Detection of Candida albicans Using a Manufactured Electrochemical Sensor.

Candida albicans is a commensal fungus that is responsible for a lot of nosocomial infections in immunocompromised people. Cell culture is currently the predominant method for diagnosing candidiasis, but it is time consuming. In this study, we developed a rapid screen procedure by devising a method for detecting C. albicans with the use of electrochemical sensors. Through this experiment, we propose a method for the detection of C. albicans in the system through the use of personal glucose meters. The hemicellulase was used to break down the cell wall of C. albicans to glucose and oligo, which can be detected by a glucose meter. The spiked samples were prepared suspending C. albicans in urine and serum, demonstrating the feasibility of the developed method in a real situation.

A flexible loop for mannan recognition and activity enhancement in a bifunctional glycoside hydrolase family 5.

BACKGROUND: An array of glycoside hydrolases with multiple substrate specificities are required to digest plant cell wall polysaccharides. Cel5E from Clostridium thermocellum and Cel5A from Thermotoga maritima are two glycoside hydrolase family 5 (GH5) enzymes with high sequence and structural similarity, but notably possess different substrate specificities; the former is a bifunctional cellulase/xylanase and the latter is a cellulase/mannanase. A specific loop in TmCel5A, Tmloop, is one of the most structurally divergent regions compared to CtCel5E and interacts with substrates, suggesting the importance for mannan recognition. METHOD: A Tmloop inserted CtCel5E and its related mutants were produced to investigate the role of Tmloop in catalysis. Crystal structure of CtCel5E-TmloopF267A followed by site-direct mutagenesis reveals the mechanism. RtCelB, a homolog with Tmloop was identified to have mannanase activity. RESULT: Tmloop incorporation enables CtCel5E to gain mannanase activity. Tyr270, His277, and Trp282 in the Tmloop are indispensable for CtCel5E-Tmloop catalysis, and weakening hydrophobic environment near the Tmloop enhances enzyme kcat. Using our newly identified loop motif to search for structurally conserved homologs in other subfamilies of GH5, we identified RtCelB. This homolog, originally annotated as a cellulase also possesses mannanase and xylanase activities. CONCLUSION: Our studies show that Tmloop enhances GH5 enzyme promiscuity and plays a role in catalysis. GENERAL SIGNIFICANCE: The study identified a loop of GH5 for mannan recognition and catalysis. Weakening the hydrophobic environment near the loop can also enhance the enzyme catalytic rate. Our findings provide a new insight on mannan recognition and activity enhancement of GH5.

Retention models and interaction mechanisms of benzene and other aromatic molecules with an amylose-based sorbent.

Stoichiometric displacement models have been widely used for understanding the adsorption mechanisms of solutes in chromatography systems. Such models are used for interpreting plots of solute retention factor versus concentrations of polar modifier in an inert solvent. However, these models often assume that dispersion forces are negligible and they are unable to account for solutes with significant aromatic interactions. In this study, a systematic investigation of the relationship between retention behavior and aromatic groups was performed using five simple aromatic molecules-benzene, naphthalene, mesitylene, durene, and toluene-with a commercially available amylose tris(3,5-dimethylphenylcarbamate)-based sorbent. The enthalpy changes of adsorption, determined from van't Hoff plots, were obtained separately in pure n-hexane and in pure isopropanol (IPA). In pure n-hexane, the solute adsorptions were driven by electrostatic interactions, favoring a T-shaped binding configuration (edge-to-face π-π interaction). The order of enthalpy change indicated the amount of effective T-shaped π-interactions. In pure IPA, solute adsorption was dominated by dispersion forces, favoring a sandwich binding configuration (face-to-face π-π interaction). The adsorption isotherms of toluene revealed that in pure IPA and in pure n-hexane, the isotherms were linear. The results suggested that the high solvent strength of IPA weakened the interactions between aromatic molecules. The retention behavior of the benzene, naphthalene, mesitylene, and durene as a function of IPA concentration was investigated. U-shaped retention curves were found for all aromatic solutes. A new retention model for monovalent aromatic solutes was developed for describing the U-shaped curves. Three key dimensionless groups were revealed to control the retention behavior. The models suggested that solvophobic interactions should be accounted for in the retention models used to investigate the retention behaviors of solutes associated with aromatic groups.

Elucidation of adsorption mechanisms of solvent molecules with distinct functional groups on amylose tris(3,5-dimethylphenylcarbamate)-based sorbent.

Although polysaccharide derivative-based sorbents have been widely used for chiral separation, for a long time it remained unclear how these CSPs interact with the molecules associated with different functional groups. In this study, six molecules were chosen for retention behavior studies: acetone (AC), tetrahydrofuran (THF), methanol (MET), isopropanol (IPA), tert-butanol (TBA), and benzene (BZN). An immobilized amylose carbamate stationary phase, amylose tris(3,5-dimethylphenylcarbamate)-based sorbent, or Chiralpak IA, was used. Van't Hoff plots of ln k versus 1/T showed that alcohol molecules may simultaneously form two H-bonds with the IA sorbent. The results of density functional theory simulations and IR spectra support this inference showing that alcohol may bind with amide groups in three possible configurations. Frontal tests of AC and IPA were performed to estimate adsorbed solute concentration. Langmuir isotherm for IPA adsorption and mass action model for IPA self-aggregation were used for analyzing the IPA frontal results. Average IPA aggregation numbers range from 1.4 to 2.3. More than fifty percent of IPA molecules were found to be in aggregate form. From the frontal test results, thermodynamic properties of the adsorptions were determined. Retention behaviors of the five solutes as a function of IPA concentration were investigated. The absolute values B of the slopes from plots of the logarithms of the retention factor versus the logarithms of the IPA concentration increase in the order THF

Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum.

We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.