Purification, characterization and partial amino acid sequences of thermo-alkali-stable and mercury ion-tolerant xylanase from Thermomyces dupontii KKU–CLD–E2–3

We investigated the properties of the low molecular weight thermo-alkali-stable and mercury ion-tolerant xylanase production from Thermomyces dupontii KKU-CLD-E2-3. The xylanase was purified to homogeneity by ammonium sulfate, Sephadex G–100 and DEAE–cellulose column chromatography which resulted 27.92-fold purification specific activity of 56.19 U/mg protein and a recovery yield of 2.01%. The purified xylanase showed a molecular weight of 25 kDa by SDS–PAGE and the partial peptide sequence showed maximum sequence homology to the endo-1,4-β-xylanase. The optimum temperature and pH for its activity were 80 °C and pH 9.0, respectively. Furthermore, the purified xylanase can maintain more than 75% of the original activity in pH range of 7.0–10.0 after incubation at 4 °C for 24 h, and can still maintain more than 70% of original activity after incubating at 70 °C for 90 min. Our purified xylanase was activated by Cu2+ and Hg2+ up to 277% and 235% of initial activity, respectively but inhibited by Co2+, Ag+ and SDS at a concentration of 5 mM. The Km and Vmax values of beechwood xylan were 3.38 mg/mL and 625 µmol/min/mg, respectively. Furthermore, our xylanase had activity specifically to xylan-containing substrates and hydrolyzed beechwood xylan, and the end products mainly were xylotetraose and xylobiose. The results suggested that our purified xylanase has potential to use for pulp bleaching in the pulp and paper industry.

Application of Plackett Burman and Box Behnken Design for the Optimization of a-glucosidase Production by Thermophillic Thermomyces dupontii

A consecutive optimization based on statistical approach was applied for a-glucosidase production by both wild and mutant T. dupontii. Plackett Burman design (PBD) with two levels was employed in order to screen the significant effect of different nutritional and physical parameters through submerged fermentation. Among all nine variables tested in PBD, incubation time, inoculum size and ammonium sulphate concentration were selected. The Box-Behnken approach was further applied for process optimization. The a-glucosidase production for both wild and mutant T.dupontii was obtained at 72 h of incubation, 1.25 mL inoculum size and 0.25% ammonium sulphate concentration with relatively 95% correlation between the experimentally predicted and observed values. The duration of maximum enzyme production in RSM was cost-saving and fast. The quadratic model was in satisfactory adjustment with the experimental data with high R2 value which describes 98.90% of response variability of the model. Moreover, the novel approach of this present work is that, consecutive optimization were applied for maximum a-glucosidase production using response surface methodology by both wild and mutant thermophillic T. dupontii. Results revealed that thermophillic mutant T. dupontii could be potential candidate for industrial applications.

Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation

ABSTRACT Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNAGly, were successfully adapted for genome editing in T. dupontii. We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii. The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus. Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents. IMPORTANCE Thermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii. In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii. Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

The Effect of Endo-1,4-β-Xylanase From Thermomyces Dupontii KKU−CLD−E2−3 on Eucalyptus Pulp Bleaching and Effluent Treatment

Endo-1,4-β-xylanase-chlorine dioxide bleaching of eucalyptus pulp and analysis of effluent was investigated. The eucalyptus pulp bleaching in D0 stage was prepared with enzyme dose 100 Unit of 10% consistency and incubated at 70 °C for 30 min. The brightness level was achieved up to 72.50 (% ISO). The kappa number and viscosity of eucalyptus pulp was found to be 1.70 and 8.90 (cp), respectively. The paper from pulp bleaching with enzyme has shown an increase in brightness, breaking length, bursting strength, and tearing resistance to be 89.60% IOS, 37.27 Nm/g, 138.81 kPa and 218.02 mN, respectively. The SEM and FTIR analysis of pulp fibers revealed a significance of morphological and structural changes. The analysis of the effluent also showed statistically-significant differences in TOC, BOD, TDS and TSS. The FTIR analysis of the effluent showed the organic compounds and chloride dioxin in cases of those treated by chemicals.