Bioprospecção de proteínas de interesse biotecnológico em folhas de Morinda citrifolia L.

Abstract

Morinda citrifolia L. (Rubiaceae), popularly known as noni, has been used by Polynesians for food and medicinal purposes for over 2000 years. Although it is a widely used species with medicinal purpose, few studies are focused on the isolation and evaluation of the biotechnological potential of its proteins. Thus, this work aimed to prospect proteins in the noni leaves and to evaluate their potential for application in areas of economic and biotechnological interest. The leaves obtained from field-grown plants were dried at room temperature for seven days and used for protein extraction with the following buffers: 50 mM sodium acetate, pH 5.0, 50 mM sodium phosphate, pH 7.0 and 50 mM Tris-HCl, pH 8.0, in the ratio of 1:10 (w/v). The most alkaline condition was able to significantly extract the highest protein content (3.642 ± 0.259 mg/g of dry leaf), with an electrophoretic profile (SDS-PAGE 12.5%) composed mainly of protein bands with apparent masses of 18, 30 and 40 kDa. The crude extract showed the presence of hemagglutinating activities (154.95 ± 3.34 HU/mg), proteolytic (197.02 ± 7.07 AU/mg), peroxidase (4.15 ± 0.17 U/mg) and mainly inhibitory trypsin (1335.01 ± 27.84 IU / mgP). Using the same alkaline condition for protein extraction from leaves obtained in vitro, it was possible to extract 6,480 ± 0,366 mgP/g of fresh leaf, as well as to detect the peroxidase (1.00 ± 0.09 U/mg), proteolytic (140,07 ± 2, 82 AU/mg) and trypsin inhibitory activities (200.00 ± 14.14 IU/mg). A protocol for the partial purification of a trypsin inhibitor of this species was delineated due to the high specific activity obtained for the inhibition of trypsin in the crude extract of the noni leaves obtained from field cultivated plants. NLTI (Noni Leaf Trypsin Inhibitor) was isolated using the steps of three-phase partition and affinity chromatography on Sepharose-4B matrix containing immobilized bovine trypsin, exhibiting specific activity of 9,000 IU/mg, an apparent molecular mass of 16 kDa, with an index of purification of 6.74 times and yield of 0.42%. The results showed that the leaves of M. citrifolia L. are a promising source of proteins with biotechnological potential, particularly trypsin inhibitors, supporting future investigations that allow the development of biotechnological products based on these macromolecules, as well as the production of these macromolecules from plant tissues grown in vitro.