01/03/2022
Purification of native enzymes from macroalgae
Enzymes are crucial biological catalysts in many industrial processes and diagnostic applications. Macroalgae are a promising but rarely investigated source for the discovery of innovative, biotechnologically relevant enzymes. However, the biochemical composition of algae complicates the purification of enzymes. In order to extract and identify particularly active redox enzymes from algae, a process cascade was developed with the goal of producing them recombinantly in different expression systems.
Marine algae as a source for the discovery of new enzymes :
Macroalgae are multicellular aquatic organisms that perform photosynthesis. They belong to three different taxonomic classes: Brown algae, green algae and red algae. Bioactive substances from algae such as polysaccharides and antioxidants are nowadays valuable ingredients in the food and cosmetics industry [1-3]. Interestingly, enzymes in macroalgae still remain largely unexplored, as the native purification of these is extremely difficult due to the high contents of polysaccharides, polyphenols, the stable cell walls, strongly fluctuating environmental parameters and subsequent inefficient extraction procedures. Nevertheless, enzymes from marine algae have a high potential for biotechnological applications, as they often have different features compared to known enzymes from land plants.
Enmes are crucial biological catalysts in many industrial processes and diagnostic applications. Macroalgae are a promising but rarely investigated source for the discovery of innovative, biotechnologically relevant enzymes. However, the biochemical composition of algae complicates the purification of enzymes. In order to extract and identify particularly active redox enzymes from algae, a process cascade was developed with the goal of producing them recombinantly in different expression systems.
Marine algae as a source for the discovery of new enzymes :
Macroalgae are multicellular aquatic organisms that perform photosynthesis. They belong to three different taxonomic classes: Brown algae, green algae and red algae. Bioactive substances from algae such as polysaccharides and antioxidants are nowadays valuable ingredients in the food and cosmetics industry [1-3]. Interestingly, enzymes in macroalgae still remain largely unexplored, as the native purification of these is extremely difficult due to the high contents of polysaccharides, polyphenols, the stable cell walls, strongly fluctuating environmental parameters and subsequent inefficient extraction procedures. Nevertheless, enzymes from marine algae have a high potential for biotechnological applications, as they often have different features compared to known enzymes from land plants.
Development of universally applicable enzyme detection methods :
For the extraction and purification of enzymes in their native form, sensitive, highly specific detection methods are required, since the enzymes are usually contained in small quantities in the tissue only.
For example, various substrates can be used for the detection of peroxidases leading to a chromogenic or fluorescent product in a redox reaction or a chemiluminescence reaction, which can be measured spectrometrically at specific wavelengths. Ideally, the assay itself can also lead to an initial functional characterization.
One of the most well-known substrates for the detection of haemperoxidases is 3,5,3',5'-tetramethylbenzidine (TMB). TMB is oxidized by haemperoxidases during the enzymatic degradation of hydrogen peroxide (H2O2). The oxidation produces radical cations that enter into complexes with the non-oxidized form in a transition state. These charge-transfer complexes produce a blue coloration detectable at 652 nm, while the completely oxidized form (diimine, yellow) has a maximum absorption at 450 nm.
The substrate Thymol blue (TB), among others, can be used for the detection of haloperoxidases. Haloperoxidases are a class of biotechnologically relevant enzymes that catalyze halogenations and thus have a potential application in chemical synthesis [5, 6]. As a prosthetic group, which is necessary for the function of the enzyme, they may possess heme or vanadium or be free of covalently bound groups [7,8]. In the presence of H2O2, haloperoxidases oxidize halides, which then halogenate substrates. The reaction with TB leads to a chromogenic reaction at a pH between 7 and 8, which is based on a shift in the pKa value and can be measured at 620 nm.
The developed assays are used as high-throughput screening methods to analyze the potential of different algal species and thus to pre-select for further analysis, as well as for evaluation during the purification process.
The difficulties in establishing an enzyme assay lie in the dependence of the enzyme activity on many parameters. For the kinetic measurements of peroxidase activities, the substrate concentration, the H2O2 concentration, the buffer conditions and possible cofactors must be optimally adjusted. In contrast to classical approaches for the extraction and identification of single enzymes, the authors focus on the simultaneous purification of numerous (redox) enzymes from different algal species.
Thus, it is particularly important to develop assay conditions that are universally applicable for the analysis of the respective species. During the optimization process of the TB assay, the relative enzyme activity was analyzed as a function of different potassium bromide concentrations and pH values (fig. 1C). The optimal conditions are a pH value at approximately 7.5 and a potassium bromide concentration of 20 mM. In preliminary experiments, the optimal H2O2 (8 mM) and TB (0.5 mM) concentrations were also evaluated with different algal extracts.
Native gel electrophoresis enables visualization of enzymatic activities :
Another important tool in the characterization and selection of new enzymes is native gel electrophoresis to visualize and identify their native forms. In this gel electrophoresis method, proteins are separated along a polyacrylamide gel depending on their size, net charge as well as their shape. In contrast to SDS-PAGE, SDS is absent in the buffer and as a consequence, proteins are not denatured. Thus, native gel electrophoresis enables in-gel activity determination and the assignment to distinct protein bands within one purification step.
Purification of peroxidases from macroalgae :
In order to extract enzymes in their native form from algae, the algae material is first homogenized in a suitable buffer. Then, the insoluble cell components are separated by centrifugation and the proteins are precipitated by adding acetone. After dissolving the protein pellet, the first chromatographic separation is carried out using an FPLC (fig. 2A). By using an alkaline buffer system, a large proportion of the proteins carry a net negative charge. Consequently, proteins bind to an anion-exchange chromatography (AEX) column to varying degrees. The elution of the proteins can be performed stepwise or via a gradient by the addition of counter ions (e.g. sodium chloride). Preliminary experiments with different buffer systems, elution methods and NaCl concentrations, have shown the benefit of stepwise elution with specific NaCl concentrations. Figure 2 displays an example of a chromatogram of an AEX with stepwise elution. The brown alga Saccharina latissima was used as crude extract. Different activity profiles for haem (B) and haloperoxidases (C) can be identified.
The resulting activity peaks were concentrated separately from each other and additionally purified using size exclusion chromatography in a second step. Active fractions were applied to a native gel and stained with TMB and TB.
Finally, the activity bands were cut out and prepared for mass spectrometry (MS) analysis. By combining a two-step chromatographic with one activity-based gel electrophoretic purification step, the probability of clearly identifying active peroxidases is very high. The transcriptome analysis of the algae species used, which was carried out by the consortium, is suitable as a comparative database for MS identification.
Outlook :
Different chromatography techniques allow the fractionation of multiple enzymes. Native gel electrophoresis additionally offers the possibility to evaluate the efficiency of purification.
However, chromatography-based methods require samples with relatively low viscosity. For this reason, purification of active enzymes from algal species with very high polysaccharide contents is difficult. At the moment, we are developing enzyme-assisted methods for the degradation of algal polysaccharides after cell disruption to reduce the viscosity of algal extracts.
Acknowledgements :
This project is part of the BMBF-funded project REA - Redox Enzymes from Algae as Innovative Tools in Bio-Industry. We would like to thank our project partners Yu-Chen Wu and Levent Piker (CRM, Kiel) for providing the macroalgae, Timo Jensen and Matthias Peipp (UKSH, Kiel) for the RNA extraction for transcriptome analysis and Dominik Bents and Steffen Hennig (CRM, Kiel) for numerous bioinformatic analyses.
