Exploitation of the Myrosinase-Glucosinolate System

Biotechnology

- Myrosinase immobilization
- Sulfatase immobilization

Myrosinase immobilization
Myrosinase was purified from Sinapis alba seeds and immobilized on different low cost solid matrices such as alumina, silica etc. (Iori et al. 1988. Biotechnol. Lett., 10, 575-578). The myrosinase was also immobilized on Nylon 6.6 that was used to build up a biosensor for total glucosinolate determination (Leoni et al. 1991, J.Agric. Food Chem., 39, 2322-2326) and a bioreactor useful to produce a number of glucosinolate derivative products (Leoni et al. 1993, Tetrahedron Lett. 34, 7967-7970; Leoni et al. 1994, Tetrahedron Asymm., 5, 1157-1160; Leoni et al. 2000. Biotechnol Bioeng. 68, 660-664).
The biological properties of the compounds produced by the bioreactor and their potentiality in fine chemistry have been investigated (Leoni et al. 2000. Tetrahedron Asymm. 10, 4775-4780; Gueyrard et al. 2000. Heterocycles 52, 827-643; Gueyrard et al. 2001. Tetrahedron Asymm. 12, 337-340; Galletti S. et al 2001. J. Agric. Food Chem. 49, 471-476).

Sulfatase immobilization
Sulfatase was also purified from crude commercial preparation of Helix pomatia and then immobilized on Nylon 6.6.
A small bioreactor was performed for analytical purpose and for preparing desulfoglucosinolates that were used as HPLC analytical standards and to determine their response factors in HPLC analysis when a UV detector was used (Leoni et al. 1996 Ind. Crops Prod., 7, 335-343; Iori et al. 1996 Euroanalysis IX, European Conference of Analytical Chemistry Bologna, Italy, Sept. 1-7, 1996. Book of abstracts TuP118; Iori et al., 1999 10th International Rapeseed Congress Canberra (Australia) 27-30 Sept. 1999 Abstract Book, pag. 121).
Finally, desulfoglucosinolates were also used as starting molecules to produce pure nitriles by a commercial recombinant b-O-glucosidase (Iori et al. 1999, 10th International Rapeseed Congress Canberra (Australia) 27-30 Sept. 1999, Abstract Book, pag. 119; Wathelet J-P et al. 2001, Biotechnol. Lett. 23, 443-446).

Contacts:
Dr. Onofrio Leoni, o.leoni@isci.it
Dr. Renato Iori, r.iori@isci.it

Nutraceuticals & Chemoprevention

Animal and cell line studies have demonstrated that Brassica vegetables like broccoli, cabbage, Brussels sprouts, etc. have a protective effect against different kind of cancers. The availability of a number of pure glucosinolates in gram-scale and also pure myrosinase has allowed performing in vitro and in vivo studies, mostly carried out in cooperation with some national and European research groups (Nastruzzi et al. 1996. J. Agric. Food Chem., 44, 1014-1021; Iori et al. X Congresso Nazionale Società Italiana di Fitochimica (SIF), Firenze, 7-10 Maggio 2000, p. 25.; Hrelia et al. 2000. Eurotox 2000, XXXVIII European Congress of Toxicology. London, 17-20 September 2000.: Nastruzzi et al. 2000. J. Agric. Food Chem., 48, 3552-3575; Fimognari et al. 2002. Carcinogenesis, 23, 581-586.; Perocco et al. 2002. Cancer Letters 184, 65-71; Fimognari et al. 2002 Cell. Mol. Life Sci. 59, 2004-2012; Pappa et al. 2003. ICMAA-VIII International Conference Pisa, Italy, 4-8 October 2003; Fimognari et al. 2003. Annals of the New York Academy of Sciences (in press); Paolini et al. 2003. Carcinogenesis (in press).

Activity

Extraction and purification of several glucosinolates such as glucoraphanin, glucoerucin, gluconasturtiin, sinigrin, glucotropaeolin, glucoraphenin, glucoraphasatin, glucomoringin and glucobrassicin. (Iori et al. 1999. Bioorg. & Med. Chem. Letters, 9 (7): pp. 1047-1048; Barillari et al. 2001 Fitoterapia, 72, 760-764; Barillari et al. 2002. 23rd IUPAC-2002 International Symposium on the Chemistry of Natural Products, 28 July-2 August 2002 Florence, Italy; Barillari et al. 2002. NutraCos Nov./Dec. 2002, 6-9. Iori et al. 2003. IENICA Symposium, Bologna - Italy, May 15 - 16, 2003).
These glucosinolates were in white powder form and are available in a gram scale useful for the in vivo and in vitro tests. The yield of purification is between 2-3% with respect to the starting materials and the purity is more than 90%.
Purification of Myrosinase for in vivo and in vitro tests (in situ production of isothicyanates).
Production and purification of isothiocyanates, available in a gram scale for in vivo and in vitro tests, from the corresponding glucosinolates.
Purification of desulfo-glucosinolates to use as standard in HPLC analysis.
Determination of dietary glucosinolates in vegetables.

Contacts:
Dr. Renato Iori, r.iori@isci.it
Dr. Jessica Bacillari, j.barillari@isci.it

Biofumigation

Biofumigation is an alternative strategy to control soil-borne pathogens with a low environmental impact using plants containing chemical compounds characterized by a biocidal activity on a wide array of soil-borne fungi, nematodes, insects and weeds. In particular, great attention is devoted to Brassicaceae and other minor families containing the glucosinolates-myrosinase system which is able to produce, after tissues damage, compounds with interesting applicative perspectives due to their well-known biocidal activity.

Activity

Preparation of pure glucosinolates hydrolysis derivative products and optimization of the analysis procedures for their determination on headspace and solution (Leoni et al., Proceedings of the 25th International Symposium on Capillary Chromatography, Riva del Garda -ITALY)
Evaluation of the biological activity of pure glucosinolate degradation products toward fungi (Manici L. M. et al. 1997. J. Agr. Food Chem. 45, 2768-2773; Manici M. L., et al. 1998. 9th Intern. Congress Pest. Chem., London 1998), nematodes (Lazzeri L. et al. 1993, J. Agr. Food Chem., 41, 825-829 ; Curto G. et al. 2002. Nematol. Medit. 30 Supplemento: 71-74) and weeds (Angelini L. et al. 1998. Seed Sci Tech. vol. 26, 3, 771-779)
Selection of several ecotypes of Brassicaceae vocated for an application as biocidal green manure crops and characterized from different agronomic and technological characteristics .
Study of biocidal green manure effect both in laboratory (Lazzeri L., Manici L.M. 2001. Hortsci. 36(7): 1283-1289) and full field trials to control fungi and nematodes mainly on strawberry (Lazzeri L.. 2003. Pest Manag. Sci. 59, 9: 983-990).
Study on biocidal plants dehydration to produce biocidal pellets both at a full field level and in industrial plant (Lazzeri L. et al., 2002. In press on Ind. Crops Prod.)
Study of the potential of producing biocidal meals starting from Brassicaceae seeds. Selection and optimization of the cultivation techniques of seed biocidal varieties.
Evaluation of the effect of biocidal meal and pellets on some soil pathogens (Patent Cerealtoscana - Lazzeri et al. N° Dep. BO2002 A 0005444) and on some post harvest applications.

Contacts:
Dr. Luca Lazzeri, l.lazzeri@isci.it
Dr. Onofrio Leoni, o.leoni@isci.it

Food technology

Post-harvest pathogens control
In the ambit of a National Project named RAISA (Advanced Researches for Agricultural System Innovation), supported by the Italian National Research Council (CNR), the correlation between chilling injury of fruit during post-harvest and the activity of some oxidasic enzymes has been studied in detail (Leoni et al., 1993, Atti Simposio "La Qualità dei Prodotti Ortofrutticoli Freschi nella Fase di Postraccolta", Viterbo, 19-20 novembre 1992, p. 371-378; Gallerani et al. 1993, Ibidem, p 361-369).
In addition, some in vitro studies on the activity of some hydrolysis glucosinolates derivative products have been carried out (Mari et al., 1993, Ann. Appl. Biol., 123, 155-164; Mari et al., 1996 Plant Pathol. 45, 753-760; Mari et al. 2002. Plant Pathol. 51, 231-236). The opportunity to apply the results of these studies in fruit protection from Monilinia laxa and Penicillium expansum during storage is now under investigation in the ambit of a National Research Project, named VALE (Valorizzazione della qualità della produzione nazionale di frutta: studio di una nuova tecnologia di conservazione post-raccolta a base di formulati vegetali funzionalizzati).

Contacts:
Dr. Onofrio Leoni, o.leoni@isci.it