Kanna et al. AMB Express 2011, 1:15 http://www.amb-express.com/content/1/1/15
3 family. xylB in A. oryzae belongs to the GH43 family (Suzuki et al. 2010). Based on amino acid sequence, Bxl1 of Trichoderma reesei RutC-30 (Margolles-Clark et al. 1996), XlnD of Aspergillus nidulans (Perez-Gonzalez et al. 1998), and Aspergillus niger (van Peij et al. 1997,) belong to the GH 3 family (Suzuki et al. 2010). Further- more, heterologous expression of xylB of A. oryzae in Escherichia coli was more stable than endogenous XylB expression in A. oryzae (Suzuki et al. 2010) or heterolo- gous expression of xlnD from A. niger strain ATCC 10864 in Aspergillus awamori, which has similar activity on 4-nitrophenyl-b-D-xyloopyranoside to A. niger XlnD (Selig et al. 2008).
Acremonium cellulolyticus is a cellulase-producing fila- mentours fungus isolated in Japan (Yamanobe et al., 1987). Repeated UV and/or nitrosoguanidine (NTG) mutagenesis of the wild strain Y-94 was used to enhance cellulase productivity, and strains with high cellulase pro- ductivity (TN, C-1, and CF-2612) have been selected. Although the productivity of cellulase is quite high in A. cellulolyticus, its hemicellulase production is not sufficient. Recently, we have sequenced the whole genome of A. cellulolyticus (unpublished data) and could deduce many genes for saccharifying enzymes. Although there are only a few reports of successful transformation in A. cellu- lolyticus, we could successfully obtain many transformants with protoplast- PEG method. Therefore, we tried to enhance b-xylosidase productivity of this fungus by intro- ducing its b-xylosidase gene under strong promoter.
Among strains of A. cellulolyticus, CF-2612 has the highest cellulase productivity (Fang et al. 2009), but it underwent random mutagenesis and may have muta- tions at every site as well as cellulase related genes. And apparently random mutagenesis affected the growth rates of CF-2612 because it grows more slowly than other strains. Therefore, we used the wild type strain, Y-94, in our study.
Materials and methods Fungal strain and culture condition A. cellulolyticus Y-94 (FERM Number BP-5826) was cultured in 10 ml of medium in 100 ml flasks at 30°C with shaking at 200 rpm. The composition of the culture medium for A. cellulolyticus was described previously (Fang et al. 2009). Sampling was performed at 1, 3, and 7 days for analysis of gene expression using real-time PCR and/or enzyme activity. A. cellulolyticus strains were cul- tured in potato dextrose (PD) medium for cloning and transformation.
Measurement of the amount of ATP, b-xylosidase and b-mannosidase activity, and saccharification efficiency The amount of ATP measured based on fluorescence using a Rucifel-250 kit (Kikkoman, Tokyo Japan) and
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Lumitester C-100 (Kikkoman) according to the manu- facturers’ instructions.
Activities for b-xylosidase and b-mannosidase were measured using 100 l of 10 mM 4-nitrophenyl-b-D- xyloopyranoside (PNP-Xyl, Sigma, MO USA) or 4-nitrophenyl-b-D-mannopyranoside (PNP-Man, Sigma) as the substrates (final concentration is 1 mM), respectively. Fifty l of enzymes were incubated with 1 mM PNP-Xyl or PNP-Man at 45°C for 10 minutes in 850 l of 50 mM acetic acid buffer (pH.5). After 10 minutes, 500 l of 1 M NaCO3 was added. Because 4- nitrophenol which will be generated from substrates by enzymatic hydrolysis is a chromogenic substance, enzyme activities were assayed by measuring absor- bance at 420 nm by UV-2550 Spectrophotometer (Shi- madzu, Kyoto, Japan). One unit of the enzyme activity is defined as the amount of enzyme that produces 1
mol of p-nitrophenol per minute. For analysis of
saccharification efficiency, culture medium was centri- fuged at 9,000 g for 10 min to collect the supernatant containing the secreted enzyme. Enzyme solutions were incubated at 45°C in 50 mM acetic acid buffer with 4% xylooligosaccharides (Wako Pure Chemicals, Osaka JAPAN). The xylose concentration was mea- sured using a high performance liquid chromatography system (JASCO, Tokyo, Japan), under the conditions described previously (Buaban et al. 2010).
Cloning b-xylosidase gene from A. cellulolyticus A putative b-xylosidase gene, bxy3A, was identified in
cellulolyticus genome sequence information using
nidulans xlnD sequence as the query for a homology
search. In silico molecular cloning (in silico biology, Yokohama Japan) which is a software for gene analysis was used for homology search. Augstus 2.2 http://augus- tus.gobics.de/ which is a program for eukaryotic genome sequence was used for the prediction of genes. The b-xylosidase coding region was amplified using A. cellu- lolyticus CF-2612 genomic DNA as the template, and the cellobiohydrolase Ι (cbh1) promoter was amplified from Y-94 genome. For the extraction of genomic DNA, cells cultured in PD medium were collected by centrifu- gation, and 3 volumes of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) with 2% sodium dodecyl sulfate (SDS) were added to the cell pellet. The cell suspension was incubated at 50°C for 1 hr. Potassium acetate (5 M) was added to the cell suspension at one-tenth of total volume, and this mixture was incubated on ice for 1 hr. The mixture was centrifuged at 13,000 g for 10 min, and the supernatant was subjected to two rounds of phenol-chloroform treatment, and ethanol precipitation was performed to obtain genomic DNA. The DNA was incubated with RNaseA (Nippon gene, Toyama, Japan) at 37°C for 1 hr to degrade contaminating RNA.