α cellulose

α-Cellulose, sulfuric acid (H₂SO₄;
95–98%), nitric acid (70%; HNO₃), cellulose acetate
(CA; 30 KDa), H₂O₂ (30
wt%), 2,9-Dimethyl-1,10-Phenanthroline (DMP) and copper(II) sulfate
were obtained from Sigma-Aldrich. Sodium hydroxide pellets were purchased
from Merck. Acetone and dimethyl sulfoxide (DMSO) were of technical
grade and purchased from VWR. Preparation of simulated sea water was
done according to ASTM standard D6691–17 without the presence
of any microorganisms rendering to average salinity of world sea water.
A phosphate buffer solution (0.1 M) was prepared from K₂HPO₄ and NaH₂PO₄ (Sigma-Aldrich)
with pH adjusted to 7.0 by H₂SO₄ (1 N, VWR)
and NaOH (1 N, VWR). All chemicals were used as-received.

Dimethyl sulfoxide (DMSO, 99.7%, anhydrous), tetrabutylammonium fluoride trihydrate (TBAF, 97%) and bis(ethylenediamine)copper(II) hydroxide solution (CED solution, 1 M in copper, molar ratio of ethylenediamine/copper of 2:1) were purchased from Sigma-Aldrich (Darmstadt, Germany). Four different samples of cellulose (one single batch each) have been studied with different average of DP (Table 1). Avicel^® (PH-101, Ph Eur, cellulose microcrystalline, batch n°BCBK2051V), α-cellulose (powder, batch n°BCBH3503V) and cotton fibers (cotton linters, medium fibers, batch n°MKBQ8042V) were purchased from Fluka (Sigma Aldrich, Darmstadt, Germany), and Vitacel^® (L600/30, Ph Eur, powdered cellulose, batch n°7120891215 X) from JRS Pharma (Rosenberg, Germany). Pullulan standards P 1720 kDa, P 970 kDa, P 636 kDa, P 318 kDa, P 184 kDa, P 100 kDa, P 45.5 kDa, P 20 kDa, P 9.2 kDa, P 5.9 kDa, P 1.1 Da and P 0.3 Da were purchased from Polymer Standards Service (PSS, Mainz, Germany).

The Escherichia coli EPI300™-T1^R clone harboring fosmid pCC1FOS carrying a chicken cecal metagenomic DNA fragment containing a xylanase gene was a gift from Dr Kenneth van Driel. All enzymes and dNTPs in this study were purchased from New England BioLabs Inc., USA, and Promega, USA. Plasmid DNA extraction and purification kit was purchased from GE Healthcare, UK. TALON Superflow Metal Affinity Resin (Clonetech) was purchased from TaKaRa (Otsu, Japan). The expression vector pET-32a (Novagen) was used for cloning and expressing the xylanase. E. coli Tuner (DE3)pLysS was used as expression host and was cultivated on Luria–Bertani medium (Difco). The enzyme substrates used were xylan from oat-spelt (Fluka), xylan from beechwood (Megazyme), α-cellulose (Sigma), carboxymethyl cellulose (Sigma), starch (Sigma), β-glucan from barley (Sigma), 4-nitrophenyl-β-D-xylopyranoside (Megazyme), 4-nitrophenyl-β-D-cellobioside (Sigma), and 4-nitrophenyl-α-D-galactopyranoside (Fluka). Molecular weight standard mix containing xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose (Megazyme) was the gift from Professor Khanok Ratanakhanokchai, KMUTT, Thailand. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

The strains used were C. uda (DSM 20108) purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and Cellulomonas sp. ATCC 21399 (DSM 20108) obtained from the American Type Culture Collection (Manassas, Virginia, USA). These two strains are identical and should both be referred to as C. uda.
The basal growth medium (referred to as BM) contained (per litre): NaCl, 1.5 g; (NH₃)₂SO₄, 6.2 g; (Na)₂HPO₄, 9.1 g; KH₂PO₄, 0.9 g; EDTA, 50 mg; MgSO₄·7H₂O, 0.2 g; ZnSO₄·7H₂O, 8 mg; FeSO₄·7H₂O, 20 mg; MnSO₄·H₂O, 15 mg; CaCl₂·2H₂O, 26 mg; MOPS, 41.8 g; and yeast extract, 300 mg. Prior to autoclaving (121 °C for 20 min), the pH was adjusted to 7.4 with 5 M NaOH. Biotin (1 mg l⁻¹) and thiamine (1 mg l⁻¹) were aseptically added to the autoclaved medium from filter-sterilized stock solutions. Cellulosic growth substrates, α-cellulose (Sigma, C-8002) and Avicel (Merck, Microcrystalline, 1.02331.0500), were added prior to autoclaving, whereas the soluble growth substrates, glucose and cellobiose, were added aseptically to the autoclaved medium through a 0.22-µm filter.

Corncobs were obtained domestically (Tainan, Taiwan) and were washed with deionized water. After drying at 105 °C, corncobs were mechanically grinded into particles and sieved through 40 mesh sieves (particle size smaller than 0.49 mm). All chemical reagents were purchased from commercial sources and used without further purification. Iron (III) chloride, o-phenanthroline, and dimethyl sulfoxide (DMSO) were purchased from Aldrich and J.T. Baker, respectively. Glucose and gluconic acid were purchased from Alfa Aesar. Hydrogen peroxide solution (35 wt% in H₂O), α-cellulose, and cellulase from Trichoderma reesei were purchased from Sigma-Aldrich. S. cerevisiae for fermentation was purchased from Algist Bruggeman.

All experiments were conducted with α-cellulose (Sigma Aldrich), D-glucose (Acros Organics) or D-fructose (Acros Organics). The reactions took place in a biphasic reaction medium consisting of an aqueous phase (demineralized water) acidified with HCl (Fisher Scientific, 37%) and an organic phase consisting of MIBK (Acros Organics, 99.5%).