Spruce (Picea abies) seeds contained a soluble β-glucosidase which was active with 4-nitrophenyl β-D-glucoside but did not catalyse hydrolysis of coniferin (conifery β-D-glucoside). In contrast, a cell wall fraction from hypocotyls and roots of spruce seedlings showed good glucosidase activity towards coniferin with a pH optinum Between 4.5 and 5.5. A number of other aryl β-glucosides were also hydrolysed by these particulate fractions. Michaelis constants and maximal velocities were determined for coniferin (Km =380 and 1300 μ for roots and hypocotyls, respectively) and other aryl glycosides. The cell wall fractions probably contain more than one glycosidase. For 4-nitrophenyl β-D-glucoside and picein (4-hydroxyacetophenone β-D-glucoside) two separate and distinct lines could be drawn for high and low substrate concintrations in Lineweaver-Burk of Eadie-Hofstee plots.
Attempts to solublize the β-glucohydrolase activity could be extracted from the hypocotyl particulate fraction by treatment with 0.6 M Nacl.
The solublized enzyme was separated on Sepharose 6 B into was fractions with glucosidase activity (glucosidase I and II). Glucosidase I was Purified to apparent homogeneity by repeated gel filtration on Sepharose 6 B. Dodecylsulfate-gel electrophoris and sedimentation equilibrium measurements showed that glucosidase I is composed of only one polypeptide chain with Mr 58 570 SS2400. The properties of glucodidase I with respect to pH and temperature optimum and to substrate specificity towards coniferin and other aryl β-glucosides are similar to those of the hypocotyol particulate fraction. In contrast, glucosidase II has a 10 times smaller ratio for the relative maximal velocities with coniferin and 4-nitrophenyl β-glucosidase than glucosidase I.
The presince of cell-well-bound β-glucosidase for coniferin in spruce seedlings is consistent with the view that coniferin participates in lignification.