Laboratory: BIOCATALYSIS AND NATURAL BIOACTIVE SUBSTANCES
The expected results, obtained by the planned research activities, will represent an additional value to the knowledge in some of the most intensively developed directions of modern Biotechnology, in particular Medical Biotechnology, Food Biotechnology and Biotechnologies for new biomaterials.
Application of interdisciplinary scientific approach for carrying out of research activities would benefits the reaching of a new knowledge for development of screening programs for searching of new enzymes and clarification of mechanisms of enzyme reactions and their application for modification and synthesis of specific biomolecules with desired activities.
The key directions in the study of biological catalysts that will lead to significant progress in bio-attention economy are:
- Synthesis of new and improvement of existing therapeutic molecules using the enzymes in the heme-enzymatic processes;
- Solving the effect of replacement of amino acid residues in the human proteins and their involvement in the event of a number of diseases such as oxidative stress, Alzheimer’s disease, and hereditary diseases;
- A study of enzymes and other proteins found in organisms that live in extreme conditions (temperature, pressure, acidity) and their application in industrial biotech processes;
- Using the methods of synthetic biology for the production of enzymes or alteration of metabolic pathways for the more economically efficient synthesis of bioactive components with new functionalities from cheaper raw materials.
- Development of new experimental systems for the application of enzymes in micro-reactors for biosensors and others.
Main goal: To create modern research infrastructure for the development of integrated research platform for constructing new enzymes to synthesize specific novel bioactive components or modify natural biomolecules in which to alter their biological properties. Multidisciplinary approach to research will plan to create a new concept for creating technology to produce enzymes with specific properties and chemo-enzymatic processes for the synthesis of new bioactive substances. Infrastructure will be used jointly by universities, institutes and companies to expand research vertical and horizontal knowledge transfer, training, development of current bio-products and international scientific partnership.
The infrastructure which would be created within this project and the planned collaborative research will attract organizations with similar research interests and activities and will serve for building national scientific bodies working in the field of biocatalysis and chemo-enzymatic modification of natural bioactive substances. The structure can be developed into a center for research and development of bioactive functional ingredients with therapeutic and preventive action based on enzyme technology for the Balkan region and to integrate into the European Research Area. The project results will provide the industry with innovative solutions in the development of bio-pharmaceutical biotechnology including biotransformation and food biotechnology.
- Creation of a gene / enzyme library based on the isolated natural sources;
- Developing of a methodology for implementation of “directed evolution” as a result of which to receive specific mutants screened on pre-set parameters of enzymatic reaction;
- Developing of a concept for constructing of a recombinant strain that produced the mutant enzymes based on the available methodology in use host cell – E. coli and L. plantarum, and development of technologies using eukaryotic host;
- Developing of a methodology and technological scheme for optimization of processes for production of recombinant enzymes in a pilot plant;
- Developing of a methodology and technological scheme for a chemo-enzymatic modification of natural or synthetic bioactive substances or synthesis of new ones at pilot plant and industrial stage.
The following tasks would be solved by realization of this WP:
- The genes/enzymes in microorganisms isolated from specific ecological habitats of the territory of Bulgaria would be estimated and systematized;
- The methodology and laboratory technological scheme for production of new mutant enzymes based on the method “directed evolution” would be elaborated;
- Pilot scale technology for production of new recombinant enzymes and their application for modification of bioactive substances with trade potential would be established.
The present proposal is related to the introduction and development of a platform for modification of enzymes by using a technique called “directed evolution,” which is based on the creation of a large number mutant genes encoding enzymes in microbial strains selected from a natural source and subsequent expression of these mutant genes and screening for selection of enzymes which fulfill the demanded properties.
The planned research aims to:
- Implementation of an integrated algorithm in the research program, which includes development of the necessary methodology and tools for creating of large enzyme libraries. The application of the technique of “directed evolution” for obtaining of new enzymes which are effective in industrial conditions;
- Developing a new model for a multidisciplinary research approach in the field of metabolic engineering and the protein engineering and bioinformatics design, as extremely powerful technologies to obtain new enzymes usable in biotechnologies based on enzymatic reactions, chemo – enzymatic cascade reactions or structured de novo metabolic pathways for synthesis of new bioactive substances;
- Developing a model of innovative technological scheme to improve the functional properties of natural bioactive substances through specific enzymatic transformation;
- Developing a new or improved methodology for production of encapsulated preparations of natural of modified bioactive substances with antimycotic action by using new carriers and nanotechnology, enabling directed effect and personal approach in the treatment of various diseases in particular mycotic infectious diseases.
The team that has over 15 years’ experience in the study of transferase enzymes applied for production of oligosaccharides with prebiotic potential and is recognizable among the international scientific community. Within several national and international projects within the department “Biochemistry and Microbiology” at the Faculty of Biology of PU, of Plovdiv “is built and operated Center for the Study of prebiotics.
Summarized examples illustrate how the accumulated genomic data for microorganisms with proven practical importance can be used for the discovery of new enzymes with potential application for the production of new bio-materials with a wide range of applications such as polysaccharides and oligosaccharides with specific chemical bounds. Research activities in this direction from a fully sequenced genomes of lactic acid bacteria to identify new genes and characterization of their products – are tasks which solution would also facilitate the accumulation of new fundamental knowledge and application possibilities.
It is known from the obtained results in recent years, that the concept of “one enzyme is responsible for one enzymatic reaction” is not valid for at least a portion of the enzymes. In various conditions of the enzymatic reaction, they appear to have additional activities, wherein the one active center of the enzymes of the same family may catalyze several different chemical reactions.
We offer development of the new hypothesis that the enzymes are multifunctional and our goal is to find correlation between phylogenetic analysis of the level of enzyme strucтure, amino acid sequence similarity and specificity and reactivity of the enzymatic reaction that govern the evolutionary adaptation of enzymes in particular and metabolic processes in the cell. This will enable to achieve a breakthrough in solving the following scientific challenges:
- Phylogenetic analysis of the enzymes of the same family, isolated from various natural sources;
- Setting and change of the molecular mechanisms of action of the new enzymes obtained after optimizing methodology of “directed evolution”;
- Screening of mutant enzymes and determination of their main characteristics (substrate specificity, pH – optimum, temperature optimum, Vmax, Km), as well as realization of specific reactions in a heterogeneous condition (durability in the presence of organic solvents);
- Optimization of the conditions for production of recombinant strain producers of the searched enzymes;
- Optimization of the principal technological scheme for the production of recombinant enzymes in laboratory and pilot plant;
- Optimization of the conditions of the enzymatic reactions of the mutant enzymes in order to reveal their cost-effective potential for the production of bio-active components.
From our previous studies, it is known that strain Leuconostoc mesenteroides URE 13 produces a complex of different glycosyltransferase enzymes among which are identified as glucosyltransferases (GH70), and fucosyltransferase (GH68) (Vasileva et al. 2009, 2012; Bivolarski et al. 2013). In addition to dextransucrase this strain produces also a high molecular glucosyltransferase with mollecular weight of 300 kDa., which is similar to DSR-E enzyme from Leuconostoc citreum NRRL B-1299. There are data that the enzyme from URE 13 synthesizes a glucan polysaccharide with a high content of linkages different from those of the classical dextran. According to the received data until now, determination of the full genomic potential for production of glycosyltransferases by strain Leuconostoc mesenteroides URE 13 is of a significant interest.
1. Molecular biological methods:
- The identification of microorganisms from different ecological habitats will be done on the basis of universal primers;
- Bioinformatics analysis of genes in microorganisms and plants of different ecological habitats encoding enzymes synthesizing and hydrolyzing the oligosaccharides and disaccharides with different glycoside bonds;
- Isolation, sequencing and cloning of the most important genes encoding enzymes with the highest activity against glycosides and oligosaccharides with different glycoside bonds;
- Isolation, sequencing and cloning of the genes encoding glucansaccharases and other transferases, which synthesized oligosaccharides and capable to modified natural bioactive substances;
- Expression of recombinant proteins;
- Preparation of mutants in the process of “directed evolution” and screening of promising mutant lines.
2. Biochemical analysis:
- Analysis of the activity of enzymes synthesizing and hydrolyzing oligosaccharides and disaccharides with various glycoside bonds, and those with glycosylation ability;
- Analysis of glycosyltransferases, β-glucosidases, β-glucuronidase, and key enzymes of the main metabolic pathways of carbohydrates by spectrophotometric and luminometric analysis of the enzymes.
3. Electrophoretic Methods:
- Standard methods for detecting PCR products and the protein profile of cellular proteins of the isolated microorganisms, and in situ assays for detection of specific enzymes of carbohydrate metabolism would be used.
4. Chromatographic methods:
- Separation and quantification of oligosaccharides and glycosides by HPLC analysis;
- Separation and quantification of the polyphenols, anthocyanins, and other low molecular weight compounds of plant origin by HPLC analysis.
- Analysis of metabolites and bioactive substances during the cultivation of the tested strains in nutritive mediums supplemented with specific ingredients.
- Determination of the concentration of lactate, acetate, ethanol, residual amounts of oligosaccharides using the HPLC system and a gas chromatograph.
5. Microbiological Methods:
- Cultivation of Microorganisms – study of kinetics of microbial growth and optimization of cultivation parameters in the presence of glycosides and oligosaccharides;
- Isolation and purification of the beta-glucosidase and phosphoglucosidases in microbial cells after cell disintegration and isolation of the enzymes
- Study of the antimicrobial activity of the isolated and purified biologically active substances of plant and microbial origin and determination of the minimum inhibitory concentrations.
6. Bioprocess engineering of recombinant strains producers of glycosyltransferase enzymes and glucohydrolases:
- Optimization of the cultivation conditions of the recombinant strain-producers in a bioreactor;
- Immobilization of enzymes in specific new carriers and study the kinetic parameters of the enzyme reaction;
- Encapsulation of natural bioactive substances and their modifications and study their functional properties – antimicrobial activity, antioxidant activity, leakage of specific enzymatic reactions and other.
- ACTIVITY 1. Study of the enzymatic synthesis of novel biopolymers by glycosyltransferases. This activity will be realized by following five steps:
- ACTIVITY 2. Evaluation of new enzymes for the synthesis of oligosaccharides. This activity will be realized within the following 6 steps:
- ACTIVITY 3. In vitro study of the mechanisms of antimicrobial action of newly isolated natural bioactive substances and newly modified and synthesized compounds. This activity 3 will be realized within the following seven steps:
- ACTIVITY 4. Encapsulation of the selected natural and modified bioactive substances and new enzymes.