The anaerobic rumen fungus N. frontalis PMA02 was obtained from the culture collection of the Department of Agricultural Science, Korea National Open University and cultivated in a modified Lowe’s medium containing a mixture of 2% glucose, cellobiose, and starch (2:1:1) as carbohydrate sources (Kwon et al., 2009). Escherichi coli (E.coli) clones containing AXE homologs were selected from the constructed EST library (Kwon et al., 2009), and inserts of larger than 1 kb were first screened by colony polymerase chain reaction (PCR) using T3 and T7 primers (Figure 1A). One of the clones (NfAXE1) which contains a putative full-length ORF with a 5′ and a 3′ UTR was selected for further study. The software programs ProtParam (http://web.expasy.org/protparam/) and Predict Protein (https://www.predictprotein.org) were used to analyze the physical and chemical parameters of NfAXE1, and SWISS-MODEL, a structural bioinformatics web-server (http://swissmodel.expasy.org), was used to determine the tertiary structure of NfAXE1 (Gasteiger et al., 2005).

The open reading frame (ORF) with 981 bp was amplified by PCR using a forward (5′-GCCATATG AGAGCCAGTATT ATT-3′) and a reverse (5′-CCGGATCC ATTTTCTTCAG CAGAA-3′) primers, incorporating the NdeI and BamHI sites (underlined), respectively. The PCR products were then inserted into the NdeI and BamHI restriction sites of the expression vector pET28a (Novagen, Merck KGaA, Darmstadt, Germany) to generate pET28a-NfAXE1. The plasmid of pET28a-NfAXE1 was transformed into E.coli BL21 (DE3) cells and cultivated in 50 mL of lysogeny broth medium containing kanamycin (50 μg/mL) at 37°C with shaking at 250 rpm. When the optical density of the culture reached 0.5 at 600 nm, expression of the recombinant protein was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and decreasing the culture temperature to 25°C. After a 12 h incubation, cells were harvested by centrifugation at 2,500×g for 5 min and re-suspended in 8 mL of native binding buffer (Invitrogen, Carlsbad, CA, USA); following the addition of 8 mg of lysozyme, the cells in suspension were incubated for 30 min on ice and then ruptured using a US-300 sonicator (Nissei Corp., Osaka, Japan; six 10 s high-intensity bursts with a 10 s cooling period between each burst). The lysates were centrifuged at 3,000×g for 15 min and the supernatants collected. Eight mL of crude lysate were applied to a Ni-NTA agarose column (Invitrogen, USA) and washed four times with 8 mL of native washing buffer containing imidazole (75 mM, pH 6.0). The protein was eluted with 10 mL of elution buffer containing imidazole (250 mM, pH 8.0) and collected in 500 μL samples.

Protein concentrations were determined by Bradford method using bovine serum albumin as a standard (Bradford, 1976). Crude proteins (109 μg samples) and purified recombinant proteins (3 μg samples) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% SDS gel. After separation, the gel was stained with Coomassie Brilliant Blue solution containing 10% (v/v) phosphoric acid, 10% (w/v) ammonium sulfate, 0.12% (w/v) Coomassie Brilliant Blue G250, and 20% (v/v) methanol. For microfluidic gel electrophoresis, 3 μg of each protein sample was loaded to a microfluidic chip and analyzed using the Experion Pro260 automated gel electrophoresis system (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.

The sample with 20 μL of volume dissolved in 0.5 M potassium phosphate buffer (pH 6.0) was mixed with 0.2 μL of protein solution and distilled water to a final total volume of 180 μL. The reaction was initiated by adding 20 μL of 10 mM p-NA dissolved in dimethyl sulfoxide and 0.5 M potassium phosphate buffer (pH 6.0). After a 15 min incubation at 40°C, the amount of p-NA released was estimated by measuring the absorbance at 420 nm using an automated microplate-based multi-detection reader (FLUOstar Optima; BMG LABTECH, Offenburg, Germany). One enzyme unit was defined as the amount of enzyme which released 1 μM of p-NA per minute at 40°C. All assays were carried out at the previously determined optimum reaction temperature and pH.

For the assay using α-naphthyl acetate as substrate, 1.34 mL of 100 mM sodium phosphate buffer (pH 7.0), 0.5 μL of purified protein solution, and 0.15 mL of 5 mM α-naphthyl acetate dissolved in methanol were incubated together for 15 min at 39°C, following which the reaction was terminated by adding 1.5 mL of 10% (w/v) SDS containing 0.01% (w/v) 4′-amino-2,3′-dimethylazobenzene and the absorbance at 560 nm measured using an UV-Vis 8453 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Alpha-naphthol was used as a standard, and one enzyme unit was defined as the amount of enzyme releasing 1 μM of α-naphthol per minute.

In order to determine the substrate specificity of the purified recombinant protein toward acetylated xylan, birchwood xylan and beechwood xylan were acetylated according to the method of Johnson et al. (1988) using acetic anhydride as an acetyl group donor. First, the purified protein solution was mixed with 100 μL of 0.5 M potassium phosphate buffer (pH 6.0) and distilled water to a final total volume of 300 μL. Second, a 200 μL sample of 10% (w/v) acetylated either birchwood xylan or beechwood xylan was added to the protein solution to initiate the enzymatic reaction. After incubation at 40°C for 20 min, 10 μL of 0.5 M sulfuric acid was added to terminate enzyme reaction, and the reactions were transferred to microcentrifuge tubes and centrifuged at 8,000×g for 3 min. The supernatant with 30 μL of volume was then injected into an Agilent 1200 HPLC system (Agilent Technologies, USA) equipped with an Agilent 1200 VWD detector (Agilent Technologies, USA) and an Aminex HPX-87 column (Bio-Rad, USA) and separated using 5 mM H₂SO₄ as the mobile phase (flow rate 0.6 mL/min) at 25°C. One enzyme unit was defined as the amount of enzyme releasing 1 μM acetic acid per minute.

For testing the synergistic effect of AXE on xylanase activity, endo-1, 4-β-xylanase from Trichoderma longibrachiatum (X2629; Sigma, St. Louis, MO, USA) was mixed with recombinant NfAXE1 protein to make a standard solution of 250 U/mL xylanase and 54 U/mL NfAXE1. The enzyme mixture with 20 μL of volume was mixed with 100 μL of 2% (w/v) acetylated birchwood xylan and incubated at 40°C for 30 min, following which 1 ml of Tetrazolium Blue solution [0.05 M potassium sodium tartrate and 0.005% (w/v) Tetrazolium Blue Chloride in 0.05 M NaOH] was added and the reaction mixture boiled for 5 min to terminate the reaction (Jue and Lipke, 1985). Absorbance at 560 nm was measured using an Agilent UV-Vis 8453 spectrophotometer (Agilent Technologies, USA), with glucose as the standard. One enzyme unit was defined as the amount of enzyme releasing 1 μM of reducing sugar per minute at 40°C.

A previously constructed expressed sequence tag (EST) library (Kwon et al., 2009) was used to screen candidate clones containing the AXE homolog of N. frontalis PMA02 (NfAXE1). Three contigs and three singletons were identified as putative AXEs, which were subsequently subjected to re-analysis using colony PCR. Among the ESTs in the library, one colony of 1,494 bp insert (Korea Patent Number: 10-098605; Figure 1A) was homologous to an AXE isolated from Orpinomyces sp. PC-2 (AxeA; AF001178.1) and from Neocallimastix patriciarum (bnaI; U66251.1) with 86% and 77% of nucleotide sequence identities, respectively. Sequence analysis of the NfAXE1 exhibited very low identity (<20%) with all other AXEs in NCBI database and was deposited in the NCBI database (accession number DI175536.1). Sequence analysis revealed that the selected colony contains an ORF of 978 bp (Figure 1B), a 152 bp 5′ untranslated region (UTR), and a 365 bp 3′ UTR (Figure 1B), accounting for 1,494 bp of AXE sequence and thereby predicting as a nearly full-length cDNA of N. frontalis. This AXE was designated NfAXE1. Sequencing of the genomic DNA sequence encoding NfAXE1 and subsequent comparison with cDNA revealed that NfAXE1 genomic DNA did not have an intron and had only one exon with domain of unknown function (DUF) (Figure 1B).

The estimated molecular weight of NfAXE1 based on its deduced amino acid sequence was 36,505.9 Da with 326 amino acid residues, of which glutamic acid was the most abundant and tryptophan the least abundant (Figure 2). In terms of protein secondary structure, 25.77% of the amino acids were composed in nine α-helixes and 15.95% in 12 β-strands, with 16 predicted protein binding regions. According to ProtParam analysis results, NfAXE1 was classified as an unstable protein based on its aliphatic index of 86.13 and instability index (II) of 40.03, leading to an estimated half-life of 20 h in the yeast cell. The calculated isoelectric point (pI) was 4.54, with 63 negatively charged residues (Asp and Glu) and 38 positively charged residues (Arg, His, and Lys). The existence of highly hydrophobic sequences at the N-terminal region suggested the possible existence of a putative signal peptide with cleavage site probability 0.559 between residues 22 and 23.

The deduced amino acid sequence of NfAXE1 cDNA was homologous to AxeA (AAC14690) of Orpinomyces sp. PC-2 and bnaI (AAB69090) of N. patriciarum with 70.21% and 48.74% similarity, respectively (Figure 3). Phylogenetic analysis (Figure 4) revealed that NfAXE1 was located in the same clade with AxeA (AAC14690.1) from the anaerobic rumen fungus Orpinomyces sp. and bnaI (AAB69090.1) from N. patriciarum, as well as with WP_014546948.1 from Fibrobacter succinogenes. Partial sequence of the other AXE from N. frontalis (AAQ10006.1) showed low sequence similarity (9.62%) with NfAXE1. Sequence similarities of AXEs from aerobic fungi, including Tricoderma reesei (CAA93247.1), Aspergillus niger (CAA01634.1), and Fusarium oxysporum (ENH75054.1), were only 2.03%, 3.24%, and 2.04%, respectively.

After a 12 h induction with IPTG, recombinant protein was harvested and purified using a Ni-NTA agarose column (Figure 5A). During purification, the enzyme activity increased by 101.2 fold, with a yield of 24.4% and a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as the substrate (Table 1). Based on the results of microfluidic gel electrophoresis, the final protein product has a purity of 90% and a molecular mass of 43 kDa (Figure 5B), slightly bigger than predicted native NfAXE1 probably because of N-terminal his-tags. In an enzyme assay using p-NA as substrate, the recombinant NfAXE1 achieved peak enzyme activity at 40°C, with activity subsequently decreasing with increasing incubation temperature (Figure 6A). The enzyme activity of recombinant NfAXE1 increased with increasing pH, reaching peak activity under slightly alkaline conditions (Figure 6B).

Various substrates were tested to determine substrate specificity of the recombinant NfAXE1 under optimum conditions (40°C, pH 8). The amount of liberated acetic acid, a marker of AXE activity, was the highest with p-NA as substrate and lowest with acetylated birchwood xylan as substrate. When NfAXE1 was mixed with endo-1,4-β-xylanase from Trichoderma longibrachiatum, the amount of xylose released from acetylated birchwood xylan increased by 1.4-fold compared to the NfAXE1-free condition (Figure 7).