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Enzymatic tailoring of protein interactions and functionalities in food matrix

Final Activity Report Summary - PRO-ENZ (Enzymatic tailoring of protein interactions and functionalities in food matrix)

Novel crosslinking enzymes were screened, purified, biochemically characterised and used for detailed reaction mechanism studies as well as for application tests as part of the PRO-ENZ project.

The enzymes were screened from different microbial sources and from microbial genome databases. The enzymes were produced, when possible, in homologous systems, or alternatively using heterologous expression systems. The PhD training of two students, i.e. Greta Faccio and Chiara Gasparetti was focussed on the production of novel enzymes.

Greta Faccio identified novel putative tyrosinase (tyr), catechol oxidases and sulfhydryl oxidases from gene sequences and subsequently expressed them in trichoderma. Sequence analysis of the available genes from the available genome sequences of filamentous fungi retrieved two groups of tyr sequences with respect to their length, ranging from 500 to 660 or 400 amino acids. The short sequences lacked the c-terminal domain, involved in the enzyme maturation. Results concerning the sequence analysis for secreted fungal tyrs, the production and the biochemical characterisation of a secreted catechol oxidase from a. oryzae produced in t. reesei were published (refer to Casparetti, Faccio et al. 2010, 'Discovery of a new tyrosinase-like enzyme family lacking a c-terminally processed domain: Production and characterisation of an aspergillus oryzae catechol oxidase', Applied Microbiology and Biotechnology 86(1):213-26).

Two novel sulfhydryl oxidase (SOX) enzymes, AoSOX1 and AoSOX2 were produced in t. reesei and biochemically characterised. Both enzymes were flavin-dependent enzymes active on reduced glutathione, dithiothreitol and in a lesser extent on d-cysteine and l-cysteine. The results from the expression and biochemical characterisation of the AoSOX1 and AoSOX2 were summarized in two publications, namely:

1. Faccio et al. 2010, 'Secreted fungal sulfhydryl oxidases: Sequence analysis and characterisation of a representative flavin-dependent enzyme from aspergillus oryzae', BMC Biochemistry 11:31 and
2. Faccio et al, 'Production and characterisation of a novel flavin-dependent sulfhydryl oxidase with good pH and temperature stability from a. oryzae', which was to be submitted by the time of the project completion.

The enzyme AoSOX1 was tested as an improver in wheat dough, in the presence of ascorbic acid. The addition of AoSOX1 resulted in softer dough, while in presence of ascorbic acid harder dough was obtained (more information in Faccio et al. 'Sulfhydryl oxidase effect on wheat dough also in presence of ascorbic acid', in the course of submission by the time of the project completion).

Chiara Gasparetti studied the reaction mechanism of the fungal tyr from t. reseei and compared it to the commercial tyr from agaricus bisporus (AbTyr). The results showed that the t. reesei tyr was different from the tyrs reported in literature, given that the formed end products from catechol and l-dopa were different. The enzyme had superior cross-linking ability on proteins in comparison to the other tyrs (refer to 'Comparison of substrate specificity of tyrs from trichoderma reseei and agaricus bisporus', Selinheimo E., Gasparetti C., Mattinen M., Steffensen C.L. Buchert J., Kruus K. 2009, Enzyme and Microbial Technology 44, 1-10.) The putative short tyr gene identified in the sequence analysis of the aspergillus oryzae genome was expressed in t. reesei, the enzyme was subsequently produced and partially characterised. The substrate specificity analysis revealed relatively high activity on catechol and only a minor activity on l-dopa, suggesting that the enzyme was rather a catechol oxidase than a tyr. The results were published as joint paper with Greta Faccio, titled 'Discovery of a new tyr-like enzyme family lacking a c-terminally processed domain: Production and characterisation of an aspergillus oryzae catechol oxidase', Gasparetti C., Faccio G., Arvas M., Buchert J., Saloheimo M., Kruus K. 2010, Applied Microbiology and Biotechnology, 86, 213-226.). Finally, crystals of the catechol oxidase from a. oryzae were obtained in collaboration with the University of Joensuu.