Analysis of G‑Block Distributions and Their Impact on Gel Properties of in Vitro Epimerized Mannuronan Olav Aarstad,* Berit Løkensgard Strand, Lise Mari Klepp-Andersen, and Gudmund Skja ̊ k-Bræk Department of Biotechnology, Norwegian University of Science and Technology, NTNU Sem Sælands vei 6-8, N-7491 Trondheim, Norway * S Supporting Information ABSTRACT: This paper reports a study of the distribution and function of homopolymeric guluronic acid blocks (G-blocks) in enzymatically modified alginate. High molecular weight mannur- onan was incubated with one native (AlgE6) and two engineered G-block generating mannuronan C-5 epimerases (AlgE64 and EM1). These samples were found to contain G-blocks with a DP ranging from 20 to approximately 50, lacking the extremely long G- blocks (DP > 100) found in algal alginates. Calcium gels from epimerized materials were highly compressible and exhibited higher syneresis and rupture strength but lower Youngs modulus than gels made from algal polymers of similar G-content. Addition of extremely long G-blocks to the epimerized alginate resulted in decreased syneresis and rupture strength and an increased Young’s modulus that can be explained by reinforcement of the cross-linking zones at the cost of length and/or numbers of elastic segments. The presence and impact of these extremely long G- blocks found in natural alginates suggest that alginate gels can be viewed as a nanocomposite material. ■ INTRODUCTION Alginates are linear glycuronans composed of (1→4) linked residues of β-D-mannuronic acid (M) and α-L-guluronic acid (G). The residues are arranged in a block-wise fashion along the polymer chain forming homopolymeric (M-blocks or G- blocks) or heteropolymeric regions (e.g., MMG, GGM, GMG). They are highly soluble in water and form gels with cations. The gel-forming capacity correlates with the content and average length of the G-blocks. 1,2 The nonrandom, non- repeating structure in alginates has been attributed to its unique biosynthesis in which the G-residues are introduced in a postpolymerization step catalyzed by mannuronan C-5 epimerases. 3,4 In a recent paper we described a new strategy for analysis of block length distribution in alginates based on specific enzyme degradation combined with chromatography and NMR. 5 When this analysis was performed on native alginates from a range of brown algae, we found to our surprise that all samples, independent of their composition, contained a fraction of 10- 15% of homopolymeric G-blocks comprising more than 100 consecutive residues. The biosynthesis and functional role of these long G-blocks in calcium alginate gels is still elusive. The alginate producing bacterium, Azotobacter vinelandii, expresses seven AlgE isoenzymes. These epimerases are modular enzymes consisting of one or two catalytic A-modules and one to seven regulatory R-modules. 4 The seven AlgE enzymes yield different residue sequences in the polymer products, 6-9 but the nature of the structure-function relation- ship is not fully understood. In this study we were using epimerases that generate G-blocks, acting either in a processive mode where the enzyme slides along the polymer, carrying out repetitive epimerizations without dissociating, or in a preferred attack mode where the affinity for the substrate increases with epimerization. Since mannuronan C-5 epimerases from the alginate producing bacteria are available in our laboratory, the aim of this work was to make alginate with less compositional heterogeneity by modifying homopolymeric mannuronan with specific epimerases, analyze the G-block length and distribution and use the material to elucidate the role of long G-blocks in alginate gels. ■ MATERIALS AND METHODS Alginates. L. hyperborea alginate (F G = 0.67 and M w ≈ 2.0 × 10 5 Da) was provided by FMC Biopolymer. A high molecular weight mannuronan (F G = 0.0 and M w ≈ 3.4 × 10 5 Da) was isolated from an epimerase negative AlgG - mutant of Pseudomonas fluorescens. 10 Purification and deacetylation was done as described elsewhere. 11 Enzymes. Three mannuronan C-5 epimerases were used in this study: the wild type epimerase AlgE6 from A. vinelandii and the two genetically engineered epimerases AlgE64 and EM1. AlgE6 was produced by fermentation of the recombinant E. coli strain SURE and partially purified by ion-exchange chromatography on Q-Sepharose FF as previously described. 6 The hybrid enzyme AlgE64 was made by combining the A-module from AlgE6 with the R-module from the highly processive AlgE4 (unpublished results). Received: May 8, 2013 Revised: August 7, 2013 Published: August 12, 2013 Article pubs.acs.org/Biomac © 2013 American Chemical Society 3409 dx.doi.org/10.1021/bm400658k | Biomacromolecules 2013, 14, 3409-3416