Isolation and physicochemical characterization of bioactive proteins and
special secondary metabolites for their use in agro-industry technology

1. Enzymes & Cofactors
2. Protease inhibitors
3. Secondary metabolites

Enzymes & Cofactors

Peroxidases

• Horse radish and tomato peroxidase, relationship with indole acetic oxidase acivity: biochemical genetics in tomato mutants and inhibition study by ascorbic acid (Palmieri et al. 1978. Physiol. Plant.42, 85-90; Palmieri & Giovinazzi F., 1982, Physiol. Plant. 56, 1-5).
• Isolation from durum wheat meal and relationship between peroxidase activity and the yellow colour of pasta (Iori et al. 1995, Cereal Chem., 72, 176-181).
  

Ascorbic acid oxidase

• Isolation from zucchini squash and characterization of its main physicochemical properties:
  - stopped flow kinetic of its aerobic reduction and (Baici et al. 1979. J. Mol. Catal. 6, 135-143)
  - crystallization and X-ray studies of the crystals (Ladenstein et al., 1979. FEBS Lett. 107, 407-408).
  

Lipoxygenase

• Isolation from germinating sunflower seeds and its molecular characterization (Leoni et al.,1985. J. Food Sci. 50, 88-92).
• Behaviour of soybean lipoxygenase in hydrocarbon micellar system (Meier et al. 1981. J. Am. Oil Chem. Soc., 601A).
• Use of lipoxygenase in enzyme technology:
  - in a coupled enzymatic assay for determining lipase activity in hydrocarbon reverse micellar system (Hochkoeppler & Palmieri 1990. Biocatalysis, 3, 357-365)
  - to measure oxygen uptake in hydrocarbon reverse micelles solutions (Hochkoeppler & Palmieri 1990. Biotechnol.Bioeng. 36, 672-677).
  

Polyphenol oxidase
Isolation and characterization of polyphenol oxidase from artichoke heads ant its relationship with browning and storage aptitude (Leoni et al. 1990, Food Chem., 38, 27-39; Lattanzio et al. 1994. Food Chem. 50, 1-7; Lattanzio et al. 1994. It. J. Food Sci. 1, 3-30)

Proteases

• Technological application of sunflower defatted meal containing proteases in milk clotting (Walde et al.,1982, Int. J. Nutr. Res., 52, 230; Palmieri et al.,1984. Proceedings International Symposium on Science and Biotechnology for an Integral Sunflower Utilization, Bari, 25 October 1984, pp. 197-214.
• Isolation and study of molecular properties of some germinating sunflower seed proteases (Walde et al., 1984. J. Agric. Food Chem. 32, 322-329).
• Isolation by hydrocarbon reverse micelles, and by traditional multi-step column chromatography, of a trypsin like enzyme from larvae midgut of European Corn Borer Ostrinia nubilalis. Study of its main properties, including the inhibition potential of some proteinic inhibitors (Bernardi & Palmieri, 1996. Biotechnol. Lett. 18, 663-666; Bernardi et al., 1996, Insect Biochem.& Mol. Biol. 26, 883-889).
  
  

Myrosinases and Cofactors
Isolation, characterization, structural studies, analytical and technological applications.

• Two plant myrosinases, -thioglucoside glucohydrolases (EC. 3.2.3.1), (from seeds of Sinapis alba and Crambe abyssinica) have been isolated, using affinity chromatography and other traditional column chromatographic techniques (chromatofocusing, ion exchange chromatography etc.). These enzymes were also characterized for their main physicochemical properties (Palmieri et al.,1982. Anal. Biochem. 123, 320-324; Palmieri et al.,1986. J. Agric. Food Chem. 34, 138-140; Pessina et al.,1990. Arch. Biochem. Biophys., 280, 383-389; Hochkoeppler & Palmieri 1992. Biotechnol. Prog. 8, 91-96; Bernardi et al., 2003. J. Agric. Food Chem. 57, 2737-2744).
• The three dimension structure and the aminoacid sequence of the main myrosinase isoenzyme, isolated from ripe seeds of Sinapis alba, was also determined by X ray crystallography (Burgmeister et al. 1997. Structure 5, 663-675).
• Myrosinase was also exploited in soluble and in immobilized form for quantitative analysis of glucosinolate in Brassicaceae tissues and extracts and to produce some of the main glucosinolate degradation products (Leoni et al.,1993. Tetrahedron Lett. 34, 7967-7970; Leoni 1994. Tetrahedron Asymm. 5, 1157-1160; Leoni et al., 2000. Tetrahedron Asymm. 10, 4775-4780; Leoni et al., 2000. Biotechnol. Bioeng. 68, 660-664; Galletti et al., 2001. J. Agric. Food Chem. 49, 471-476; Finiguerra et al., 2001. J. Agric. Food Chem. 49, 840-845; Gueyrard 2001. Tetrahedron Asymm.12, 337-340.
• The myrosinase cofactor, Epithio-Specifier Protein (ESP) has been isolated from ripe oilseed rape seeds. Almost 80% of its primary structure has been determined. (Bernardi et al., 2000. FEBS Letters 467, 296-298).
  

Protease inhibitors

A new family of trypsin inhibitors has been isolated and characterized in the seeds of Brassicaceae family. Particular attention has been addressed to:

MTI 1
A new trypsin inhibitor of Kunitz type (18 kD) was isolated from ripe seeds of white mustard (Sinapis alba L.): The stoichiometry of the trypsin-inhibitor complex was 1 to 1, with a dissociation constant of 2.2 x 10-9 M. (Menegatti et al., 1985. J. Agric. Food Chem., 33, 784-789).

MTI 2
A new serine proteinase inhibitor (MTI 2) was isolated from ripe white mustard seeds. MTI 2 inhibits bovine trypsin and chymotrypsin with dissociation constants (Kd) of 1.6 x 10^-10 and 5 x 10^-7 respectively at pH 8.0 and 21°C, with a stoichiometry of 1 to 1. The amino acid sequence was also determined (Menegatti et al., 1992. FEBS Lett. 301, 10-14).

RTI
- A new serine proteinase inhibitor (RTI) was isolated from ripe oilseed rape seeds. RTI inhibits bovine trypsin and chymotrypsin with dissociation constants (K_d) of 3.0 x 10^-10 and 4.1 x 10^-7 respectively at pH 8.0 and 21°C, with a stoichiometry of 1 to 1. The amino acid sequence and the reactive site was also determined at position Arg²⁰-Ile²¹. Ceciliani et al., 1994. FEBS Lett., 342, 221-224.
- The trypsin inhibitory activity (TIA) was measured in six oilseed rape cv of double high and "00" genotypes: TIA was 3.9 IU g^-1and 6.9 IU g^-1 respectively. Three different inhibitors were also characterized, especially as resistance against thermal denaturation. Genotypes "00" show also a higher thermostable TIA (Visentin et al. 1992. Phytochemistry 31, 3677-3680).
- TIA distribution and some molecular properties of the inhibitors were also studied not only in oilseed rape but also in some minor crucifers (Iori et al., 1991. GCIRC Eighth International Rapeseed Congress, July 9 -11, 1991 Saskatoon, Saskatchewan, Canada Vol 3, pp. 911-916; Iori et al. 1995. GCIRC Eighth International Rapeseed Congress, July 4-7 1995, Cambridge, UK)

Secondary metabolites

Glucosinolates are one of the most important compounds of this class of molecules. More than 20 different glucosinolates were isolated from different Brassica ripe seeds, including some glucosinolates not previously purified and characterized such as 4-methyl-tio-butenyl-glucosinolate and glucobrassicin, using an improved outline of purification (Visentin et al.1992. J.Agric. Food Chem., 40, 1687-1681; Barillari J., et al., 2001. Fitoterapia, 72, 760-764). Pure glucosinolates were characterized by HPLC and NMR spectrometry and used as substrates with different kind of myrosinases for their kinetic characterization. These glucosinolates, and white mustard myrosinase, were used to catalyze their hydrolysis for determining the in vitro cytotoxicity of the correspondent degradation products (isothiocyanates, nitriles, thiones, etc.) on some human tumoral cell lines (Nastruzzi et al. 1996. J.Agric. Food Chem. 44, 1014-1021; Leoni et al.,1997. Bioorg. Med. Chem. 5, 1799-1806). This enzymatic system was also used to evaluate the cytotoxicity towards different kind of plant pathogens (fungi, nematodes, insects etc.) (Mari et al. 1996. Plant Pathol. 45, 753-760; Lazzeri L. 1993. J. Agric. Food Chem., 41, 825-829; Manici et al. 1997. J. Agric. Food Chem. 45, 2768-2773; Palmieri S., 1998. 9th Int. Cong. Pesticide Chemistry, London 2-7 August 1998).

Contacts:
Dr. Sandro Palmieri, s.palmieri@isci.it
Dr. Renato Iori, r.iori@isci.it