Isolation and enzyme characterization of xylanase-producing thermophilic fungi

S. Boonlue1, T. Aimi2, T. Morinaga3 1 Faculty of Science, Khon Kaen University, 2Faculty of Agriculture, Tottori University, 3Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima

Abstract Xylanases are well known xylan-degrading enzymes that have great potential applications in the food, beverages, animal feeding, and paper and pulp bleaching industries. Therefore, in order to search for a new producer of xylanolytic thermophilic fungi and characterize the molecular and enzyme feature of the selective isolates, the thermophilic fungi were screened in Japanese soil at 50C. The Scytalidium thermophilum Af101-3 strain was found to be the best isolate that produced higher xylanase and corncob decomposing activity than the other strains isolated in Japanese soil. Phylogenetic analysis revealed the S. thermophilum strain isolated in this study is a genetically new isolate. A G/11 family xylanase-encoding gene was investigated in S. thermophilum Af101-3 which consisted of 1,613 bp and containing an intron of 72 bp long in an open reading frame of 708 bp. In addition, the xylanase production and the expression of xylanase-encoding gene by reverse transcription PCR (RT-PCR) of this fungus were investigated under carbon catabolite repression (CCR), and it was exhibited that its transcription of gene and xylanase production were not affected with the presence of 2% glucose. The xylanase from S. thermophilum Af101-3 was purified and characterized. Keywords: xylanase, G/11 family, characterization, corn cob, thermophilic fungi, Scytalidium thermophilum, xylanase encoding gene, carbon catabolite repression Objective In this study, we aimed to search for a new producer of xylanolytic thermophilic fungi and characterize a molecular and enzyme feature of the selective isolate. Conclusion Scytalidium thermophilum Af101-3 is the best xylanase-producing thermophilic fungus among all strains isolated from Japanese soil. The phylogenetic analysis showed the genetic divergence as a new isolate (Fig. 1) and found to be the first report on xylanase-producing fungus. The xylanase encoding gene in this fungus was investigated to be the G/11 family consisting of 1,613 bp in size which interrupted with an intron of 72 bp long in an open reading frame of 708 bp (Fig 2). The xylanase production and its gene expression were observed under carbon catabolic repressor (2% glucose) by RT-PCR approach. The result showed the both of transcription of gene and xylanase production were not affected with the presence of glucose (Fig. 3). The xylanase from the S. thermophilum Af101-3 was purified by the following methods consisting of ammonium sulfate precipitation, cation exchange chromatography, and polyacrylamide gel electrophoresis (PAGE) extraction (Table 1). The molecular mass of purified xylanase was 25.55 kDa which was determined by SDS-PAGE. Isoelectric focusing (IEF) showed pI of purified xylanase at approximately 9.3. The optimal pH and temperature for the action of the enzyme were at pH 8.0 and 60C, respectively. The purified xylanase was stable at more than 70% of maximum level at pH 5.0-7.0 and temperature between 40 and 60C. The activity of enzyme was strongly stimulated by Ca2+, and inhibited by Na+ and Zn2+. The Km and Vmax values of purified xylanase were 35.47 mg of xylan/ml and 1.05

mol/min/mg of protein, respectively. The xylose, xylobiose, xylotriose, and xylotetraose were main products of hydrolysis of various xylans (oat spelt, beechwood, birchwood) by crude supernatant. A similar result was obtained from hydrolysis of different xylans by purified xylanase except for xylose that could not be detected.
Talaromyces emersonii AF96-6

Th. lanuginosus (AJ131864) 100 Th. lanuginosus AF64-2 100 Th. lanuginosus AF81-3

S. thermophilum AF101-3 99 S. thermophilum AF74-1 100 S. thermophilum AF17 95 S. thermophilum AF64-1 100 S. thermophilum (AJ131855)

100

S. thermophilum (AJ131862)

S. thermophilum (AJ131861)

S. thermophilum (AJ131863)

H. grisea var thermoidea (AJ131856)

S. thermophilum (AJ131860)

H. insolens (AJ131857)

S. thermophilum (AJ131858)

S. thermophilum (AJ131859) 0.1

Fig. 2. Nucleotide and deduced protein sequence of the xylanase gene from S. thermophilum Af101-3 strain. Intron sequences are given in lowercase letters. The deduced protein sequence is given below the nucleotide sequence. Polyadenylation start site is underlined. The consensus sequence of CreA binding (5-SYGGGG-3) is double underlined. Arrowheads indicate directions of primers for amplification by PCR.

Fig. 1. NJ phylogenic tree from the nucleotide sequence analysis of rDNA-ITS. Scale bar shows distance values under tree and bootstrap values greater than 60 are shown in the tree. The tree is rooted by the nucleotide sequence of the Ta. emersonii Af96-6 strain isolated in this study. All strains isolated in this study are underlined.

Table 1. Summary of purification of xylanase from S. thermophilum Af101-3

Purification step Total activity (U) Culture supernatant Ammonium sulfate CM-Toyopearl 650M PAGE extraction 536.75 137.80 6.10 0.13 Total protein (mg) 3548.25 549.64 4.15 0.04 0.15 0.25 1.47 3.25 100 25.67 1.14 0.02 1.00 1.67 9.80 21.67 Sp. activity (U/mg) Recovery (%) Purification (fold)

28S

rRNAs
18S +Glucose 6 hrs A X A 8 hrs X A 10 hrs X -Glucose A X

bp
600 500 400

RT-PCR

Fig. 3. RT-PCR analysis of xylanase and actin gene in S. thermophilum Af101-3 Abbreviations: X, xylanase cDNA; A, actin cDNA

References: Boonlue, S., Aimi, T. and Morinaga, T. (2003) Molecular characterization of a xylanase-producing thermophilic fungus isolated from Japanese soil, Current Microbiology 47, 119-124. Boonlue, S., Aimi, T. and Morinaga, T. (2007) Nucleotide sequence of a G/11 family xylanase encoding gene in Scytalidium thermophilum, DNA Sequence, (Accepted).