Indole3carboxylate decarboxylase

Scheme 8

The highest indole-3-carboxylate decarboxylase activity was found in A. nico-tianae F11612 isolated from the soil sample through conventional enrichment culture using indole-3-carboxylate as a sole carbon source.44 The occurrence of the enzyme was also found in Fusarium oxysporum IAM5009 and Gibberella fujikuroi IFO6605 from culture collections. The enzyme of A. nicotianae FI1612 was inducibly formed by the addition of 0.05% (w/v) indole-3-carboxylic acid, and 3-cyanoindole and L-tryptophan also induced the enzyme activity slightly. No activity was found without the addition of inducers. In the screening for indole-3-carboxylate decarboxylase, its weak activity was frequently lost during aerobic cultivation. However, after the optimization of culture conditions of A. nicotianae FI1612, increased activity of whole cells was found even under aerobic conditions. The highest total activity of about 500 nmol ml-1 min-1 was observed after 32 h at 28°C.

When a cell extract prepared from A. nicotianae FI1612 cells was stored without the addition of sulfhydryl-protecting reagents, 80% of the initial activity was lost after storage at 4°C for 4 days. The enzyme activity was stabilized by the addition of a mixture of 1 mM dithiothreitol, 50 mM Na2S2O3, and 20% (v/v) glycerol to the purification buffer. However, much loss of activity seemed inevitable during enzyme purification. Due to its lability, the enzyme could not be purified to be homogeneous; however, the molecular mass of the enzyme was estimated to be 258 kDa by gel permeation. The decarboxylation activity was maximal at 50°C and pH 7.0. The A. nicotianae FI1612 enzyme did not require any cofactor for its activity, although the pyrrole-2-carboxylate decarboxylase activity of B. megaterium PYR2910 was completely dependent on an organic acid such as acetic acid.43

The carboxylation of indole into indole-3-carboxylate was observed by the purified indole-3-carboxylate decarboxylase as well as by the whole cells. For the carboxylation reaction, temperatures over 30° C were not appropriate. The activities at 10, 20, and 30° C were about the same. The activity was maximal at pH 8.0 (Tris-HCl buffer, 100 mM). As shown in Fig. 10, the resting cells of A. nicotianae FI1612 also catalyzed the carboxylation of indole efficiently in the reaction mixture containing 20 mM indole, 3 M KHCO3, 100 mM potassium phosphate buffer (pH 6.0) in a tightly closed reaction vessel. By 6 h, 6.81 mM indole-3-carboxylic acid accumulated in the reaction mixture with a molar conversion yield of 34%. Compared to the carboxylation of pyrrole by pyrrole-2-carboxylate decarboxylase, the lower value compared might derive from the lower solubility of indole in the reaction mixture.

Resting cells of A. nicotianae FI1612 also catalyzed the carboxylation of 2-methylindole and quinoxaline. The activities toward 2-methylindole and quinoxaline were 37 and < 1% of the activity toward indole, respectively. The reaction products of the reverse carboxylation, indole-3-carboxylic acid and 2-methylindole-3-carboxylic acid, were isolated and identified through physico-chemical analyses with the authentic compounds as reference.

g 10

g 10

Figure 10: Carboxylation of indole by whole cells of Arthrobacter nicotianae

FI1612.

Figure 10: Carboxylation of indole by whole cells of Arthrobacter nicotianae

FI1612.

Was this article helpful?

0 0

Post a comment