*2.6. Expression and Substrate Specificity of HcdB Dioxygenase*

HcdB dioxygenase was expressed from the plasmid pTHPPDO in *E. coli* BL21. All attempts to purify HcdB aerobically resulted in the loss of the enzymatic activity, even in the presence of organic solvents, such as glycerol, ethanol, and acetone that were known as stabilizers for similar enzymes [30–32]. The addition of dithiothreitol and ferrous sulfate to the aerobically purified protein did not restore its activity, although it had been shown to activate and/or stabilize other extradiol dioxygenases [30–34].

Therefore, due to the highly unstable nature of the HcdB enzyme, all the activity measurements were carried out in vivo using the whole cells of *E. coli* BL21 transformed with the pTHPPDO plasmid. The bioconversion of pyrocatechol, 3-methylcatechol, 3-methoxycatechol, 4-methylcatechol, and caffeic acids in each case produced yellow products with the absorbance maxima expected for the proximal *meta*-cleavage products of these catechols, as shown by the solid line in Figure 5 [35–37]. A weak yellow color was visible in bioconversion mixture containing 3-(2,3-dihydroxyphenyl)-propionic acid, and no changes in color took place, but the absorbance maxima shifts were observed during reactions with pyrogallol, gallacetophenone, 2 ,3 -dihydroxy-4 -methoxyaceto-phenone hydrate, 3,4-dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, and 2,3,4-trihydroxy-benzophenone, as shown in Figure S11 in the Supplementary Material. No activity was observed with 1,2,4-benzenetriol and 6,7-dihydroxycoumarin. The *E. coli* cells without the *hcdB* gene showed no activity towards the aforementioned compounds. The expected shift of peaks of the UV-VIS spectra of the reaction products to a shorter wavelength were observed after acidification of the reaction mixtures containing pyrocatechol, 3-methylcatechol, 3-methoxycatechol, 4-methylcatechol, and caffeic acid, as shown by the dashed line in Figure 5 [38,39]. These findings revealed that *hcdB* encodes an extradiol dioxygenase, which can utilize a number of differently substituted catechols.

Furthermore, the HcdB dioxygenase was co-expressed with the HcdA hydroxylase in *E. coli* BL21 cells. The activity of those cells towards 3-(2,4-dihydroxyphenyl)-propionic acid was analyzed. No coloration occurred during the incubation of over 72 h, compared to the formation of a reddish bioconversion product by *E. coli* cells containing the *hcdA* gene only. Products of 3-(2,4-dihydroxyphenyl)-propionic acid conversion by HcdA and HcdB were analyzed by HPLC-MS. Ions with masses 181.00, 195.00, 197.00 ([M−H]−) were not detected showing a complete

conversion of substrate and its hydroxylated forms. Also, none of the expected ions ([M−H]<sup>−</sup> 229.00 or [M+H]<sup>+</sup> 231.00) were observed for a presumed product of the oxidative cleavage of 3-(trihydroxyphenyl)-propionic acid.

**Figure 5.** Biotransformations of pyrocatechol (**A**), 3-methylcatechol (**B**), 3-methoxycatechol (**C**), 4-methylcatechol (**D**), and caffeic acid (**E**) by whole cells of *E. coli* BL21 containing *hcdB* gene. Biotransformations were carried out in 50 mM potassium phosphate buffer pH 7.5 (solid line) at 30 ◦C with 0.5–1 mM of substrate. Incubation time is shown in min. Dashed lines indicate peak shifts to a shorter wavelength after acidification of reaction mixtures.
