Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein.

South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary structural and physicochemical characteristics of partial but not full-length begomovirus proteins have been elucidated to date. The full-length codon-optimised SACMV BC1 gene was cloned into a pET-28a (+) expression vector and transformed into expression host cells E. coli BL21 (DE3). The optimal expression of the full-length BC1-encoded movement protein (MP) was obtained via induction with 0.25 mM IPTG at an OD600 of ~0.45 at 37 °C for four hours. Denatured protein fractions (dialysed in 4 M urea), passed through an IMAC column, successfully bound to the nickel resin, and eluted using 250 mM imidazole. The protein was refolded using stepwise dialysis. The molecular weight of MP was confirmed to be 35 kDa using SDS-PAGE. The secondary structure of SACMV MP presented as predominantly ß-strands. An ANS (1-anilino-8-naphthalene sulphonate)-binding assay confirmed that MP possesses hydrophobic pockets with the ability to bind ligands such as ANS (8-anilino-1-naphthalenesulphonic acid). A 2' (3')-N-methylanthraniloyl-ATP (mant-ATP) assay showed binding of mant-ATP to MP and indicated that, while hydrophobic pockets are present, MP also exhibits hydrophilic regions. Intrinsic tryptophan fluorescence indicated a significant conformational change in the denatured form of BC1 in the presence of ATP. In addition, a phosphatase assay showed that MP possessed ATPase activity.

Starch Hydrolysis, Polyphenol Contents, and In Vitro Alpha Amylase Inhibitory Properties of Some Nigerian Foods As Affected by Cooking.

The effect of cooking on starch hydrolysis, polyphenol contents, and in vitro α-amylase inhibitory properties of mushrooms (two varieties Russula virescens and Auricularia auricula-judae), sweet potato (Ipomea batatas), and potato (Solanum tuberosum) was investigated. The total, resistant, and digestible starch contents of the raw and cooked food samples (FS) ranged from 6.4 to 64.9; 0 to 10.1; and 6.4 to 62.7 g/100 g, respectively, while their percentages of starch digestibility (DS values expressed as percentages of total starch hydrolyzed) ranged from 45.99 to 100. Raw and boiled unpeeled potato, raw and boiled peeled potato, raw A. auricula-judae, and sweet potato showed mild to high α-amylase inhibition (over a range of concentration of 10-50 mg/mL), which was lower than that of acarbose (that had 69% inhibition of α-amylase over a range of concentration of 2-10 mg/mL), unlike raw R. virescens, boiled A. auricula-judae, and boiled sweet potatoes that activated α-amylase and boiled R. virescens that gave 0% inhibition. The FS contained flavonoids and phenols in addition. The significant negative correlation (r = -0.55; P = 0.05) between the α-amylase inhibitory properties of the raw and cooked FS versus their SD indicates that the α-amylase inhibitors in these FS also influenced the digestibility of their starches. In addition, the significant positive correlation between the α-amylase inhibitory properties of the raw and cooked FS versus their resistant starch (RS) (r = 0.59; P = 0.01) contents indicates that the RS constituents of these FS contributed to their α-amylase inhibitory properties. The study showed the usefulness of boiled unpeeled potato, boiled potato peeled, and raw sweet potato as functional foods for people with type 2 diabetes.