*2.4. Crude ALDH Extraction from Banana Pulps, and Functional Characteristics of the Enzyme* 2.4.1. Crude Protein Extraction

Ten grams of banana pulps were macerated together with 10 mL of Tris buffer (0.2 M, pH 8.8), 0.8 g of Polyclar-VT, 1.0 mg of DTT, and 3 g of quartz sand with a mortar and pestle in an ice bath for 5 min. The final homogenates were adjusted to pH 8.5. From this point, the procedures were conducted under 4 ◦C in an ice bath. The homogenates were centrifuged at 8300× *g* for 15 min, and supernatants were collected. The pellets were carefully resuspended into 30 mL of Tris buffer (0.1 M, pH 8.8), and centrifuged again in the same condition. The first and second supernatants were merged for ALDH

activity test (protein-S). The pellets were resuspended into 30 mL of Tris buffer (0.1 M, pH 8.8, containing 1.0 mg of DTT) with the assistance of ultrasonic wave for 10 min. Then, centrifugation (8300× *g* for 15 min) was applied. The supernatants were used for pellet ALDH activity test (protein-P).

For each of the above supernatants (protein-S and protein-P), solid ammonium sulfate was added to the supernatants (80% saturation: 5.61 g 10 mL<sup>−</sup>1). After stirring for 2 h, the precipitations were separated by centrifugation at 25,000× *g* for 15 min. The precipitations were solubilized by 10 mL of Tris buffer (0.01 M, pH 8.8) with DTT (1 mg) and dialyzed by using a total of 2 L of Tris buffer (0.01 M, pH 8.8) for 3 h where the buffer was renewed twice. After dialysis, the insoluble pellets were removed by centrifugation at 25,000× *g* for 15 min, and the supernatants were collected as crude protein-S and protein-P, respectively.

A crude protein-PT sample was also prepared to test whether Triton X-100 nonionic surfactant improves protein extraction and enzyme activity of crude protein-P. The procedures were similar to the above for protein-P, and the only change was to add 0.1% Triton X-100 to the Tris buffer during the ultrasonic treatment.

The crude proteins were stored at −20 ◦C until enzyme characteristics analysis. The activity was stable for at least one month under DTT condition.
