Immobilization of mutated xylanase from Neocallimastix patriciarum in E. coli and application for kraft pulp biobleaching

As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Improving the Thermostability of a Fungal GH11 Xylanase via Fusion of a Submodule (C2) from Hyperthermophilic CBM9_1-2

Xylanases have been applied in many industrial fields. To improve the activity and thermostability of the xylanase CDBFV from Neocallimastix patriciarum (GenBank accession no. KP691331), submodule C2 from hyperthermophilic CBM9_1-2 was inserted into the N- and/or C-terminal regions of the CDBFV protein (producing C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2) by genetic engineering. CDBFV and the hybrid proteins were successfully expressed in Escherichia coli BL21 (DE3). Enzymatic property analysis indicates that the C2 submodule had a significant effect on enhancing the thermostability of the CDBFV. At the optimal temperature (60.0 °C), the half-lives of the three chimeras C2-CDBFV, CDBFV-C2, and C2-CDBFV-C2 are 1.5 times (37.5 min), 4.9 times (122.2 min), and 3.8 times (93.1 min) longer than that of wild-type CDBFV (24.8 min), respectively. More importantly, structural analysis and molecular dynamics (MD) simulation revealed that the improved thermal stability of the chimera CDBFV-C2 was on account of the formation of four relatively stable additional hydrogen bonds (S42-S462, T59-E277, S41-K463, and S44-G371), which increased the protein structure’s stability. The thermostability characteristics of CDBFV-C2 make it a viable enzyme for industrial applications.

Expression of Xylanase Gene from Pyromyces finnis in Pichia pastoris and Characterization of Recombinant Protein

The heterologous expression and characteristics of a new xylanase from Pyromyces finnis have been described. The endo-l,4-β-xylanase XylP (EC 3.2.1.8) consists of 223 amino acids and 19 residues of a putative signal peptide in the N-terminal region. The amino acid sequence of the mature protein has the greatest homology with the sequence of the native catalytic N-terminal domain of Neocallimastix patriciarum endo-l,4-β-xylanase (84%). A synthetic nucleotide sequence encoding a mature XylP protein was expressed in Pichia pastoris. The purified recombinant enzyme showed activity with birch xylan and arabinoxylan. When using birch xylan as a substrate, the optimum pH for the enzyme was 5.0, and the optimum temperature was 50 °C. The specific activity of the xylanase was 4700 U/mg protein, and Km and Vmax were equal to 0.51 mg/mL and 7395.3 umol/(min∙mg), respectively. The recombinant XylP protein showed moderate thermal stability and high pH stability, resistance to digestive enzymes and protein inhibitors of grain xylanases. It was also shown that the Mg2+, Co2+ and Li+ ions have a positive effect on the enzyme activity. xylanase, xylan, feed enzyme, Pichia pastoris, Pyromyces finnis The work was performed with the financial support of the Ministry of Education and Science of Russia (Unique Project Identifier RFMEFI60717X0180) using the Unique Scientific Installation -National Bioresource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA

Catalytic Efficiency Diversification of Duplicate β-1,3-1,4-Glucanases from Neocallimastix patriciarum J11

ABSTRACTFour types of β-1,3-1,4 glucanase (β-glucanase, EC 3.2.1.73) genes, designatedbglA13,bglA16,bglA51, andbglM2, were found in the cDNA library ofNeocallimastix patriciarumJ11. All were highly homologous with each other and demonstrated a close phylogenetic relationship with and a similar codon bias toStreptococcus equinus. The presence of expansion and several predicted secondary structures in the 3′ untranslated regions (3′UTRs) ofbglA16andbglM2suggest that these two genes were duplicated recently, whereasbglA13andbglA16, which contain very short 3′UTRs, were replicated earlier. These findings indicate that the β-glucanase genes fromN. patriciarumJ11 may have arisen by horizontal transfer from the bacterium and subsequent duplication in the rumen fungus. β-Glucanase genes ofStreptococcus equinus,Ruminococcus albus7, andN. patriciarumJ11 were cloned and expressed byEscherichia coli. The recombinant β-glucanases cloned fromS. equinus,R. albus7, andN. patriciarumJ11 were endo-acting and had similar substrate specificity, but they demonstrated different properties in other tests. The specific activities and catalytic efficiency of the bacterial β-glucanases were also significantly lower than those of the fungal β-glucanases. Our results also revealed that the activities and some characteristics of enzymes were changed during the horizontal gene transfer event. The specific activities of the fungal β-glucanases ranged from 26,529 to 41,209 U/mg of protein when barley-derived β-glucan was used as the substrate. They also demonstrated similar pH and temperature optima, substrate specificity, substrate affinity, and hydrolysis patterns. Nevertheless, BglA16 and BglM2, two recently duplicated β-glucanases, showed much higherkcatvalues than others. These results support the notion that duplicated β-glucanase genes, namely,bglA16andbglM2, increase the reaction efficiency of β-glucanases and suggest that the catalytic efficiency of β-glucanase is likely to be a criterion determining the evolutionary fate of duplicate forms inN. patriciarumJ11.