IN SILICO RATIONAL ENGINEERING OF NOVEL ENZYMES IRENE HNL: Hydroxy Nitrile Lyase catalyzes the hydrolysis of C-CN bonds
IRENE >>Project summary

Project summary

The IRENE project

IRENE is an FP7 Collaborative project for Specific International Cooperation Actions (SICA), financially supported by the European Commission and the Russian Federal Agency for Science and Innovation (FASI). IRENE project is developing computational methods and strategies which are applied to rationally design and produce efficient biocatalysts for industrial applications. The consortium gathers a multidisciplinary group of European, Russian and Uzbek scientists with complementary expertise, covering all the major aspects of this front line research in a novel field of biocatalysis.


Beneficiary number

Beneficiary name

Beneficiary short name


1 (coordinator)

Università degli Studi di Trieste




Kungliga Tekniska Hoegskolan




Københavns Universitet




Technische Universiteit Delft




Novozymes A/S




The National University of Uzbekistan




Belozersky Institute of Physicochemical Biology




B.P.Konstantinov Petersburg Nuclear

Physics Intitute - Russian  Academy of Science




Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry – Russian Academy of Science




Molecular Technologies




Bio/ Techologies, Innovations, Researches LTD





The ultimate aim of IRENE project: expanding the use of sustainable bio-catalysts in industry

Enzymes are increasingly used to perform a range of chemical reactions. These catalysts from nature are sustainable, selective and efficient, and offer a variety of benefits such as environmentally friendly manufacturing processes, reduced use of solvents, lower energy requirement, high atom efficiency and reduced cost. However, natural biocatalysts are often not optimally suited for industrial applications. To boost the use of enzymes in industrial processes, it is important to expand the range of reactions catalyzed by enzymes and to improve their properties for industrial applications. Therefore in the last years a lot of efforts have been made for developing techniques and strategies for conferring new properties to enzymes, thus making them more suitable for applications at industrial scale. According to a recent survey among industry experts in the field of biocatalysis, the major bottlenecks for replacement of conventional synthetic routes with biocatalysts in industry and to the full exploitation of enzymes’ catalytic potential can be identified in the following points:

· requirement of multidisciplinary expertise for the implementation of the whole process, which cannot be afforded by most part of small and medium size enterprises.

· longer time for new process development.

· the still highly empirical nature of catalyst selection.


The concept: how IRENE is facing the problem

IRENE wants to overcome the previously mentioned bottlenecks by the convergence of different expertise for developing computational methods and strategies for rationally design and produce the next generation of efficient biocatalysts for industrial applications.

Due to the strong interaction between theoretical groups and experimentalists all computational tools used in this project are validated by experiments. Failures and successes are used for methods’ evaluation, correction, tuning, comparison and combination, in an iterative process that is leading to the development of new methods and strategies, but also to the definition of practical guidelines, for any specific enzyme design issue.

IRENE is pursuing these objectives by:

· taking advantage of computational strategies used in different disciplines and integrating them in a unified concept for studying enzyme catalysis.

· creating synergies and complementarities between experimental and computational approaches in order to transform information into knowledge.


The subjects of IRENE current activities

The experimental and computational work is focused on different specific enzymes, which are expected to have strong impact on the chemical, pharmaceutical and food industry, as well as in the applications to biotransformations and biomass conversions.

From the conceptual point of view, IRENE has three major design subjects:

· the introduction of new activities in specific enzyme scaffolds (reaction promiscuity).

· the improvement of catalytic activity towards specific targets (substrate promiscuity).

· the redesign of enantioselectivity.

The work on each different design subject involves more than one enzyme and is proceeding in parallel, thus limiting the risks of not achieving the methodological project objectives because of the failure of one single goal.


Scientific and technological impact of the computational methods already developed and under-development within the IRENE consortium

The platform of computational tools developed so far is directed to scientists and industrial operators for being applied to:

· rational design of efficient biocatalysts to be produced through engineering.

· fast and efficient in silico screening of available enzymes/mutants to fully exploit catalytic potential of existing biocatalysts and providing quantitative parameters describing enzyme’s efficiency.

· fast substrate-screening and rational substrate engineering.

· understanding molecular basis of biocatalyst’ action and properties.


The structure of IRENE project

IRENE work plan is composed by five work-packages dedicated to RTD activities and one horizontal work package (WP) for management and dissemination activities.



The entire project is organised as a combination of mutually interconnected approaches to meet the goal of rational design of enzymes. It is based on molecular modelling of mutants and in silico screening of massive libraries of mutant enzymes followed by production of most promising mutants and experimental validation of their catalytic properties. The different Tasks have been approached thanks to multiple computational expertise (QM, MM, MD, Multivariate Statistics, 3DQSAR) which ultimately converge inside the horizontal computational platforms of WP1.


Results achieved so far

The work on each different design subject involved more than one enzyme and is proceeding in parallel. This approach has allowed to develop and to validate successfully the required novel computational methods for each work package, despite the unavoidable technical difficulties faced in some cases . This has avoided the risk of not achieving the methodological project objectives because of the failure of one single goal.


Advances in development of computational approaches for enzyme engineering


Methods developed for enzyme engineering and function-activity correlations

The computational methods applied so far in the different tasks (sometimes in parallel) can be summarized as follows:

1 Molecular simulation, visualization and conceptualization

2 Conceptualization based on extraction of molecular descriptors

3 Energy based design and screening

4 Bioinformatics analysis to identify relevant motifs and correlations between specific domains

5 Multivariate statistical analysis and 3D QSAR

6 Automatic mutants generation based on “multiobjective optimization softwares” able to integrate all methods listed above to construct a high-throughput scheme (under development)

Overall, these methods appear as robust and in most cases have been already experimentally validated. Their integration proceed smoothly towards the design and production of second generation of mutants for target enzymatic activities. No specific contingency is envisaged, due to an intense flow of information and input among Partners.


Methods for in silico screening of enzymes and substrates

Computational methods were adapted and refined to be used for the in silico screening. Since quantitative methods are expected to be developed, experimental validation was accomplished in each case. The families of methods can be summarized as follows:

1 Specific enzyme mutants generation and virtual screening based on docking software

2 Virtual library generation and autodock based screening approach

3 3D-QSAR approaches

4 High-throughput screening of automatic generated mutants based on “multiobjective optimization softwares” (under development)


Methods for rational solvent selection

In addition, new methods for solvent screening and prediction of solvent effect are under development, with the aim of being integrated in the methods previously reported, so to be able to simulate more precisely the relevant experimental conditions:

· an algorithm was developed to calculate the effect of medium composition on biocatalysed reactions equilibrium.

· a study is on going on the effect of solvent on enzyme enantioselectivity by explicit solvent simulation for to find correlations between the solvent properties and enantioselectivity.



The overall evaluation of the methods and models shows that they are representative of the specific enzymatic targets, and suitable to be applied within the work-plan for experimental validation. No specific risk can be pointed out, also because the IRENE project structure is based on the study of different enzymes, so that computational methods can be adapted and validated as experimental data are produced by partners. There is no need for actions to tackle any contingency.


Advances in software development

· The existing version of the QM/MM method which is based on the EFP formalism, has been improved, generalized and implemented in the GAMESS-US package.

· A preliminary faster, but still accurate, version of the EFP QM/MM method has been developed, keeping the main idea of the EFP QM/MM approach.

· Graphical User Interface (GUI) was developed based on the Avogadro program, to improve the user-friendliness of the softwareAn approach based on high-performance docking-based calculations of structures and substrate specificity of mutant enzymes has been developed.

· A specific module has been integrated into the program LEAD (which performs fast and extra precise protein-ligand docking).

· A new python version of PROPKA has been released.



Overall, the advances on software are in line with the DOW and no deviation from the original work-plan is envisaged.


Advanced in the engineering of specific enzymes of industrial interest

The following list of biocatalysts has been rationally designed and is under development or already under optimization within the IRENE project:

· amide forming enzymes with higher efficiency and broader specificity as compared to the known proteases to be used in fine chemistry and in polymer chemistry (first generation ready, second generation under study).

· nitrilases endowed with higher stability as compared to the natural nitrilases and able to accept a wider array of substrates (designed first set of models, first library under evaluation, more models under study).

· esterase able to enlarge the application of ester-form enzymes to bulky substrates-(models delivered, first generation cloned and to be purified).

· Lipases to be used in the production of environmentally interesting and compostable polymers (models developed, first generation mutants produced and successfully tested).

· amidases with increased synthetic efficiency and improved regioselectivity for more cost effective enzymatic synthesis of beta lactam antibiotics (models designed, first generation produced and successfully tested).

· glycoside hydrolases with enhanced synthetic efficiency for the production of glycoconjugates of relevance and to be used in the biotransformation of “reluctant” oligosaccharides in food industry (models designed, first generation produced and successfully tested).

· enteropeptidases with higher activity and higher selectivity for biological applications (models designed, first generation produced and successfully tested).

· Penicillin acylase mutants with improved enantioselectivity to be applied in specific enantioresolutions and cascade reaction (models designed, mutants produced and successfully tested).

· CALB mutants endowed with different or inverted enantioselectivity to be applied in in convergent stereo selective transformation and DKR (provided by one of IRENE Partners, quantitative predicting model for stereoselectivity prediction developed).

· Hydroxynitrilyase to be applied in DKR and cascade reactions (designed, cloned, to be purified).

Finally, a number of lipases able to accept structurally different fatty acids for food applications has been produced, characterized and tested.


What will be impact of IRENE project at industrial and economic level?

At present European companies supply about 70% of the world enzymes. To maintain and strengthen the European leadership in the biocatalysis sector, different routes must be pursued to make enzymes readily available for practical applications, thus making biocatalysis competitive as compared to conventional organic chemistry.


What will be the impact of IRENE project at political and social level?

· Promoting the cohesion of the scientific community at European, Russian level, involving also Uzbekistan, thus fostering the principles of the bio-economy.

· Boosting the utilization of renewable resources in a sustainable manner.

· Improving the environmental sustainability of productive processes as well as the safety aspects associated to workers’ health in industry.

· Contributing to European policies for the construction of a “Knowledge based” society.

· Attracting and training young researchers, in particular women, to form a new skilled generation.

· Accelerating the implementation of the European bio-based economy.

· Boosting international co-operation with Russia and Uzbekistan in crucial scientific and technological areas.


IRENE is a project funded by the European Commission under the Seventh Framework Programme
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