Characterization of a Salt-Tolerant and Cold-Adapted Xylanase from Bacillus cellulosilyticus

Abstract

A xylanase (Xyn10A) gene from the saline-alkali-tolerant microorganism Bacillus cellulosilyticus DSM 2522 was cloned and expressed in Escherichia coli BL21 (DE3). The open reading frame was composed of 1008 base pairs, and it encoded 335 amino acid residues belonging to glycosyl hydrolase family 10. The optimal temperature and pH of the purified Xyn10A were 40 °C and 8.0, respectively. The Xyn10A was sensitive to heat and showed obvious cold-adapted activity, retaining 38.3%, 55.7%, and 82.9% of the optimal activity at 4, 20, and 30 °C, respectively. Xyn10A also showed a high level of NaCl tolerance. The highest activity was observed with 1.5 M NaCl. The specific enzyme activity of Xyn10A was as much as 163.8 U/mg. Kinetic assays showed that Km, Vmax, and Kcat were 2.56 mg/mL, 202.5 μM/min/mg, and 132.6 /s, respectively. Additionally, the main hydrolysis products using birchwood xylan as substrate were xylobiose, xylotriose, and xylotetraose, as determined by thin layer chromatography analysis. As a cold-adapted and salt-tolerant enzyme, Xyn10A is an ideal candidate for further research and biotechnological applications.

High-Level Heterologous Expression of Endo-1,4-β-Xylanase from Penicillium citrinum in Pichia pastoris X-33 Directed through Codon Optimization and Optimized Expression

“…The K m(app) , V max and k cat were calculated to be 2.8 mg/mL, 310.7 µmol/min and 243 s −1 , respectively. The Pc Xyn11A k cat was higher than that previously reported for some other xylanases, such as those from Malbranchea cinnamomea and Bacillus cellulosilyticus with k cat values of 4.26 and 165.8 s −1 , respectively [50,51]. In comparison to other family 11 β-1,4 xylanases, the K m(app) of the Pc Xyn11A of this study was lower than that of the native Aspergillus niger xylanase (Native XylA), recombinant A. niger xylanase produced in Pichia pastoris (reXylA), Nonomuraea flexuosa xylanase produced in Trichoderma reesei (Nf Xyn11A) and P. citrinum xylanase produced in Y. lipolytica (re-xynA) [37,52,53], as shown in Table 3.…”

Identification and Characterization of a Novel, Cold-Adapted d-Xylobiose- and d-Xylose-Releasing Endo-β-1,4-Xylanase from an Antarctic Soil Bacterium, Duganella sp. PAMC 27433

“…Interestingly, rXylR was not only almost completely inhibited in the presence of 1 mM Ca 2+ or Cu 2+ but also noticeably suppressed by 1 mM Ni 2+ , Zn 2+ , or Fe 2+ , although it was partially inhibited by 1 mM Mg 2+ , Ba 2+ , or Co 2+ . The significant inhibitory effects of the indicated divalent cations on the rXylR activity were very comparable to the inhibitory or stimulatory effects of the same compounds on the biocatalytic activity of some cold-active GH10 endo-β-1,4-xylanases [ 14 , 16 , 18 , 27 , 30 ]. For example, the metal ions (each 1 mM), Ca 2+ and Cu 2+ , had no inhibitory effect on the activity of Psychrobacter sp.…”

“…strain HY-13 [ 26 ] and a bi-modular GH10 endo-β-1,4-xylanase with a ricin-type β-trefoil lectin domain-like domain from Luteimicrobium xylanilyticum HY-24 [ 22 ] are examples of enzymes that were formed as inactive protein aggregates in the recombinant cells. However, similar to other non-modular GH10 endo-β-1,4-xylanases [ 17 , 18 , 21 ], rXylR comprised of only a single catalytic GH10 domain was overproduced in an active form in E. coli BL21. Thus, the enzyme could be simply purified to electrophoretic homogeneity by basic affinity chromatography using a HisTrap HP column.…”

“…The molecular size (42.0 kDa) of rXylR was larger than that (37.0 kDa) of a thermolabile GH10 endo-β-1,4-xylanase from the Antarctic mold Cladosporium sp. [ 13 ] and that (39.0 kDa) of a cold-adapted GH10 endo-β-1,4-xylanase from Bacillus cellulosilyticus [ 18 ], however, its molecular size was similar to that (43.0 kDa) of a cold-adapted GH10 endo-β-1,4-xylanase from the Antarctic yeast Cryptococcus adeliae [ 11 ] and that (43.0 kDa) of a cold-active GH10 endo-β-1,4-xylanase from the marine bacterium Echinicola rosea sp. nov. JL3085 T [ 27 ].…”

“…Accordingly, like other microbial hemicellulases [ 2 ], many mesophilic and thermophilic endo-β-1,4-xylanases with different molecular structures and substrate specificities have also been identified and characterized [ 4 , 10 ]. However, only four cold-adapted or cold-active endo-β-1,4-xylanases from bacterial and fungal species in Antarctica have been genetically and functionally characterized to date [ 11 , 12 , 13 , 14 ], although some such enzymes from non-Antarctic organisms have been characterized at a molecular level [ 15 , 16 , 17 , 18 ]. In particular, 19 xylanolytic filamentous fungi were recently isolated from marine and terrestrial Antarctic environments [ 19 ] but the molecular properties of their related enzymes have not been characterized to date.…”