Isolation and partial purification of beta-galactosidases from cotyledons of two cowpea cultivars

Abstract

-Three isoforms of β-galactosidases were isolated and partially purified from the cotyledons of quiescent seeds of Vita 3 and Vita 5 cowpea [Vigna unguiculata (L.) Walp.] cultivars differing in water and salt stress tolerance. The purification procedure consisted of ammonium sulfate fractionation, acid precipitation, ion exchange chromatography through DEAE-sephadex and affinity chromatography through Lactosyl-sepharose columns. The three isoforms isolated from the two cultivars showed the same chromatographic patterns, same optimum of temperature for enzyme activity assay (60ºC), identical thermal stability up to 50°C, and similar pH optima (3-4). However, they differed from each other in sensitivity towards metal ions and certain chemical agents presents in the assay medium. The results have shown that the observed differences in β-galactosidases from the cotyledons of quiescent seeds were not sufficient to relate them to stress tolerance. ADDITIONAL INDEX TERMS:cotyledons, isozymes, Vigna unguiculata, Vita 3 and Vita 5 cultivars, salt tolerance. ISOLAMENTO E PURIFICAÇÃO PARCIAL DE β-GALACTOSIDASES DE COTILÉDONES DE DOIS CULTIVARES DE FEIJÃO-DE-CORDARESUMO -Três isoformas da enzima β-galactosidase foram isoladas e purificadas parcialmente a partir de cotilédones de sementes quiescentes dos cultivares Vita 3 e Vita 5 de feijão-de-corda [Vigna unguiculata (L.) Walp.], os quais diferem em tolerância aos estresses hídrico e salino. O processo de purificação consistiu de fracionamento com sulfato de amônio, precipitação ácida, seguida pela associação de cromatografia de troca iônica em coluna de DEAE-Sephadex com cromatografia de afinidade em coluna de Lactosyl-Sepharose. As três isoformas isoladas dos dois cultivares mostraram os mesmos padrões cromatográficos, mesma temperatura ótima de ensaio para atividade enzimática (60ºC), idêntica estabilidade térmica até 50ºC e apresentaram ótimos de pH semelhantes (3-4). Entretanto, diferiram umas das outras em suas sensibilidades a íons metálicos e a certos agentes químicos presentes no meio de reação. Os resultados mostraram que as diferenças observadas nas β-galactosidases de cotilédones de sementes quiescentes foram insuficientes para correlacioná-las com a tolerância ao estresse.

β-galactosidases from cowpea stems: properties and gene expression under conditions of salt stress

“…The optimum test temperature for the -gal I, II and III enzymes was 55 °C, with a considerable drop in activity from 60 °C. The range seen for the optimum test temperature is similar to those found for other -galactosidases purified from plants (CHILAKA;OKEKE;ADAIKPOH, 2002;ENÉAS-FILHO et al, 2000;ENÉAS-FILHO, 2001;SUDÉRIO et al, 2011b). In the study of thermostability, -gal I showed a reduction in activity when incubated at just 40 °C, with all enzymes showing a sharp drop in activity at 60 °C until complete inactivation at 70 °C (Figure 2).…”

“…The activities of the cytosolic -galactosidases ( -gal I and II) and cell wall -galactosidase ( -gal III) as a function of the pH of the reaction medium, showed an increase from pH 2.5 until reaching maximum activity at pH 4.0, with a progressive decrease to pH 6.5 (Figure 1). The optimal pH activity range demonstrated by -gal I, II and III is common to a wide variety of plant species, especially legumes (ALCÂNTARA et al, 2006;BISWAS;KAYASTHA;SECKLER, 2003;CHILAKA;OKEKE;ADAIKPOH, 2002;ENÉAS-FILHO et al, 2000;ENÉAS-FILHO et al, 2001;SUDÉRIO et al, 2011b). The optimum test temperature for the -gal I, II and III enzymes was 55 °C, with a considerable drop in activity from 60 °C.…”

“…The elution of the peaks retained on the column was done with a salt gradient (0.2-1.0 M NaCl in a buffer of 100 ml 25 mM Tris-HCl pH 7.2). (ENÉAS-FILHO et al, 2000).…”

“…Elution was performed with an equilibration buffer containing 100 mM lactose and 0.5 M NaCl. Those fractions rich in -galactosidase activity were pooled, dialysed against deionised water for 24 hours at 4 °C and stored at -20 °C (ENÉAS-FILHO et al, 2000).…”

Isolation of the promoter of a cotton β-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants

“…Thus, it was concluded that the natural substrate of β-galactosidase is the storage xyloglucan of the cotyledonary cell walls [44] . Furthermore, the cotyledons of several plants were often used to purify β-galactosidase including Vigna unguiculata, Copaifera langsdorffii, and Hymenaea courbaril [45][46][47] . Our results are coincident with those of the studies described above and suggest that the plant β-galactosidase has a critical role in the cotyledons of the germinated seeds.…”