*3.3. Extraction and Functional Characteristics of ALDH*

As shown in materials and methods, crude proteins were extracted in three ways: from supernatant (protein-S), or from pellets by sonication-assisted extraction with or without Triton X-100 (protein-PT and protein-P, respectively). The protein-S possessed more than 95% of ADH activity, while both protein-P and protein-PT had very little ADH activity (Figure 5a). On the other hand, ALDH activity was evidenced in all protein extractions (Figure 5b,c).

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**Figure 5.** ADH and ALDH activity in crude proteins extracted from different fractions and effect of Triton X-100 (protein extraction enhancer) and 4-methyl pyrazole (ADH inhibitor). Proteins were extracted by ammonium sulfate precipitation from Tris buffer extraction of banana pulp tissues at the "yellow with brown flecks" stage. The activity was presented based on fresh sample mass (**a**,**b**) or protein mass (**c**). Protein-S: extraction from supernatant merged from 2 × Tris buffer extractions; protein-P: extraction from pellets was washed 2 × Tris buffer and solubilized by sonication; protein-PT: extraction from pellets after 2 × Tris buffer washing and solubilized by sonication with addition of Triton X-100. Vertical line at each column shows average ± SD (n = 3).

When based on weight of pulp tissue, calculated ALDH activity distribution for protein-P was slightly more than for protein-S; however, the distribution at pellet extraction was increased when Triton X-100 was used (protein-PT) (Figure 5b). When 4-methyl pyrazole was added into the reaction mixture, ALDH activity distribution at protein-PT was about 60%, and at protein-S, it was about 40% (Figure 5b). However, when calculation was based on protein weight, the highest ALDH activity was found in protein-P, followed by protein-PT and then protein-S. This was partially because Triton X-100 caused more extraction in non-ALDH proteins (protein-PT), and protein-S contained more non-ALDH proteins (Figure 5b,c). ALDH-PT contained more yellow pigments as contaminants. Protein-S had the lowest ALDH activity at protein basis (Figure 5). Thus, protein-P was chosen as crude extract of ALDH in the following experiments.

ALDH activity was very low when the phosphate buffer concentration was less than 20 mM (Figure 6a). The activity increased slightly until the buffer concentration reached saturation at 100 mM (Figure 6a). Optimum pH of the ALDH was 8.8 as shown in Figure 6b, and at pH 8.5–9.2, ALDH activities were kept > 80% capacity (Figure 6b). Km values of butanal and NAD+ of the ALDH were about 250 μM and 25 μM, respectively, from the Lineweaver–Burk plot (data not shown). When replacing NAD<sup>+</sup> by NADP+, the ALDH activity reduced to about one third in comparison to NAD+ (data not shown).

**Figure 6.** Activity of crude ALDH. (**a**) Optimum concentration of phosphate buffer; (**b**) Optimum pH; and (**c**) Substrate specificity to C2–C6 branched and straight chain aldehydes. Horizontal line at each column shows average ± SD (n = 3).

Substrate specificity of the ALDH was shown in Figure 6c. Lower molecular weight straight chain aldehydes, except ethanal, had high affinity to ALDH, while poor affinity was detected for branched chain aldehydes (Figure 6c).
