Starch-degrading enzymes from the brown-rot fungus Fomitopsis palustris

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Highlights

  • Two starch degrading enzymes were purified from a brown-rot fungus.

  • FpAmy13A showed higher activity at acidic conditions but was unstable.

  • FpGLA15A was active at neutral conditions with high thermal stability.

  • Different roles of the enzymes in the starch degradation were suggested.

Abstract

Brown-rot fungi preferentially degrade softwood and cause severe breakdown of wooden structures. At the initial stage of the brown-rot decay, penetrating hyphae of the fungi are observed in ray parenchyma. Since starch grains are known to be present in the ray parenchyma of sapwood, investigation of the functions and roles of the starch-degrading enzymes is important to understand the initial stage of brown-rot decay. We purified and characterized two starch-degrading enzymes, an α-amylase (FpAmy13A) and a glucoamylase (FpGLA15A), from the brown-rot fungus, Fomitopsis palustris, and cloned the corresponding genes. The optimal temperature for both enzymes was 60 °C. FpAmy13A showed higher activity at a broad range of pH from 2.0 to 5.0, whereas FpGLA15A was most active at pH 5.0–6.0. Notable thermal stability was found for FpGLA15A. Approximately 25% of the activity remained even after treatment at 100 °C for 30 min in sodium phosphate buffer at pH 7.0. These different characteristics imply the different roles of these enzymes in the starch degradation of wood.

Introduction

Wood-decay fungi, particularly basidiomycetes, strongly degrade wood components, and are classified into white-rot fungi and brown-rot fungi according to their wood-decay mechanisms. Brown-rot fungi are especially important to the carbon cycling of coniferous forests, as many brown-rot fungi preferentially degrade softwood. In brown-rot decay, it is proposed that the amorphous wood-polysaccharides are enzymatically depolymerized, while the primary degradation of crystalline cellulose is non-enzymatic [1]. Recently, Zhang et al. investigated the gene expression of wood-degradation enzymes during brown rot [2]. This research indicated that most glycoside hydrolases (GHs), such as endoglucanases degrading amorphous cellulose and hemicellulases, were up-regulated in the later stages of brown rot. On the other hand, GHs acting on pectin and starch were up-regulated in the initial stages of brown rot, suggesting that pectin and starch degradation is important in the initial stage of brown rot.

Softwood has a relatively uniform structure when compared with hardwood, and mainly consists of tracheids (90–95%) and ray parenchyma cells (5–10%). Tracheids provide for mechanical strength as well as water transport. Microscopic observation of brown-rotted wood revealed that the hyphae of the fungi initially go through bordered pits on the surface of tracheid [1]. The bordered pit has a torus containing pectin; thus, degrading pectin was suggested to be important for facilitating access by brown-rot fungi [3,4]. In our previous study, we purified a polygalacturonase, which is one of the pectin degrading enzymes [5]. In addition, the degradation of starch in ray parenchyma is also important in the initial stage of brown rot. Fungal hyphae were observed to be distributed in ray parenchyma, indicating the fungal degradation of starch to gain nutrients [6,7]. This process seemed to be involved in the initial loosening of wood cell walls and colonization through the growth of brown-rot fungi into wood. However, as far as we know, there are few studies on the enzymatic degradation of wood starch.

Starch generally consists of two kinds of polysaccharides with different structures; amylose and amylopectin. Amylose is a linear α-1,4-linked glucan, and amylopectin is an α-1,4-linked glucan with branches of short α-1,4-linked glucan by α-1,6-linkage. Many starch degrading enzymes have already been purified from archaea, bacteria and fungi, and α-amylase, β-amylase and glucoamylase are known to cleave α-1,4-linkages of starch and to produce several types of oligosaccharide, maltose and glucose, respectively.

Several starch-degrading enzymes from wood-decay fungi have been reported and registered on the Carbohydrate-active enzymes (CAZy) database (http://www.cazy.org/): α-amylase (GH family 13, EC 3.2.1.1) from Phanerochaete chrysosporium [8], glucoamylases (GH family 15, EC 3.2.1.3) from Fomitopsis palustris [9] and glucoamylases Lentinula edodes [10]. However, the enzymatic properties of these enzymes, including the relationship between wood-decay and starch degradation, have not been fully elucidated.

F. palustris is a typical brown-rot fungus, which has been used as a standard fungus for the performance evaluation of wood preservatives in Japan. In this study, two starch degrading enzymes, a GH family 13 α-amylase (FpAmy13A) and a GH family 15 glucoamylase (FpGLA15A) were purified from F. palustris and characterized to investigate their functions and roles in starch degradation during brown-rot decay.

Section snippets

Fungal strain and regents

F. palustris, strain FFPRI 0507 from Forest Products Research Institute of Japan, was used in this study. All reagents, unless otherwise mentioned, were obtained from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan) or Kanto Chemical Co., Inc. (Tokyo, Japan).

Growth conditions

F. palustris grown on potato dextrose agar (potato starch 4.0 g/L, dextrose 20 g/L and agar 15 g/L) was precultured in potato dextrose broth (potato starch 4.0 g/L and dextrose 20 g/L) at 26 °C for 7 days. The mycelia were recovered by

Purification of FpAmy13A and FpGLA15A

F. palustris was cultured in liquid medium containing soluble starch, and starch degradation enzymes were purified from the culture supernatant with 4 steps of column chromatography. When applying to TOYOPEARL Phenyl-650M column (the second step of the enzyme purification process), two peaks containing the hydrolytic activity toward soluble starch were observed (Fig. 1). The active fractions eluted at 15% (corresponding FpAmy13A) and approximately 8% (corresponding FpGLA15A) saturation of (NH4)2

Discussion

In the present study, a GH13 α-amylase (FpAmy13A) and a GH15 glucoamylase (FpGLA15A) from the brown-rot fungus F. palustris were purified and characterized, and the corresponding genes were identified. Brown-rot fungi powerfully degrade wood structures by both enzymatic and non-enzymatic reactions [16]. In the initial stage of brown rot, most hyphae are observed at the ray parenchyma [6,7]. These reports suggest that brown-rot fungi initially attack the starch grain in ray parenchyma, and

CRediT authorship contribution statement

Yuki Tanaka: Methodology, Investigation, Writing - original draft, Visualization, Funding acquisition. Naotake Konno: Methodology, Writing - review & editing, Funding acquisition. Tomohiro Suzuki: Methodology, Data curation. Naoto Habu: Methodology, Writing - review & editing, Supervision, Project administration.

Acknowledgments

This work was supported by a research grant from Utsunomiya University (http://www.utsunomiya-u.ac.jp/en/index.php) to the UU-COE. This research also was supported by a Grant-in-Aid for Scientific Research (No. 19K06166) from the Japan Society for the Promotion of Science (JSPS) to N. K. and a Research incentive for Research Fellowship for Young Scientists from JSPS to Y. T.

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    These authors contributed equally to this work (Co-first authors).

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