Molecular characterization and secreted production of basidiomycetous cell-bound β-glycosidases applicable to production of galactooligosaccharides

Cell-bound β-glycosidases of basidiomycetous yeasts show promise as biocatalysts in galactooligosaccharide (GOS) production. Using degenerated primers designed from Hamamotoa singularis (Hs) bglA gene, we newly identified three genes that encode cell-bound β-glycosidase from Sirobasidium magnum (Sm), Rhodotorula minuta (Rm), and Sterigmatomyces elviae (Se). These three genes, also named bglA, encoded family 1 glycosyl hydrolases with molecular masses of 67‒77 kDa. The BglA enzymes were approximately 44% identical to the Hs-BglA enzyme and possessed a unique domain at the N-terminus comprising 110 or 210 amino acids. The Sm-, Rm-, and Se-BglA enzymes as well as the Hs-BglA enzyme were successfully produced by recombinant Aspergillus oryzae, and all enzymes were entirely secreted to the supernatants. Furthermore, addition of some nonionic detergents (e.g. 0.4% [v/v] Triton-X) increased the production, especially of the Hs- or Se-BglA enzyme. Out of the BglA enzymes, the Se-BglA enzyme showed remarkable thermostability (∼70°C). Additionally, the Sm- and Se-BglA enzymes had better GOS yields, so there was less residual lactose than in others. Accordingly, the basidiomycetous BglA enzymes produced by recombinant A. oryzae would be applicable to GOS production, and the Se-BglA enzyme appeared to be the most promising enzyme for industrial uses.

Tergitol-15-S-9 inactivates SARS-CoV-2 and boosts immunoassay signals

Inactivation of SARS-CoV-2 virus is necessary to mitigate risk but may interfere with diagnostic assay performance. We examined the effect of heat inactivation on a prototype SARS-CoV-2 antigen immunoassay run on the ARCHITECT automated analyzer. Recombinant full-length SARS-CoV-2 nucleocapsid protein and virus lysate detection was reduced by 66 and 31%, respectively. Several nonionic detergents were assessed as inactivation alternatives based on infectivity in cultured Vero CCL81 cells. Incubation of SARS-CoV-2 in 0.1% Tergitol 15-S-9 for 10 min significantly reduced infectivity and increased the immunoassay signal for cultured lysate and patient specimens. Tergitol 15-S-9 can inactivate SARS-CoV-2 while preserving epitopes on the nucleocapsid protein for enhanced detection by immunoassay antibodies.

Purification of a Thermostable β-mannanase from Paenibacillus Thiaminolyticus - characterization and its Potential Use as a Detergent Additive

Endo-1, 4- β- D-mannanase (EC 3.2.1.78) is a glycoside hydrolase involved in random cleavage of β-1, 4- D-manno-pyranosyl linkages within mannans and heteromannans and generates branched and linear oligosaccharides. A β-mannanase was purified from a thermotolerant bacterium Paenibacillus thiaminolyticus isolated from a soil sample. Enzyme was purified to homogeneity with specific activity of 8812 U/mg protein. Sodium dodecyl sulfate (SDS) and native poly-acryl amide gel electrophoresis indicated that the purified mannanase is a monomeric protein with a molecular mass of 38 kDa. The purified enzyme was found to be maximally active at temperature and pH of 60°C and 7.0, respectively. It was stable at 55°C for 24 h and maintained more than 50 % activity up to 3 h at 60°C. The enzyme was very stable in the pH range of 5.0-9.0. Purified β-mannanase demonstrated high stability after 1 h of pre-incubation with most of the tested organic solvents. Enzyme retained significant stability in the presence of various detergent additives, commercially available detergents and dish washing liquids. The high compatibility and substantial stability in the presence of nonionic detergents and dishwashing liquids confirmed its utility as an additive to dish washing liquids and laundry detergents. Enzyme exhibited efficacious de-staining of heteromannan based stains of chocolate ice cream and salad dressing in the wash performance test for detergent application. It also exhibited anti-soil redeposition effect on cotton swatches treated with tennis court clay and heteromannans.

Potential of bacteriophages as disinfectants to control of Staphylococcus aureus biofilms

Background Staphylococcus aureus is the causative agent of chronic mastitis, and can form a biofilm that is difficult to completely remove once formed. Disinfectants are effective against S. aureus, but their activity is easily affected by environmental factors and they are corrosive to equipment and chemically toxic to livestock and humans. Therefore, we investigated the potential utility of a bacteriophage as a narrow-spectrum disinfectant against biofilms formed by S. aureus. In this study, we isolated and characterized bacteriophage vB_SauM_SDQ (abbreviated to SDQ) to determine its efficacy in removing S. aureus biofilms. Results SDQ belongs to the family Myoviridae and consists of a hexagonal head, long neck, and short tail. This phage can sterilize a 109 CFU/mL culture of S. aureus in 12 h and multiply itself 1000-fold in that time. Biofilms formed on polystyrene, milk, and mammary-gland tissue were significantly reduced after SDQ treatment. Fluorescence microscopy and scanning electron microscopy showed that SDQ destroyed the biofilm structure. Moreover, the titer of SDQ remained relatively high after the lysis of the bacteria and the removal of the biofilm, exerting a continuous bacteriostatic effect. SDQ also retained its full activity under conditions that mimic common environments, i.e., in the presence of nonionic detergents, tap water, or organic materials. A nonionic detergent (Triton X-100) enhanced the removal of biofilm by SDQ. Conclusions Our results suggest that SDQ, a specific lytic S. aureus phage, can be used to control biofilm infections. SDQ maintains its full activity in the presence of nonionic detergents, tap water, metal chelators, and organic materials, and can be used in combination with detergents. We propose this phage as a narrow-spectrum disinfectant against S. aureus, to augment or supplement the use of broad-spectrum disinfectants in the prevention and control of the mastitis and dairy industry contamination caused by S. aureus.

Use of substitute Nonidet P-40 nonionic detergents in intracellular tubulin polymerization assays for screening of microtubule targeting agents

Shell Chemical Company Nonidet P-40 has been used for decades in many biochemical assays as a nonionic, nondenaturing detergent; however, Shell no longer manufactures this product. Four commercially available substitutes were investigated and their activities titrated in an intracellular tubulin polymerization assay. Although claimed by the supply companies to be identical to the Shell Nonidet P-40, all four substitutes were about 10-fold more potent and needed to be diluted accordingly. As microtubule targeting drugs are a major class of anticancer agent, and many researchers use the intracellular tubulin polymerization assay, this information is important to help troubleshoot assay development with the new substitutes. As the Shell Nonidet P-40 has been used in many biochemical buffers, these results will be of general interest to the biochemical, cell, and molecular research community.