Yeast GH30 Xylanase from Sugiyamaella lignohabitans Is a Glucuronoxylanase with Auxiliary Xylobiohydrolase Activity

Abstract

Xylanases are the enzymes that catalyze the breakdown of the main hemicellulose present in plant cell walls. They have attracted attention due to their biotechnological potential for the preparation of industrially interesting products from lignocellulose. While many xylanases have been characterized from bacteria and filamentous fungi, information on yeast xylanases is scarce and no yeast xylanase belonging to glycoside hydrolase (GH) family 30 has been described so far. Here, we cloned, expressed and characterized GH30 xylanase SlXyn30A from the yeast Sugiyamaella lignohabitans. The enzyme is active on glucuronoxylan (8.4 U/mg) and rhodymenan (linear β-1,4-1,3-xylan) (3.1 U/mg) while its activity on arabinoxylan is very low (0.03 U/mg). From glucuronoxylan SlXyn30A releases a series of acidic xylooligosaccharides of general formula MeGlcA²Xyl_n. These products, which are typical for GH30-specific glucuronoxylanases, are subsequently shortened at the non-reducing end, from which xylobiose moieties are liberated. Xylobiohydrolase activity was also observed during the hydrolysis of various xylooligosaccharides. SlXyn30A thus expands the group of glucuronoxylanases/xylobiohydrolases which has been hitherto represented only by several fungal GH30-7 members.

Keywords: GH30-7 subfamily; Sugiyamaella lignohabitans; glucuronoxylanase; glycoside hydrolase family 30; xylan; xylanase; xylobiohydrolase; yeast.

Figure 1

Amino acid sequence alignment of SlXyn30A with GH30-7 xylanases Talaromyces cellulolyticus TcXyn30B (GAM36763), TcXyn30C (GAM40414.1), TcXyn30A (GAM43270), Thermothelomyces thermophila TtXyn30A (AEO55025), Talaromyces purpureogenus (Penicillium purpurogenum) TpXynC (AKH40280), Bispora sp. BXylD (ADG62369.1), Trichoderma reesei TrXynVI (EGR45006.1), T. reesei TrXynIV (AAP64786.1), Acremonium alcalophilum AaXyn30A [7], and Talaromyces leycettanus TlXyn30A [10]. The secondary structure elements and numbering of TcXyn30B are shown on top, numbering of SlXyn30A is at the bottom. Arginine suggested to be responsible for the recognition of MeGlcA substitution is shown in blue and is marked by a blue up triangle. Longer β2-α2 loop in xylobiohydrolases is highlighted in yellow and the residue interacting with Xylp moiety occupying the −2a subsite is brown and marked by a brown down triangle.

Figure 2

Effect of temperature (a) and pH (b) on activity of SlXyn30A.

Figure 3

TLC analysis of hydrolysis products released from GX, Rho and AraX by SlXyn30A after 2 min, 1 h, 5 h, 24 h and after treatment with β-xylosidase (x). St—standards of linear XOs.

Figure 4

MALDI-ToF MS analysis of 5-day hydrolysate of GX by SlXyn30A.

Figure 5

HPLC analysis of hydrolysis of XOs by SlXyn30A. (a) Time course of Xyl₄ hydrolysis; (b) Analysis of hydrolysis products generated from Xyl₃-Xyl₆ after 30 min of reaction.

Figure 6

TLC analysis of hydrolysis products released from Xyl₄, MeGlcA³Xyl₄ and Xyl₃-NP by SlXyn30A after 5 min, 1 h, 5 h and 24 h. St—standards of linear XOs, St2—standard of 4-nitrophenyl β-d-xylopyranoside.

Figure 7

Various XOs tested as the substrates for SlXyn30A. The site of cleavage is denoted by an arrow, X marks compounds which were not attacked.