Research Article: Study of enzymatic properties of phenol oxidase from nitrogen-fixing Azotobacter chroococcum

Date Published: June 24, 2011

Publisher: Springer

Author(s): Susanne Herter, Marlen Schmidt, Mark L Thompson, Annett Mikolasch, Frieder Schauer.

http://doi.org/10.1186/2191-0855-1-14

Abstract

Azotobacter chroococcum is a widespread free-living soil bacterium within the genus of Azotobacter known for assimilation of atmospheric nitrogen and subsequent conversion into nitrogenous compounds, which henceforth enrich the nitrogen content of soils. A. chroococcum SBUG 1484, isolated from composted earth, exhibits phenol oxidase (PO) activity when growing under nitrogen-fixing conditions. In the present study we provide incipient analysis of the crude PO activity expressed by A. chroococcum SBUG 1484 within comparative analysis to fungal crude PO from the white-rot fungus Pycnoporus cinnabarinus SBUG-M 1044 and tyrosinase (PPO) from the mushroom Agaricus bisporus in an attempt to reveal desirable properties for exploitation with future recombinant expression of this enzyme. Catalytic activity increased with pre-incubation at 35°C; however 70% of activity remained after pre-treatment at 50°C. Native A. chroococcum crude PO exhibited not only strong preference for 2,6-dimethoxyphenol, but also towards related methoxy-activated substrates as well as substituted ortho-benzenediols from over 40 substrates tested. Presence of CuSO4 enhanced crude phenol oxidase activity up to 30%, whereas NaN3 (0.1 mM) was identified as the most inhibiting substance of all inhibitors tested. Lowest inhibition of crude PO activity occurred after 60 minutes of incubation in presence of 15% methanol and ethanol with 63% and 77% remaining activities respectively, and presence of DMSO even led to increasing oxidizing activities. Substrate scope and inhibitor spectrum strongly differentiated A. chroococcum PO activity comprised in crude extracts from those of PPO and confirmed distinct similarities to fungal PO.

Partial Text

Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2.) and related copper-containing proteins have been widely described in a considerable number of eukaryotes including fungi, plants and animals, especially insects and partially mammals. Research concerning their presence in microorganisms, physiological functions, structural characteristics and feasible biotechnological applications has tended to focus on phenol oxidases (POs) of several fungi, especially white-rot fungi [Morozova et al. 2007,Rodriguez-Couta and Toca-Herrera 2006]. In contrast the expression of POs and structurally related non-enzymatic blue multicopper protein structures in prokaryotes has not been so widely investigated [Claus 2003]. As the majority of phenol oxidases described in literature have been isolated from higher fungi, the cellular function for these oxygen-requiring enzymes in eukaryotic systems was typically related to oxidative polymerization and depolymerisation of lignin [Kawai et al. 1988,O’Malley et al. 1993], but also to formation of carposomes linked with synthesis of cell wall-associated pigments [Thurston 1994], sporulation [Leonowicz et al. 2001] and plant pathogenesis [Bar-Nun and Meyer 1989]. Similarities to the occurrence of prokaryotic phenol oxidases can also be considered [Faure et al. 1994] reported prokaryotic PO activity in Azospirillum lipoferum which lives, comparable to several soil fungi, in association with the plant rhizosphere and promotes plant growth. This bacterial PO was determined to be expressed in combination with physiological processes like cell pigmentation and the activation of phenolic plant ingredients. Within our previous studies, nitrogen-fixing cultures of the non-symbiotic Azotobacter chroococcum SBUG 1484, isolated from composted earth, exhibited PO activity when growing with nutritional deficiencies, especially depletion of exogenous nitrogen sources [Herter et al. 2011]. Interestingly, cell-associated PO production in A. chroococcum cells appeared in conjunction with an increased formation of a brown-black pigment identified as melanin. These observations were made concurrently with morphological alteration during the life-cycle of A. chroococcum SBUG 1484, in which cell bodies shortened, encapsulated and development of cysts occurred.

In the present study, an incipient approach on general properties exhibited by the cell-associated bacterial PO comprised within crude PO preparations of nitrogen-fixing cells from the strain A. chroococcum SBUG 1484 was performed. As prior attempts in preparation of purified enzyme were not successful, the experiments performed herein focussed on an initial understanding of basic enzymatic properties of this prokaryotic crude phenol oxidase, such as substrate scope and the influence of inhibitors, metals and solvents.

The authors declare that they have no competing interests.

 

Source:

http://doi.org/10.1186/2191-0855-1-14

 

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