Purification and Characterization of Linamarase from Lakan-1 and Java Variety Cassava (Manihot esculenta Crantz)

Linamarase was extracted from fresh Lakan-1 and Java variety cassava leaves and petioles through homogenization and ammonium sulfate extraction. Gel filtration chromatography (GFC) results showed increased purification and activity of enzymes but a decrease in the yield. The enzyme-specific activity was at its highest in pooled GFC Java samples at 0.7754 U/mg protein, whereas pooled GFC Lakan-1 samples have a specific activity of 0.6704 U/mg protein. Crude extracts of Java and Lakan-1 variety cassava have a total protein concentration of 10.0209 and 10.2052 mg, respectively. On the other hand, GFC Java and Lakan-1 samples have protein concentrations of 0.1413 and 0.1634 mg, respectively, which is significantly lower compared to the crude extracts. Electrophoresis results confirmed the presence of linamarases, with bands at 67 kDA comparable to the theoretical values of 62.5 and 65 kDA for its alpha and beta variants, respectively. These bands were excised from the gel and were analyzed by LC/ MS-MS. The LC/MS-MS results showed peptide fragment FGLYYVDYK, which is found in the 491–499 amino sequence of a β-glucosidase, the family of linamarase. Arginine (R) was the most abundant amino acid in the purified linamarase, with the Java variety having 98.927% arginine and Lakan-1 having 98.390% arginine. The other amino acids present in minute amounts were glutamic acid (E), histidine (H), threonine (T), lysine (K), and glycine (G), which can also be found in the linamarase sequence.