Characterization and mode of action of enzymes degrading galactan structures of arabinogalactans

Agricultural biomass consisting mainly of cellulose, hemicellulose and lignin, is a renewable source of fuels and chemicals. An interesting option is enzymic conversion of biomass to readily usable material. To improve the overall economics of enzymic conversion of biomass not only cellulose but also hemicelluloses have to be degraded to monomeric sugars (saccharification). The aim of the work presented in this thesis was to study saccharification of arabinogalactans, a subgroup of the hemicelluloses.

Arabinogalactans (AGs) have been found in numerous higher plants. In most plants these arabinogalactans occur only in small amounts, with exception of Larix and Acacia species. Their role in maintaining cell wall rigidity is discussed in chapter 1).

Chapter 2 discusses structural features of AGs, subdivided in arabino-β(1->4)-galactans (type I) and arabino-β(1->3,1->6)galactans (type II), and gives a brief overview of enzymes degrading galactan structures of AGs.

In chapter 3 the alkaline extraction of type I AGs from potato fibre, onion powder and citrus pomace is described. The extracts appeared to be mixtures of various polysaccharides. The presence of arabinan in all of these extracts is likely. By means of graded ethanol precipitation a major fraction enriched in type I AG could be precipitated in 40% v/v ethanol (denoted as F40). Results indicated that the 1,4-linked galactan backbone of onion F40 was substituted at C ₆ with single unit galactose side-chains and for potato F40 also with 1,5-arabinans. Citrus TF40, (F40 treated with an endoglucanase for removal of contaminating xyloglucan), was suggested to contain a 1,4-linked galactan substituted at C ₆ with short arabinose or highly branched arabinan side-chains and single unit galactose side-chains. A type I AG extracted from soy meal with alkali may be substituted at C ₆ to a small extent with appendages of arabinose and single unit galactose side-chains.

A type II AG from green coffee beans was indicated to consist of 1,3- linked galactan backbone substituted at C ₆ with sidechains of arabinofuranose and galactose (chapter 3). The presence of terminal mannose possibly substituted on the sidechains, indicates a more complex structure for this polysaccharide. Analysis of a commercially available type II AG from larch (stractan) showed that the side-chains consisted also of arabinopyranose residues and this AG was a more heavily branched polymer than coffee AG.

Endo-1,4-β-D-galactanases involved in the bioconversion of type I AG were purified from experimental enzyme preparations derived from Aspergillus niger and A. aculeatus (chapter 4). Their molecular weights were 42-43 kD and maximal activities were measured at pH 4.0- 4.3 and 50-55 °C on de-arabinosylated potato AG. In absence of substrate the A. aculeatus endogalactanase showed less thermal stability than the A. niqer endo-galactanase. Both endo-galactanases, which were similar in their mode of action, were suggested to degrade type I AG according to a multiple attack mechanism. It appeared that a combination of endo-galactanase and endo-1,5-α-L-arabinanase exerted synergistic effects in the initial stage of degradation of the potato AG. The action of these enzymes resulted in an increase in the downward shift of the molecular weight distribution of the digest and increased amounts of galactose, galactobiose and galactotriose (chapter 4). No synergism was observed for a combination of endo- galactanase and arabinofuranosidase B.

Chapter 5 describes the purification of a β-D-galactopyranosidase. This β-galactosidase showed maximal activity on PNP-β-D-galactopyranose at pH 5 and 50 °C and was stable up to 50 °C and in the range of pH 3.5 to 7. It released non-reducing terminal galactose residues from type I AGs but not from type II AGs. With respect to polymeric substrates the enzyme showed highest activity towards 1,4- linkages but was also able to release 1,6-linked single unit galactopyranose side-chains.
Chapter 6 describes that the differences in structural features of type I AGs were reflected in the combinations of enzymes which exerted synergistic effects in degradation. In the degradation of onion and potato F40 synergism occurred in the initial stage of degradation for the endo-galactanase/β-D-galactosidase combination. In the degradation of potato F40 the endo-galactanase/endo-arabinanase combination exerted also synergistic effects. The β-D-galactosidase released single unit galactose side-chains from both substrates thereby improving the affinity for endo-galactanase. These results were consistent with the structural features of these substrates reported in chapter 3.

The activity of β-D-galactosidase on oligomeric reaction products released by endo-galactanase also enhanced degradation of potato and onion F40. This synergism occurred only in degradation of soy AG and citrus TF40. In the degradation of citrus TF40 synergistic effects were exerted also by the endo-galactanase/arabinofuranosidase B and endo-galactanase/endo-arabinanase combinations.

An exo-1,3-β-D-galactanase purified from an experimental enzyme preparation derived from A. niger preferentially degraded 1,3-β-D-galactans (chapter 7). Mainly galactose and 1,6-galactobiose were released as reaction products from partly de-arabinosylated coffee AG. Hydrolysis of coffee AG by this exo-galactanase was accompanied by formation of small amounts of several arabinogalacto-oligomers. This indicated a limited capability of this enzyme of bypassing branching points. Optimal activity was measured at pH 5.0 and 40 °C and thermal stability was found in the pH range of 2.5 to 7.5 and up to 45 °C

In the enzymic degradation of coffee bean AG a combination of exo-galactanase and α-L-arabinofuranosidase B exerted synergistic effects. For a combination of exo-galactanase and endo-arabinanase no enhancement in degradation of this substrate occurred. None of these combinations showed activity towards a type II arabinogalactan from larch wood (chapter 7).

In chapter 8 discusses the isolation of type I and II AGs, the purification procedure of the endo-1,4-β-D-galactanases, the properties and mode action of the purified galactan degrading enzymes, their role in saccharification of AGs and other fields of possible application of galactan degrading enzymes.