Improvement of food safety applied biosensors by protein engineering

Project Information

BIOIMPROVE

Grant agreement ID: 834952

DOI

10.3030/834952

Project terminated on 31 January 2022

EC signature date 29 March 2019

Start date 1 May 2019

End date 7 April 2022

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 172 932,48

EU contribution

€ 172 932,48

Coordinated by

BIOLAN MICROBIOSENSORES SL
Spain

Periodic Reporting for period 1 - BIOIMPROVE (Improvement of food safety applied biosensors by protein engineering)

Reporting period: 2019-05-01 to 2021-04-30

Summary of the context and overall objectives of the project

Determination of the histamine content is of paramount importance for the food industry as a parameter of the hygienic quality and freshness of food, as well as to prevent scombroid fish poisoning, an allergy-like food poisoning. At present, more sensitive and user-friendly methods for histamine monitoring are needed to early detect products and raw materials susceptible of becoming hazardous for consumers.

BIOIMPROVE aims to develop an improved and highly sensitive portable biosensing device for the quantification of histamine in the lower concentration range (<5 mg/l). Such device is expected to ensure histamine contamination to be identified at all stages of the food chain, ultimately guaranteeing food safety for European consumers.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Aiming to develop an improved biosensor for histamine quantification, a multidisciplinary work to improve the enzymes applied to the biosensor has been carried out, including molecular dynamic simulations (secondment at CIC bioGUNE), protein engineering assays (e.g. development of activity screening methods in combination with the mutagenesis work), X-crystallographic studies (secondment at CIC bioGUNE), in silico searches of novel histamine dehydrogenases, cloning expression and purification of different candidate enzymes, as well as the optimization of the biosensor response (e.g. study of redox mediators, working potential). As a result, important achievements that have been (or will be) exploited at BIOLAN have been obtained. From the industrial perspective, the most important one is the development of a novel expression and purification system for the histamine enzyme production that has been successfully implemented at BIOLAN. In addition, novel screening methods have been designed that will help in the identification of novel enzyme specificities and/or evolved improved variants at BIOLAN in future. However, these joint efforts did not result in an evolved enzyme with a higher sensitivity as most of the obtained variants showed lower activity than the parental enzyme. The complexity of the enzyme, together with the lack of a crystal structure of the enzyme in complex with its histamine substrate limited the proposed molecular dinamic simulations. Although attempts to obtain the crystal structure of the histamine bound enzyme were performed, only poorly diffracting crystals were produced. At this point the main difficulty found was that the X-difraction facility assigned priority to the projects related to the SARS-CoV-2 virus, and consequently the analysis of the enzyme crystals was significantly delayed. This delay provided other opportunities to improve or develop new biosensor based on enzyme engineering. Specifically, the ER worked on the development of an improved sulphite biosensor and a novel caffeine biosensor with excellent results.

Overall, her work has resulted in new enzyme production procedures that were successfully implemented at BIOLAN significantly reducing the cost and time for biosensor manufacturing. Moreover, she has designed a promising solution for the caffeine determination in the food sector. Although the COVID-19 crisis has provided some difficulties for presenting the project outcomes, the main results were presented using social media tools, BIOLAN web page, a research-poster, seminars and public engagement events. Most importantly, the application of the new histamine enzyme to the portable biosensor for detecting histamine has led to the launch of a new generation of the portable biosensor. In this new version of the biosensor, apart from the applied enzyme with improved yield, a higher sensitivity and the miniaturization of the electronics was achieved and also connectivity with a cloud platform was enabled. The launching was promoted in different media:

https://mmaingenieria.es/biolan-lanza-un-biosensor-de-analisis-con-herramientas-de-digitalizacion/
https://parke.eus/es/bio-7000-el-innovador-dispositivo-de-analisis-de-biolan-portatil-versatil-e-inteligente-que-revolucionara-el-control-analitico/

The improvements obtained on the histamine biosensor are not patentable or publishable. However, this project has increased the value of the company in terms of its IPR. Apart from this, the patentability of the proposed solution for caffeine quantification is being evaluated.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

BIOIMPROVE has resulted in the launching of a new generation of portable biosensors designed to quantify histamine in the field thanks to their ultra-lightweight equipment, and equipped with digital connectivity to a platform that facilitates traceability and data analysis, as well as the management of the analytical process. This device responds to the main needs of the seafood sector, which was demanding improved solutions that could be applied to histamine monitoring in field (fishing boats, retailers, wholesaler markets, inspections) to early detect products and raw materials susceptible of becoming hazardous after processing (cooking, canning, smoking, etc.). Remarkably, the new histamine biosensor exhibits a linear response to histamine in the range of 5-50 mg/, and a limit of quantification of 5 mg/, showing higher sensitivity than the biosensor previously developed at BIOLAN, allowing the rapid and effective identification of histamine at all stages of the food chain. Apart from its wide linearity and higher sensitivity, the improved biosensor shows excellent analytical properties in terms of fabrication, selectivity, and stability. This device is expected to be implemented in more than 200 food industries in the following years. The establishment of such an effective and accurate histamine monitoring system in the food industry wil not only guarantee safety for consumers, but also reduce product waste and increase throughput during food production.

Apart from this, other enzyme based biosensors were improved or developed for sulphite and caffeine quantification. The new sulphite oxidase obtained by the expression system developed during this project was tested on the biosensor platform with excellent results. Currently, this new enzyme production protocol for biosensor manufacturing is being validated, and is expected to replace the previous one before the end of this year, resulting in a significantly reduction of the cost of the strips associated to this biosensor. In the case of the caffeine biosensor, the enzymes needed for caffeine detection were identified and produced in active forms. At this moment, their application on a biosensor platform is under investigation. Caffeine quantification in beverages is time consuming and requires expensive laboratory equipment and skilled personnel. The new strategy designed provides a promising solution for caffeine quantification in the food sector.

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