Locust bean galactomannan

For ScAGal substrate specificity characterization, substrates PNPG, melibiose, raffinose, stachyose and locust bean galactomannan from Sigma Aldrich, and substrates Gal¹Man₃ and Gal^3,4Man₅ together with Helix pomatia β-mannosidase acquired from Megazyme, were used. The hydrolysis products were analysed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The α-Gals of Aspergillus niger and Cyamopsis tetragonoloba from Megazyme were used for a comparative study of the hydrolysis products. Each reaction and the corresponding controls were prepared in triplicate.

Besides barley β-glucan, other substrates including sodium carboxyl methyl cellulose (CMC-Na), 4-nitrophenyl α-d-galactopyranoside (pNPG), 4-nitrophenyl β-cellobioside (pNPC), birchwood xylan, Avicel, phosphoric acid swollen cellulose (PASC) and locust bean galactomannan were all purchased from Sigma-Aldrich. Lichenin was purchased from Megazyme (Wicklow, Ireland). Whatman Grade 1 filter paper was purchased from General Electric Company, United Kingdom. The enzyme activities of Cel7A towards various substrates (1.0%, w/v) were assessed by using the standard methods. Except for filter paper that required a longer incubation period (30 min), other substrates were incubated with Cel7A for 10 min. All enzyme activity measurements were carried out three times.
The kinetic parameters K_m and V_max were determined in 100 mM McIlvaine buffer (pH 5.0) containing 0.5−10.0 mg ml⁻¹ barley β-glucan at 60°C for 5 min. The K_m and V_max values were calculated according to the Lineweaver-Burk method.