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

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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 Fe3+ or Fe2+ enhanced the activity of Y64 C1,2O while other compounds such as Ca2+, 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.

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Abbreviations

BH:

Bushnell Haas

bp:

Basepair

C1,2O:

Catechol 1,2-dioxygenase

C2,3O:

Catechol 2,3 dioxygenase

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

K m :

Michaelis constant

PCR:

Polymerase chain reaction

QFF:

Q Sepharose® Fast Flow

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

V max :

The maximum rate of reaction

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  1. Discipline of Microbiology, School of Life sciences, University of KwaZulu-Natal, Westville Campus, Private Bag X54001, Durban, 4001, South Africa

    J. Lin & R. N. Milase

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  1. J. Lin
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  2. R. N. Milase
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Correspondence to J. Lin.

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Lin, J., Milase, R.N. Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants. Protein J 34, 421–433 (2015). https://doi.org/10.1007/s10930-015-9637-7

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