Elsevier

Food Chemistry

Volume 136, Issue 1, 1 January 2013, Pages 164-169
Food Chemistry

Phenylalanine ammonia lyase (PAL) enzyme activity and antioxidant properties of some cyanobacteria isolates

https://doi.org/10.1016/j.foodchem.2012.07.119Get rights and content

Abstract

In the present study, six cyanobacteria isolates were evaluated for the PAL enzyme activity, and their methanol extracts were assessed for the total phenolic amount and other antioxidant parameters. Synechocystis sp. BASO444 and Synechocystis sp. BASO673 isolates with high levels of total phenols (66.0 ± 1.2 μg/mg, 78.1 ± 1.8 μg/mg, respectively) also showed high levels of PAL activities (20.5 ± 3.1 U/mg protein, 17.2 ± 2.3 U/mg protein, respectively) and strong antioxidant activities. To understand the effect of l-phenylalanine (l-phe) on the PAL activity, total phenolic amount, and phenolic constituents, isolates were evaluated with 100 mg/l l-phe. While PAL activities exhibited no significant change with l-phe addition, total phenolic amount of the isolates significantly increased. HPLC analysis revealed gallic acid, trans-cinnamic acid, p-coumaric acid, and ferulic acid as the main compounds. Results suggested that the two isolate mights be an important source for the l-phe inducible phenolic compounds.

Highlights

Antioxidant properties and PAL activity of cyanobacterial isolates evaluated together. ► Microalgae have the possibility of usage as natural antioxidant and PAL sources. ► l-phe induces the total phenolic accumulation in cyanobacteria. ► Cyanobacteria may have the potential to be used in the treatment of phenylketonuria.

Introduction

Phenylalanine ammonia lyase (PAL) catalyses the non-oxidative deamination of phenylalanine to trans-cinnamate and directs the carbon flow from the shikimate pathway to the branches of phenylpropanoid metabolism. It is a secondary metabolic pathway that is operative in higher plants and some other organisms which are mainly involved in defense mechanisms (MacDonald & D’ Cunha, 2007). The properties, regulation, expression, and cellular distributions of PAL have been extensively studied, and the crystal structures of PAL from parsley and the yeast Rhodosporidium toruloides have been determined (Reichert, He, & Dixon, 2009). PAL is an inducible enzyme, and the incorporation of l-phe in the fermentation medium has been indicated as significantly inducing PAL activity (MacDonald & D’ Cunha, 2007).

The first described PALs from cyanobacteria were Anabaena variabilis and Nostoc punctiforme. In comparison to eukaryotic homologues, the cyanobacterial PALs are 20% smaller in size, but it is indicated that they share similar substrate selectivity and kinetic activity toward l-phe compared with l-tyrosine. Recombinant PAL is currently being evaluated for use in humans as enzyme substitution therapy for the treatment of the inherited metabolic disease phenylketonuria (PKU) resulting from the loss or impairment of phenylalanine hydoxilase, an enzyme that metabolizes l-phe (Moffitt et al., 2007).

The general phenylpropanoid metabolism generates various common known secondary metabolites such as lignin or flavonoid and other aromatic metabolites such as coumarins, phenolic volatiles, or hydrolyzable tannins. The genes of the shikimate pathway have partly originated from the prokaryotic ancestor of cyanobacterial origin (Vogt, 2010).

The chemical activities of polyphenols in terms of their reducing properties as hydrogen or electron donating agents predict their potential for action as free-radical scavengers (antioxidants). The activity of an antioxidant is determined by its reactivity as a hydrogen or electron donating agent (which is related to its reduction potential). Polyphenols possess ideal structural chemistry for free radical-scavenging activities and have been shown to be more effective antioxidants in vitro than vitamins E and C on a molar basis (Rice-Evans, Miller, & Paganga, 1997).

Cyanobacteria are prokaryotic organisms that have served as important model organisms for studying oxygenic photosynthesis and have played a significant role in the Earth’s history as primary producers of atmospheric oxygen. Synechocystis sp. PCC 6803 was the first cyanobacteria that had its genome sequenced in 1996 (Nakao et al, 2010). In contrast, limited screening programs have been conducted on cyanobacteria due to the scarcity of expertise in physiological research and industrial production (Natrah, Yusoff, Shariff, Abas, & Mariana, 2007).

Plants possess many compounds, but their rapid production and usage for industrial and medical purposes is not easy, because plant cultivation is dependent on local soil and climatic conditions, and their attainment of optimal growth takes a longer time. In this study, we aimed at investigating some cyanobacteria isolates for the relationship between their PAL enzyme activity, total phenolics, and antioxidant activities for the first time. Determination of a cyanobacteria isolate that has a high and inducable PAL activity would create new evaluation areas in its industrial usage, and also cyanobacteria can serve as a potential resource in rapid production of the PAL enzyme.

Section snippets

Culture conditions and microorganisms

The isolates representing filamentous and non-filamentous cyanobacteria forms from different water sources were obtained from the Gazi University Faculty of Science, Biotechnology Laboratory culture collection. A confirmatory sequence analysis also was achieved for the study.

Isolation and purification of the isolates were performed by dilution and plating water samples. The isolates were cultured in BG11 medium (Rippka, Deruelles, Waterbury, Herdman, & Stanier, 1979) at 25 °C with a light/dark

Results and discussion

Current enzyme replacement therapies for PKU are focused on PAL, which has many advantages compared with phenylalanine hydroxylase (PAH) enzyme that is produced in the body to degrade l-phe, and recently, cyanobacterial PALs are evaluated for the design and construction of an effective protein-based treatment for PKU (Moffitt et al. 2007).

In the present study, PALs from the cyanobacteria are particular to Synechocystis, Leptolyngbya, and Oscillatoria species and are described for the first

Conclusions

Antioxidant and PAL activity of cyanobacterial isolates was reported for the first time together. The results of the study also showed that the microalgae have the possibility of usage as natural antioxidant sources. PALs isolated from different organisms so far exhibit problems related to administration, immunogenecity, and stability. With such screen studies, we may have the chance to find a candidate cyanobacteria that has the potential to be used as a PAL source in the tratment of

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