LuciSTARÇ bioluminescence system for complex chemical environments

• Grant number: 22/13678-4
• Support Opportunities: Research Grants - Innovative Research in Small Business - PIPE
• Duration: April 01, 2023 - May 31, 2024
• Field of knowledge: Interdisciplinary Subjects
• Convênio/Acordo: SEBRAE-SP
• Principal Investigator: Mona das Neves Oliveira
• Grantee: Mona das Neves Oliveira
• Host Company: Biolinker Biologia Sintética Eireli
• CNAE: Pesquisa e desenvolvimento experimental em ciências físicas e naturais
• City: Cotia
• Associated scholarship(s): 23/08580-8 - LuciSTARÇ Bioluminescence System for Complex Chemical Environments, BP.TT

Abstract

The scope of this work is the technical validation of an enzymatic formulation to stabilize LuciStar, an enzyme developed by BIOLINKER in AMBEV Line products. Biolinker's proposal consists in subjecting a luciferase to directed evolution in order to make it functional under the following conditions: pH 2.5-4.5; temperature 3-5 °C; presence of 4-8 % alcohol. These approximate the environments found in the target product line. The proposal is justified from both commercial-financial and scientific points of view. The former is due to the impact that luminescence can give to the product, highlighting brands that apply such technology. Biolinker, the developer of the current product, through an acceleration program with AMBEV found this demand of the partner that seeks to co-create products with new experiences for the user. From a scientific point of view, this project aims at the improvement of proteins to be applied in food products, as well as to generate a more stable luciferase that could be used in research and diagnostics under the described conditions where the wild enzyme is not very active. We performed a technical validation with the enzymes in our portfolio, identifying which would be the best candidates for the proposed use and verifying the feasibility and limitations of uses. The luciferases use different substrates and give rise to light with distinct colors. A screening of different luciferases of different luciferases and as attractiveness is a key factor in this project, the one that gave the most commercially interesting color and intensity of light in buffered cell lysate was chosen cell lysate. In this case was the ngluz luciferase from the Brazilian fungus Neonotophanus gardneri (N. gardneri)which produces an intense green light color. Having determined the target luciferase, an expression and purification protocol was developed in S. cerevisae. To our knowledge, we were the first to achieve this. It was possible to obtain a purityof approximately 65 % with one purification step by hydrophobic interaction chromatography. Previous work to ours has been done from cell lysate or natural extract of N.gardneri. However, with this protocol, we obtained only 1 mg ngLuz per liter of culture medium. This yield is far below what was expected. We calculated the production price, which includes reagents, culture media and researchers' salary, at 5000.00 R$ per mg of protein produced. The present proposal aims to perform a more robust technical validation, studying a more efficient protocol for expression and purification in E. coli that will decrease the production costs to approximately 2.0. R$ per mg of protein. To move forward with the project, it is of utmost important that the bioluminescence system does not make the final product too expensive. Using purified ngLight, assays of the enzymatic activity of luciferase were made by visual evaluation of light emission. Previous enzyme activity results were conducted in different environments. The most efficient catalysis was observed in product 2 while in other environments the light emission reaction was extremely low. For example, in product 4 the enzyme showed practically no activity. Did not show any activity. By comparison, in neutral pH buffered medium the light generation was in the range of 30000000 RLU, while in the most luminescent product 2, this was only 400000 RLU, i.e. 100 times lower. In light of these results, we conclude that for the enzyme to be used in a wide range of products and confer a more striking visual effect by emitting a larger amount of photons, catalytic parameters of this magnitude require adaptation to the desired medium. In summary, we seek in this call, support to optimize this formulation and make possible commercial application in the production line. (AU)