce them.