Beechwood xylan

Restriction enzymes, DNA polymerases, and T4 DNA ligase were purchased from Thermo Fisher Scientific (USA), and endoglycosidase H from New England Biolabs (USA). The plasmid pJET1.2/Blunt Cloning Kit (Thermo Fisher Scientific) was used for the cloning of the xylanase gene. Plasmids pET28c(+) (Novagen, UK) and pPICZαA (Invitrogen, USA) were employed to construct expression and shuttle vectors. E. coli strains DH5α and ArcticExpress (DE3) RP were bought from Thermo Fisher Scientific and Novagen (Merck4Biosciences, France), respectively, whereas P. pastoris X-33 cells from Invitrogen. Birchwood xylan, cellulose, carboxymethyl cellulose, starch (Sigma-Aldrich, St. Louis, MO, USA), beechwood xylan (Megazyme, Ireland), and pullulan (Tokyo Chemical Industry, Tokyo, Japan) served as substrates. The chemical reagents used in this work were of molecular biology or pure analytical grade and purchased from Sigma-Aldrich and AppliChem (Darmstadt, Germany). All primers used in this study are listed in Table 1.

The substrate was 1% beechwood xylan (Megazyme, Wicklow, Republic of Ireland). Enzymes were dissolved in PC buffer (pH 6.0) at a final concentration of 0.5 μg/mL. The substrates were dissolved either in PC buffer with different pH values ranging from pH 3.0–8.0 or at different concentrations of NaCl. After reacting at 65°C for 10 min, the amount of reducing sugar was determined using the dinitrosalicylic acid reagent method (Miller, 1959 (link)), and the absorbance was measured at 540 nm using a SpectraMax M5 Microplate Reader (Bihe, Shanghai, China). One unit of xylanase activity (U) corresponded to the amount of enzyme that released 1 μmol of reducing sugar equivalent from xylan per minute.

The selected strains of xylose utilizing LAB from Eri silkworm midgut were investigated for their abilities to utilize the selected polysaccharides as the sole carbon source. Briefly, individual LAB isolate was spiked on MRS agar containing 0.5% (w/v) of single carbon source of individual polysaccharides including starch (Fisher Scientific, Loughborough, Leicestershire, UK), pectin (Fisher Scientific, Loughborough, Leicestershire, UK), beechwood xylan (Megazyme International, Bray, Co. Wicklow, Ireland), locust bean gum (Sigma Aldrich, St. Louis, MO, USA) or carboxymethyl cellulose (Sigma Aldrich, St. Louis, MO, USA) supplemented with bromocresol purple (0.04%, w/v). After incubation at 37 °C for 24 h, the bacterial growth and the yellow halo-formed surrounding colonies were observed. Further duplicated experiment was also carried out, but the bromocresol purple was replaced with trypan blue (0.01%, w/v) for detection of extracellular activities of amylase, pectinase, xylanase, β-mannanase, and cellulase, respectively. The clear zone formed surrounding colonies were observed after incubation at 37 °C for 24 h.

The hydrolytic activity of the commercial enzyme cocktails and TrMan5A was determined by separately incubating 0.1 mg/mL (protein concentration) of either preparation with different substrates. The β-mannanase activity was determined with 0.5% low viscosity locust bean gum (lvLBG) (Megazyme) as the substrate at pH 5.0 (50 mM acetate buffer) by DNS assay to detect the produced reducing sugars [42 (link),79 (link)]. Xylanase activity was determined similarly, but with 1% beechwood xylan (Megazyme) as the substrate in the same acetate buffer [42 (link)]. α-Galactosidase, β-glucosidase, β-xylosidase, and β-mannosidase activities were determined based on p-nitrophenol release from 1 mM pNP-α-D-galactopyranoside, pNP-β-D-glucopyranoside, pNP-D-xylopyranoside, and pNP-β-D-mannopyranoside, respectively, using the same acetate buffer [42 (link)]. The stability of Viscozyme L and TrMan5A at 0.1 mg/mL protein concentration was studied by incubating the enzyme (s) at 40 °C and pH 5.0 (100 mM acetate buffer) over 48 h and analyzing the residual enzyme activities at different time points (0, 4, 8, 24 and 48 h). In the enzyme stability measurements, the detected enzyme activities were normalized to those at 0 h and reported as % residual activity. The reactions were carried out in triplicate and mean values ± SD are presented.

T. asperellum ND-1 (GenBank no: MH496612) was selected from the soil by our group. pPICZαA vector, P. pastoris X-33, and Escherichia coli DH5α (Invitrogen, America) were applied for β-mannanase heterologous expression, respectively. p-Nitrophenyl (pNP)-β-d-galactopyranoside (pNPG), p-nitrophenyl-α-l-arabinofuranoside (pNPAf), p-nitrophenyl-β-d-cellobioside (pNPC), sodium carboxymethyl cellulose (CMC-Na), locust bean gum (LBG), and guar gum (GG) were purchased from Sigma-Aldrich (USA). Restriction enzyme SacI and PNGase F were purchased from New England Biolabs (Ipswich, MA, USA). Mannohexaose (M6), mannopentaose (M5), mannotetraose (M4), mannotriose (M3), mannobiose (M2), and beechwood xylan were purchased from Megazyme (Wicklow, Ireland). 3,5-Dinitrosalicylic acid (DNS), d-mannose, and commercial α-galactosidase (EC 3.2.1.22, Aspergillus niger) were purchased from Shanghai Aladdin Biochemical Technology (Shanghai, China).