Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants

Abstract

This study intends to purify and characterize catechol 1,2-dioxygenase (C1,2O) of phenol-degrading Acinetobacter sp. Y64 and of E. coli transformant. Acinetobacter sp. Y64 was capable of degrading 1000 mg/L of phenol within 14 ± 2 h at 30 °C, 160 rpm and pH of 7. One C1,2O of 36 kDa was purified using ammonium sulphate precipitation and Hitrap QFF column chromatograph with 49% recovery and a 10.6-fold increase in purity. Purified Y64 C1,2O had temperature and pH optimum at 37 °C and pH 7.7 respectively with the Michaelis constant of 17.53 µM and the maximal velocity of 1.95 U/mg, respectively. The presence of Fe(3+) or Fe(2+) enhanced the activity of Y64 C1,2O while other compounds such as Ca(2+), and EDTA had an inhibitory effect. 80% of C1,2O activity remained using 4-nitrocatechol as substrate while 2% remained using 3-methylcatechol compared with that using catechol. Y64 catA gene encoding C1,2O was amplified using PCR cloned into pET22b vector and expressed in Escherichia coli BL21 DE3 (pLysS) after transformation. Purified and cloned Y64 C1,2O show no significant differences in the biochemical properties. The phylogenetic tree based on the protein sequences indicates that these C1,2Os possess a common ancestry.

Catechol 1,2-Dioxygenase is an Analogue of Homogentisate 1,2-Dioxygenase in Pseudomonas chlororaphis Strain UFB2

“…For example, the optimum activity for C12O was recorded at pH 8 and 37 °C for Acinetobacter sp. Y64 strain [22]; pH 7 and 30 °C for Gordonia polyisoprenivorans [11]; and pH 7.5 and 35 °C for Pseudomonas putida strain N6 [23]. However, an optimum temperature of 40 °C has been reported for C12O in Pseudomonas aeruginosa KB2 and Candida albicans TL3 strains [14,24].…”

“…In another study, 3-methylcatechol resulted in the induction of very low activity in Acinetobacter sp. Y64 (2% as compared to catechol), 1,2,4-benzenetriol, and 4-nitrocatechol, but 4-methylcatechol was able to induce high enzyme activity (80% as compared to catechol) [22]. In Aspergillus awamori , 2,4-dichlorophenol could not induce C12O [28].…”

Degradation Mechanism of 2,4-Dichlorophenol by Fungi Isolated from Marine Invertebrates

“…C12O enzymes are known to be expressed by various microorganisms able to degrade phenolic pollutants. Bacteria able to assimilate 3-hydroxybenzoate [17], phenol [18], benzoic acid [19], and α-naphthol [20] express such enzymes that were characterized. On the contrary, the reports about C12O expression by fungi, mostly related to their biochemical characterization, are scarce.…”

“…One unit (U) of catechol 2,3-dioxygenase (C23O) activity is defined as the amount of enzyme that produces 1 µmol 2-hydroxymuconic semialdehyde per minute under the assay conditions. The products of C12O and C23O were detected at 260 nm and 375 nm, respectively, and they were quantified according to Lin and Milase [18] and Hupert-Kocurek et al [31].…”

Genetic diversity of catechol 1,2-dioxygenase in the fecal microbial metagenome

“…Many C12O have been cloned and characterized from a variety of different bacteria, such as Acinetobacter , Arthrobacter , Corynebacterium , Pseudomonas , Rhodocococcus , Stenotrophomonas , and Streptomyces . Additionally, global microbial genome and environmental metagenome sequencing efforts are also contributing ever‐increasing genetic information to develop C12O.…”