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Biocatalyst for 1-naphthol production

Country of Origin: Spain
Reference Number: TOES20160418001
Publication Date: 18 April 2016

Summary

A Spanish public research organisation has generated a genetic variation of the peroxigenase from the A. aegerita fungi, with enhanced properties of catalytic activity, stability and heterologous expression capacity. The mutant enzyme has been designed to be applied in aromatic hydroxylation, especially for naphthol synthesis, being a green alternative to the chemical process. Companies interested in the enzymatic production of naphthol using this biocatalyst, under patent license, are sought.

Description

1-naphthol is an important intermediate in the chemical industry. The production of this organic chemical typically implies the use of highly contaminating catalysts; high energetic consumption; high production costs and high amount of polluting wastes. In this context, enzymes with oxygenase activity can provide an alternative process, less contaminant than the traditional one.  

The oxygenase enzyme of this invention was obtained applying genetic engineering to the unspecific peroxigenase (UPO) secreted by the edible mushroom Agrocybe aegerita. Starting with the wild-type UPO, some mutations where introduced with the directed evolution technique, resulting in new mutant proteins with increased peroxygenase (mono-oxygenase) activity compared with peroxidase activity, making it ideal for being used un selective hydroxylation process and decreasing by-products generation in the reaction medium. Moreover this new mutants can be expressed in industrial commonly used yeast, as Saccharomyces cerevisae or Pichia pastoris. 

Some test reactions were carried out with the new enzyme for the synthesis of 1-naphthol from naphthalene. The regioselectivity obtained for the 1-naphthol was around 97%, which is similar to the values found in the wild type UPO, but the final yield was 156% higher than expected for a UPO. Moreover, the mutant enzyme was applied to other substrates confirming that the peroxidase activity had decreased between 3 and 11, that is the reason of the low yield found for the by-products in the synthesis of 1-naphthol (values of 1,4-naphtoquinone below 1%).

The Spanish research center is looking for enzyme producers or chemical companies that can be interested in the production or the use of enzyme for the synthesis of 1-naphthol, under a patent license. The enzyme has only been tested at a laboratory scale, but it can be easily adapted to an industrial scale and can be produced in enough amounts to be applied in industrial reactors.   

The Spanish public entity has broad experience in the development of novel catalyst for innovative industrial processes and in the development of novel enzymes using directed evolution technique.

Advantages and Innovations

• The enzyme catalyses the naphthalene selective hydroxylation in one step, without co-factors, at ambient temperature and pressure and in aqueous medium.

• The mutant can be expressed in S. Cerevisiae y P. Pastoris reaching levels of production up to 0.2 g/L, which is suitable for its industrial use. 

• Shows a high thermal stability, with a value of Tª50 of 59.7º C.

• The enzyme is stable in presence of organic solvents, which are necessary for increasing the solubility of the naphthalene in aqueous mediums. 

• The total turnover number (TTN) is around 50,000, similar to those enzymes commercially available.

• The catalytic efficiency for naphthalene is up to 6.2 x 105 s-1 M-1.

• The enzyme shows other additional industrial uses in organic synthesis (oxy-functionalisation of hydrocarbons, drugs synthesis or bioremediation) and in the development of biosensors

Stage Of Development

Under development/lab tested

Requested partner

The Spanish research center is looking for enzyme producers or chemical companies that can be interested in the production or the use of enzyme for the synthesis of 1-naphthol, under a patent license.