2.4.2. Functional Characteristics of ALDH

Substrate specificity: reaction mixture (2 mL) included 1660 μL of phosphate buffer (0.2 M, pH 8.8), 100 μL of low C2–C4 aldehydes (50 mM), 100 μL of crude protein extraction (protein-S, -P or -PT), 40 μL of NAD<sup>+</sup> (50 mM), with or without 100 μL of 4-methyl pyrazole (100 mM). For C5–C6 aldehydes, 500 μL (10 mM) was added with reduced volume of buffer (1260 μL). ALDH activity was determined by monitoring the increase in NADH at 340 nm at 35 ◦C by a spectrophotometer with a 10 mm path length UV cuvette. Km value of NAD+ was measured with butanal as the substrate. An extinction coefficient of 6.2 mM−<sup>1</sup> cm−<sup>1</sup> was used for the activity calculation.

Optimum pH and buffer concentration: ALDH activity was determined by monitoring the increase in NADH at 340 nm at 35 ◦C by a spectrophotometer, as described above. Butanal was used as the substrate. For the optimum pH searching, bicine buffer (0.1 M) was used at a pH range of 6.5–10.0. For the optimum buffer concentration searching, phosphate buffer (pH 8.8) was used at a range of 0–200 mM.

Protein contents in the crude ALDH extraction: Lowry method [21,22] was adapted to determine protein content with Folin and Ciocalteu's phenol reagent and bovine serum albumin was used as the standard.

#### 2.4.3. Activity of ADH in Crude Protein Extractions and Inhibition by 4-Methyl Pyrazole

To confirm the purity of ALDH, and exclude the potential influence from other enzymes, especially ADH, the ADH activity in the crude protein extraction listed in Sections 2.4.1 and 2.4.2 was determined by monitoring the decline in NADH at 340 nm at 35 ◦C by a spectrophotometer in a mixture comprised of 1660 μL of phosphate buffer (0.2 M, pH 8.8), 100 μL of butanol (50 mM), 100 μL of crude protein extraction, and 40 μL of NAD<sup>+</sup> (50 mM) with or without 100 μL of 4-methyl pyrazole (100 mM), where the total volume was 2 mL (balanced with water to make the volume). An extinction coefficient of 6.2 mM−<sup>1</sup> cm−<sup>1</sup> was used for the activity calculation [11,19].
