Substrates belonging to seven different compound classes were equally tested in a 5 mM concentration (2 mM for syringaldazine), which in some instances may lead to substrate inhibition of the tested enzymes. buffer (100 mM, pH 5) at temperatures ranging from 25-50C. Error bars refer to standard deviation by means of four replicates. 2191-0855-1-14-S2.DOC (26K) GUID:?61F2E17E-B848-4D30-B02F-4C45D695FCF0 Abstract em Azotobacter chroococcum /em is a widespread free-living soil bacterium within the genus of em Azotobacter /em known for assimilation of atmospheric nitrogen and subsequent conversion into nitrogenous compounds, which henceforth enrich the nitrogen content of soils. em A. chroococcum /em 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 em A. chroococcum /em SBUG 1484 within comparative analysis to fungal crude PO from the white-rot fungus em Pycnoporus cinnabarinus /em SBUG-M 1044 and tyrosinase (PPO) from the mushroom Rabbit polyclonal to ACK1 em Agaricus bisporus /em in an attempt to reveal desirable properties for exploitation with future recombinant expression of this enzyme. Catalytic activity increased with pre-incubation at 35C; however 70% of activity remained after pre-treatment at 50C. Native em A. chroococcum /em crude Cot inhibitor-2 PO exhibited not only strong preference for 2,6-dimethoxyphenol, but also towards related methoxy-activated substrates as well as substituted em ortho /em -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 Cot inhibitor-2 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 em A. chroococcum /em PO activity comprised in crude extracts from those of PPO and confirmed distinct similarities to fungal PO. strong class=”kwd-title” Keywords: Bacterial phenol oxidase, laccase, tyrosinase, em Pycnoporus cinnabarinus /em , em Agaricus bisporus /em , nitrogen fixation, cysts, melanin, oxygen protection Introduction Laccases (benzenediol:oxygen oxidoreductases, EC 18.104.22.168.) 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 em Azospirillum lipoferum /em 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 em Azotobacter chroococcum /em 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 em A. chroococcum /em 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 em A. chroococcum /em SBUG 1484, in which cell bodies shortened, encapsulated and development of cysts occurred. Morphologic alterations, formation of dormant stages (particularly spore formation) or production of melanin-like pigments within simultaneous expression of POs or PO-like proteins have also been described for several prokaryotic soil-dwelling bacteria belonging to the genera em Bacillus /em [Hullo et al. 2001], em Streptomyces /em [Endo et al. 2002], em Pseudomonas /em [Mellano and Cooksey 1988], but also em Escherichia /em [Kim et al. 2002] and the melanogenic marine bacterium em Marinomonas /em [Sanchez-Amat et al. 2001]. Despite common occurrence of Cot inhibitor-2 these enzymes in similar physiological processes in both prokaryotic and eukaryotic organisms, there still exist remarkable differences with regard to enzymes characteristics and reaction preferences between enzymes not only from both domains, but even POs from species belonging to the same genus. In particular, some prokaryotic POs reveal substrate scopes that overlap with PO-related copper-containing oxidoreductases (tyrosinase) showing polyphenol oxidase (PPO) characteristics, for example the polyphenol oxidase of.