Heterologous Expression, Purification and Characterization of an Alkalic Thermophilic ß-Mannanase CcMan5C from Coprinopsis cinerea.

A endo-1,4-ß-mannanase (CcMan5C) gene was cloned from Coprinopsis cinerea and heterologously expressed in Pichia pastoris, and the recombinant enzyme was purified by Ni-affinity chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). CcMan5C hydrolyzed only locust bean gum galactomannan (LBG) but not α-mannan from S. cerevisiae or Avicel cellulose, oat spelt xylan, or laminarin from Laminaria digitata. CcMan5C exhibited distinctive catalytic features that were different from previously reported ß-mannanases. (1) CcMan5C is the first reported fungal ß-mannase with an optimal alkalic pH of 8.0-9.0 for hydrolytic activity under assay conditions. (2) CcMan5C is the first reported alkalic fungal ß-mannase with an optimal temperature of 70 °C for hydrolytic activity under assay conditions. (3) The organic solvents methanol, ethanol, isopropanol, and acetone at concentrations of 10% or 20% did not inhibit CcMan5C activity, while 10% or 20% isopropanol and acetone even enhanced CcMan5C activity by 9.20-34.98%. Furthermore, CcMan5C tolerated detergents such as Tween 20 and Triton X-100, and its activity was even enhanced to 26.2-45.6% by 1% or 10% Tween 20 and Triton X-100. (4) CcMan5C solution or lyophilized CcMan5C exhibited unchanged activity and even increasing activity after being stored at -20 °C or -80 °C for 12 months and retained above 50% activity after being stored at 4 °C for 12 months. These features make CcMan5C a suitable candidate for the detergent industry and paper and pulp industry.

An Aspergillus nidulans endo-ß-1,3-glucanase exhibited specific catalytic features and was used to prepare 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose with high antioxidant activity from barley ß-glucan and laminarin, respectively.

An endo-ß-1,3(4)-glucanase AnENG16A from Aspergillus nidulans shows distinctive catalytic features for hydrolysis of ß-glucans. AnENG16A hydrolyzed Eisenia bicyclis laminarin to mainly generate 3-O-ß-gentiobiosyl-d-glucose and hydrolyzed barley ß-glucan to mainly produce 3-O-ß-cellobiosyl-d-glucose. Using molecular exclusion chromatography, we isolated and purified 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose, respectively, from AnENG16A-hydrolysate of barley ß-glucan and E. bicyclis laminarin. Further study reveals that 3-O-ß-cellobiosyl-d-glucose had 8.99-fold higher antioxidant activity than barley ß-glucan and 3-O-ß-gentiobiosyl-d-glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin. Notably, 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose exhibited 148.9% and 116.0% higher antioxidant activity than laminaritriose, respectively, indicating that ß-1,4-linkage or -1,6-linkage at non-reducing end of ß-glucotrioses had enhancing effect on antioxidant activity compared to ß-1,3-linkage. Furthermore, 3-O-ß-cellobiosyl-d-glucose showed 237.9% higher antioxidant activity than cellotriose, and laminarin showed 5.06-fold higher antioxidant activity than barley ß-glucan, indicating that ß-1,4-linkage at reducing end of ß-glucans or oligosaccharides resulted in decrease of antioxidant activity compared to ß-1,3-linkage.

Accumulation and cross-linkage of ß-1,3/1,6-glucan lead to loss of basal stipe cell wall extensibility in mushroom Coprinopsis cinerea.

The mature basal stipe of mushroom Coprinopsis cinerea loses wall extensibility. We found that an endo-ß-1,3-glucanase ENG from C. cinerea could restore mature basal stipe wall extensibility via pretreatment such that the ENG-pretreated basal stipe walls could be induced to extend by chitinase ChiIII. ENG pretreatment released glucose, laminaribiose, and 3-O-D-gentiobiose-D-glucose from the basal stipe walls, consistent with ENG-digested products of ß-1,6-branched ß-1,3-glucan. Different effects of endo-ß-1,3-glucanase ENG and exo-ß-1,3-glucanase EXG pretreatment on the structure, amount and ratio (ß-1,3-glucoside bonds to ß-1,6-glucoside bonds) of products from the basal stipe and the apical stipe cell walls, respectively, and on the cell wall extensibility and the cell wall ultra-architecture of the basal stipes were analyzed. All results demonstrate that the more accumulation and cross-linkage of ß-1,6-branched ß-1,3-glucan with wall maturation lead to loss of wall extensibility of the basal stipe regions compared to the apical stipe cell walls.

A novel endo-ß-1,6-glucanase from the mushroom Coprinopsis cinerea and its application in studying of cross-linking of ß-1,6-glucan and the wall extensibility in stipe cell walls.

We previously reported that chitinases reconstituted heat-inactivated stipe cell wall extension in a steady and continuous extension profile by cleaving chitins cross-linked to various polysaccahrides, whereas, endo-ß-1,3-glucanases reconstituted heat-inactivated stipe wall extension in a profile of an initially fast extension and subsequent termination of extension due to its degradation of ß-1,3-glucan but not other polysaccharides such as ß-1,6-glucans cross-linked to chitins. Thus, a novel endo-ß-1,6-glucanase, GH30A, from Coprinopsis cinerea was cloned and characterized to study cross-linking of ß-1,6-glucan and wall extensibility in stipe walls. GH30A had higher activity and better thermophilicity than reported ß-1,6-glucanases. GH30A hydrolyzed pustulan having ß-1,6-linkages but not other polysaccharides without ß-1,6-linkages; GH30A did not cleave gentiobiose and single ß-1,6-linkage branches in laminarin from Laminaria digitata but cut consecutive ß-1,6-linkage branches in laminarin from Eisenia bicyclis. GH30A reconstituted heated-inactivated stipe cell wall extension with release of glucose and gentiobiose, indicating that ß-1,6-glucans were present and cross-linked to chitins in stipe walls, and cleaving ß-1,6-glucans cross-linked to chitins by GH30A led to wall loosening for extension. However, GH30A individually or in combination with endo-ß-1,3-glucanase reconstituted-stipe wall extension profile was similar to individual endo-ß-1,3-glucanase's, exploring that chitins were also cross-linked to other polysaccharides besides ß-1,3-glucans and ß-1,6-glucans.

NYD-OP7/PLC regulatory signaling pathway regulates deltamethrin resistance in Culex pipiens pallens (Diptera: Culicidae).

BACKGROUND: Investigation of insecticide resistance mechanisms is considered a vital first step towards the creation of effective strategies to control resistant mosquitoes and manage mosquito-borne diseases. Our previous study revealed that NYD-OP7 may be associated with deltamethrin resistance in Culex pipiens pallen. However, the precise function of NYD-OP7 in deltamethrin resistance is still unclear. In this study, we investigated the role of NYD-OP7 in the molecular mechanisms underlying pyrethroid resistance. RESULTS: Knockdown of NYD-OP7 not only increased the susceptibility of the mosquitoes to deltamethrin in vivo but also simultaneously repressed both expression and enzyme activity of its downstream effector molecule, phospholipase C (PLC) and expression of several insecticide resistance-related P450 genes. Knockdown of PLC also sensitized the mosquitoes to deltamethrin and reduced the expression of the P450 genes. CONCLUSIONS: Our results revealed that NYD-OP7 and its downstream effector PLC contribute to deltamethrin resistance by regulating the expression of P450s in Cx. pipiens pallens.

Preliminary characterization of putative structural cuticular proteins in the malaria vector Anopheles sinensis.

BACKGROUND: The insect cuticle protects against environmental stresses such as insecticides, physical injury, dehydration and pathogenic microorganisms. Structural cuticular proteins (CPs) are the primary components of the cuticle, and are of interest for physiology-based pest management methods. Anopheles sinensis CPs are poorly characterised at present, and therefore we performed whole-genome sequence analysis and re-analysed available transcriptome data to identify potential insecticide resistance-associated CPs. RESULTS: Genome analysis revealed 238 putative CPs belonging to 11 different families; 136 proteins possessing a Rebers and Riddiford motif (CPR), 12 CPs analogous to peritrophins with a single chitin-binding domain (CPAP1), eight CPs analogous to peritrophins with three chitin-binding domains (CPAP3), four proteins with a 44 amino acid motif (CPF), five CPF-like (CPFL) proteins, nine Tweedle proteins, three proteins of low complexity with alanine residues (CPLCA), 26 proteins of low complexity with conserved glycine residues (CPLCG), seven proteins of low complexity with invariant W residues (CPLCW), 27 proteins of low complexity with proline residues (CPLCP), and one protein with two or three copies of a C-X -C motif (CPCFC). Two CPs belonging to the CPR and CPAP3 families were over-expressed in a deltamethrin-resistant strain. CONCLUSION: These results establish an information framework for An. sinensis CPs that provides a basis for further molecular research. © 2017 Society of Chemical Industry.