Inhibitory effect of lignin on the hydrolysis of xylan by thermophilic and thermolabile GH11 xylanases

BACKGROUND: Enzymatic hydrolysis of lignocellulosic biomass into platform sugars can be enhanced by the addition of accessory enzymes, such as xylanases. Lignin from steam pretreated biomasses is known to inhibit enzymes by non-productively binding enzymes and limiting access to cellulose. The effect of enzymatically isolated lignin on the hydrolysis of xylan by four glycoside hydrolase (GH) family 11 xylanases was studied. Two xylanases from the mesophilic Trichoderma reesei, TrXyn1, TrXyn2, and two forms of a thermostable metagenomic xylanase Xyl40 were compared. RESULTS: Lignin isolated from steam pretreated spruce decreased the hydrolysis yields of xylan for all the xylanases at 40 and 50 °C. At elevated hydrolysis temperature of 50 °C, the least thermostable xylanase TrXyn1 was most inhibited by lignin and the most thermostable xylanase, the catalytic domain (CD) of Xyl40, was least inhibited by lignin. Enzyme activity and binding to lignin were studied after incubation of the xylanases with lignin for up to 24 h at 40 °C. All the studied xylanases bound to lignin, but the thermostable xylanases retained 22–39% of activity on the lignin surface for 24 h, whereas the mesophilic T. reesei xylanases become inactive. Removing of N-glycans from the catalytic domain of Xyl40 increased lignin inhibition in hydrolysis of xylan when compared to the glycosylated form. By comparing the 3D structures of these xylanases, features contributing to the increased thermal stability of Xyl40 were identified. CONCLUSIONS: High thermal stability of xylanases Xyl40 and Xyl40-CD enabled the enzymes to remain partially active on the lignin surface. N-glycosylation of the catalytic domain of Xyl40 increased the lignin tolerance of the enzyme. Thermostability of Xyl40 was most likely contributed by a disulphide bond and salt bridge in the N-terminal and α-helix regions. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13068-022-02148-4

High level secretion of cellobiohydrolases by Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of <it>Saccharomyces cerevisiae </it>for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.</p> <p>Results</p> <p>We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by <it>S. cerevisiae</it>. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of <it>cbh1 </it>and <it>cbh2 </it>genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing <it>S. cerevisiae </it>strains with the addition of beta-glucosidase.</p> <p>Conclusions</p> <p>Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.</p

Piloting gender-oriented colorectal cancer screening with a faecal immunochemical test: population-based registry study from Finland

Objective To assess the feasibility and evaluate the performance of a relaunched colorectal cancer (CRC) screening programme with different cut-offs for men and women. Design Population-based registry study. Setting Nine municipalities in Finland which started CRC screening with faecal immunochemical test (FIT) in April 2019 with cut-off levels 70 mu g Hg/g faeces for men and 25 mu g Hg/g faeces for women. Participants Men (n=13 059) and women (n=14 669) aged 60-66 years invited to screening during the first programme year. Outcome measures Participation rates, positivity rates, detection rates of CRC and advanced adenoma (AA), and positive predictive values (PPV) of FIT for CRC and AA. Results Altogether 21 993 invitees returned stool samples. The participation rate of women (83.4%; 95% CI 82.8 to 84.0) was significantly higher than that of men (74.7%; 95% CI 73.9 to 75.4). The positivity rates were 2.4% (2.2 to 2.7) and 2.8% (2.5 to 3.1), respectively. In total, 37 CRCs and 116 AAs were detected. The detection rates of CRC and AA per 1000 participants were 1.8 (1.1 to 2.9) and 7.2 (5.6 to 9.1) for men and 1.6 (0.9 to 2.4) and 3.8 (2.8 to 5.0) for women. The PPVs per 100 positive tests were 6.6 (4.0 to 10.3) and 25.7 (20.6 to 31.4) for men and 6.4 (3.9 to 9.8) and 15.5 (11.6 to 20.2) for women. Conclusions The chosen FIT strategy narrowed the gap in the diagnostic performance between men and women especially in the detection of CRC. The participation rates were excellent. The levels of positivity and detection rates were moderate and need further action. The results indicate that gender-specific protocols can be introduced to organised CRC screening. It is yet to be seen whether they are more effective than a uniform screening protocol.</p

Modular Protein Architectures for pH-Dependent Interactions and Switchable Assembly of Nanocellulose

Protein engineering shows a wide range of possibilities for designing properties in novel materials. Following inspiration from natural systems we have studied how combinations or duplications of protein modules can be used to engineer their interactions and achieve functional properties. Here we used cellulose binding modules (CBM) coupled to spider silk N-terminal domains that dimerize in a pH-sensitive manner. We showed how the pH-sensitive switching into dimers affected cellulose binding affinity in relation to covalent coupling between CBMs. Finally, we showed how the pH-sensitive coupling could be used to assemble cellulose nanofibers in a dynamic pH-dependent way. The work shows how novel proteins can be designed by linking functional domains from widely different sources and thereby achieve new functions in the self-assembly of nanoscale materials.</p